The present application claims priority from U.S. provisional patent application No. 61/358,188, filed 24/2010, the disclosure of which is incorporated herein by reference in its entirety.
A nucleotide/amino acid sequence listing in computer readable form, filed concurrently herewith and identified as follows, by reference to which it is incorporated in its entirety: a 957KB ACII (text) file named "sequence table 213134" was created at 21, 6/2010.
Peptide-based drugs are very potent drugs with relatively short duration of action and variable therapeutic index. The present disclosure relates to peptide-based prodrugs, wherein prodrug derivatives are designed to delay onset and prolong drug half-life. A delay in onset is advantageous because it allows systemic distribution of the prodrug prior to its activation. Thus, administration of the prodrug eliminates complications caused by peak activity at the time of administration and increases the therapeutic index of the parent drug.
Receptor recognition and subsequent processing of peptide and protein agonists are the major pathways for many peptide and protein-based drug degradation. Thus binding of a peptide drug to its receptor will cause a biological stimulus, but will also cause subsequent inactivation of the peptide/protein-induced pharmacological effect by enzymatic degradation of the peptide or protein. In accordance with the present disclosure, prodrugs that extend the biological half-life of a peptide or protein may be prepared based on strategies that inhibit the recognition of the prodrug by the corresponding receptor.
The prodrugs disclosed herein will eventually be chemically converted to a structure that is recognized by the receptor, where the speed of such chemical conversion will determine the time of onset and duration of the biological effect in vivo. The molecular design disclosed in this application relies on intramolecular chemical reactions that do not rely on other chemical additives or enzymes.
Preproglucagon is a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different glucagon-derived peptides, including glucagon involved in a variety of physiological functions including regulation of glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, as well as food intake (SEQ ID NO: 701), glucagon-like peptide-1 (GLP-1; amino acids 7-36 as provided in SEQ ID NO: 703 and SEQ ID NO: 704), glucagon-like peptide-2 (GLP-2; SEQ ID NO: 708), and oxyntomodulin (OXM; SEQ ID NO: 706).
Glucagon is a 29 amino acid peptide, corresponding to amino acids 33-61 of preproglucagon, while GLP-1 produced is a 37 amino acid peptide, corresponding to amino acids 72-108 of preproglucagon. GLP-1(7-36) amide (SEQ ID NO: 704; arginine amide at the C-terminus) or GLP-1(7-37) acid (SEQ ID NO: 703; glycine at the C-terminus) is a biologically active form of GLP-1 with essentially equal activity at the GLP-1 receptor.
Glucagon is a life-saving drug used for acute treatment of severe hypoglycemia. Oxyntomodulin is reported to have pharmacological ability to suppress appetite and reduce body weight. Clinical studies with GLP-1 receptor agonists or stable GLP-1 analogs demonstrate that this family of peptides is an effective treatment for type II diabetes. In addition, it may be intrinsically safer than insulin therapy because its glucose-dependent action precludes the possibility of hypoglycemia. Structure-activity relationship studies have shown that the N-terminal histidine of each of the three peptides (glucagon, GLP-1, and oxyntomodulin) is particularly important for the complete effect, and that the N-terminal extension form severely impairs biological potency.
Other peptides are known that are similar in structure to glucagon and GLP-1 and have similar activity. For example, exendin-4 is a peptide present in the saliva of the sheilandin (Gila monster) that is structurally similar to GLP-1 and, like glucagon and GLP-1, increases insulin release.
In addition, Gastric Inhibitory Peptide (GIP), also known as glucose-dependent insulinotropic peptide, is a member of the secretin family of hormones. GIP is derived from a 153 amino acid preprotein encoded by the GIP gene and circulates as a biologically active 42 amino acid peptide (SEQ ID NO: 707). The GIP gene is expressed in the small intestine as well as in salivary glands, and is a weak inhibitor of gastric acid secretion. In addition to its inhibitory effect in the stomach, GIP increases insulin release by islet beta cells when administered at physiological doses in the presence of glucose. GIP is believed to act as an intestinal factor that stimulates insulin release from the pancreas, and may play a physiological role in maintaining glucose homeostasis.
Osteocalcin (SEQ ID NO: 709) is a non-collagenous protein found in bone and dentin. It is secreted by osteoblasts and is thought to play a role in mineralization and calcium ion homeostasis. Osteocalcin is reported to act as a hormone in the body, causing beta cells in the pancreas to release more insulin, while directing adipocytes to release the hormone adiponectin, which increases sensitivity to insulin.
One disadvantage associated with the therapeutic use of bioactive peptides such as osteocalcin, GIP, glucagon, GLP-1 and oxyntomodulin is their extremely short half-life in plasma (about 2 minutes for glucagon and GLP-1). Thus, in order to obtain proper glycemic control, it is necessary to continuously administer the native glucagon related peptide for a long period of time. The short half-life of glucagon and GLP-1 related peptides is caused by the rapid degradation of dipeptidyl peptidase IV (DPP-IV), which cleaves between the second and third amino acids. This cleavage not only inactivates the native peptide, but in the case of glucagon and GLP-1, the shortened form may be a functional antagonist at its corresponding receptor. Thus, there is a need for longer acting variants of GIP, glucagon, GLP-1, and oxyntomodulin peptides, as well as related peptides, to achieve the full therapeutic potential of these drug mechanisms of action.
Detailed description of the preferred embodiments
The present disclosure describes formulations of prodrug derivatives of biologically active polypeptides that are useful for the treatment of diseases such as diabetes, obesity, and the like. More specifically, the prodrugs disclosed herein are formulated to prolong the half-life of the parent bioactive peptide or protein, while allowing the prodrug to be subsequently activated by non-enzymatic degradation mechanisms. The ideal prodrug should be soluble in water under physiological conditions (e.g., pH 7.2 and 37 ℃) and its powder form should be stable over long term storage. It should also be immunosilent and should have low activity compared to the parent drug. In some embodiments, the prodrug should exhibit no more than 10% of the activity of the parent drug. In some embodiments, the prodrug exhibits less than about 10%, less than about 5%, about 1%, or less than about 1% activity compared to the parent drug. In addition, the prodrug should be converted to the active drug in a defined amount over a defined period of time when injected into the body. As disclosed herein, applicants provide general techniques to produce prodrugs of known biologically active polypeptides selected from the glucagon superfamily peptides (including glucagon related peptides and osteocalcin, as well as analogs, derivatives and conjugates of such polypeptides) that meet each of these objectives.
More specifically, provided are chemically useful compositions comprising a sequence of a glucagon superfamily peptideA reverse prodrug, the glucagon superfamily peptide including, for example, a glucagon related peptide or osteocalcin or an analog, derivative or conjugate thereof, and modified to have a dipeptide prodrug element covalently bound to the peptide through an amide bond. Covalent attachment of the dipeptide prodrug element to the active site of the glucagon superfamily peptide inhibits the activity of the polypeptide until the dipeptide prodrug element is cleaved. In some embodiments, there is provided a "non-enzymatically active half-life" (t) under physiological conditions1/2) Between about 1 to about 720 hours of the prodrug. Physiological conditions disclosed herein are meant to include a temperature of about 35-40 ℃ and a pH of about 7.0-about 7.4, more typically a pH of 7.2-7.4 and a temperature of 36-38 ℃.
Advantageously, the rate of cleavage, and hence the activation of the prodrug, depends on the structure and stereochemistry of the dipeptide prodrug element. The prodrugs disclosed herein are ultimately chemically converted to structures recognized by the drug's natural receptors, where the rate of such chemical conversion determines the onset and duration of biological action in vivo. The molecular design disclosed in this application relies on intramolecular chemical reactions that are independent of other chemical additives or enzymes. The rate of conversion is controlled by the chemical nature of the dipeptide substituent and the cleavage of the dipeptide substituent under physiological conditions. Since physiological pH and temperature are tightly controlled within well defined ranges, the rate of conversion from prodrug to drug has high intra-and inter-patient reproducibility.
Prodrugs disclosed herein are provided that have an extended half-life as a result of being in the prodrug form for at least about 1 hour and in some embodiments greater than about 20 hours. In some embodiments, the half-life of the prodrug is about 1, 6, 8, 12, 20, 24, 48, or 72 hours. In some embodiments, the half-life of the prodrug is about 100 hours or more, including half-lives of up to about 168, 336, 504, 672, or 720 hours, and it is converted to the active form under physiological conditions by a non-enzymatic reaction driven by inherent chemical instability. In some embodiments, the non-enzymatic activation of the prodrug, t1/2For a period of between 1 and 100 hours, more usually between 12 and 72 hours, e.g.T between 12 and 48 hours and between 48 and 72 hours, and in some embodiments, measured by incubating the prodrug in a phosphate buffered solution (e.g., PBS) at 37 ℃ and pH 7.21/2Between 24 and 48 hours. In another embodiment, the non-enzymatic activation of the prodrug, t1/2The time is between 1 and 6 hours, for example about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours. In another embodiment, the non-enzymatic activation of the prodrug, t1/2The time is between 6 and 24 hours. Using the formula t1/20.693/k, where 'k' is the first order rate constant for degradation of the prodrug, to calculate the half-life of each prodrug. In some embodiments, activation of the prodrug occurs after cleavage of the amide bond linked dipeptide and formation of the diketopiperazine or the diketomorpholine and release of the active polypeptide drug. Certain dipeptides consisting of natural non-coding and/or synthetic amino acids have been shown to facilitate intramolecular breakdown under physiological conditions to release the active polypeptide.
According to some embodiments, there is provided a prodrug comprising a glucagon superfamily peptide of structure a-B-Q or osteocalcin or an analog, derivative or conjugate thereof. In this embodiment, Q is a peptide, A is an amino acid or hydroxy acid, and B is an N-alkylated amino acid. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. A and B together represent a dipeptide prodrug element linked to Q through the formation of an amide bond between the amines of A-B and Q. In some embodiments, at least one of amino acids A, B or Q to which a-B is attached is a non-coding amino acid. Further, in some embodiments, the dipeptide prodrug element is selected wherein chemical cleavage of a-B from Q is complete by at least about 90% in PBS under physiological conditions in about 1 hour to about 720 hours. In yet another embodiment, the amino acids of the dipeptide are selected wherein the cleavage half-life of A-B from Q is no more than 2-5 times the cleavage half-life of A-B from Q in a solution comprising a DPP-IV protease (including, e.g., human serum) under physiological conditions and in PBS.
According to some embodiments, the aliphatic amino group (e.g., primary amine) of Q, including, for example, the N-terminal amine or the amino group of the amino acid side chain, is modified by covalent attachment of the dipeptide prodrug element via an amide bond. In some embodiments, the dipeptide prodrug element is linked, directly or through a linking moiety, to a side chain amino group of an amino acid present in Q. In some embodiments, the linking moiety comprises an acyl or alkyl group bearing an amine. In some embodiments, a glucagon superfamily peptide, such as a glucagon related peptide, is provided comprising an acyl group or an alkyl group covalently linked to an amino acid at position 10 or 20 of the glucagon superfamily peptide, wherein the acyl group or the alkyl group further comprises a dipeptide prodrug element linked to the acyl group or the alkyl group by an amide bond. For example, embodiments contemplate a prodrug attached directly or through a linking group to the amino group of Q, and an acyl or alkyl group is attached directly or through a linking moiety to the prodrug.
In some embodiments, the dipeptide prodrug element is directly attached to an amino acid side chain, wherein the amino acid has the following general structure:
wherein n is an integer from 1 to 4.
Alternatively, the dipeptide prodrug element may be linked to an amino substituent present on an aromatic ring of an aromatic amino acid, including, for example, an aromatic amino acid selected from the group consisting of amino-Phe, amino-naphthyl (napthyl) alanine, amino tryptophan, amino-phenyl-glycine, amino-homophenylalanine, and amino tyrosine. In some embodiments, the dipeptide prodrug element is attached to an aromatic amino group of an amino acid having the general structure:
Wherein m is an integer of 0 to 3. In some embodiments, the dipeptide prodrug element is linked to the 4-amino group of an amino acid having the general structure:
wherein m is an integer of 0 to 3. In some embodiments, the dipeptide prodrug element is attached to a side chain amino group of lysine or an aromatic amino group of 4-aminophenylalanine (replacing the native phenylalanine or tyrosine residue of the biologically active peptide). In some embodiments, the dipeptide prodrug element is linked to a primary amine present on an internal amino acid of a glucagon superfamily peptide including a glucagon related peptide or osteocalcin or an analog, derivative or conjugate thereof.
In some embodiments, the dipeptide prodrug element has the general structure a-B, where a is an amino acid or a hydroxy acid and B is an N-alkylated amino acid that can be bound to a primary amino group of such a peptide through an amide bond to produce the corresponding prodrug of the peptide. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. In some embodiments, a and B are selected such that when the a-B dipeptide is bound to the primary amine of such peptide via an amide bond, chemical cleavage of a-B from the peptide is complete by at least about 90% in about 1 hour to about 720 hours in PBS under physiological conditions. In some embodiments, a and/or B is an amino acid in the D stereoisomer configuration. In some exemplary embodiments, a is an amino acid in the D stereoisomer configuration and B is an amino acid in the L stereoisomer configuration. In some exemplary embodiments, a is an amino acid in the L stereoisomer configuration and B is an amino acid in the D stereoisomer configuration. In some exemplary embodiments, a is an amino acid in the D stereoisomer configuration and B is an amino acid in the D stereoisomer configuration.
According to some embodiments, the dipeptide prodrug element may be further modified to include a hydrophilic moiety. In some embodiments, the hydrophilic moiety is a polyethylene glycol chain. According to some embodiments, a 40k or greater polyethylene glycol chain is covalently bonded to the side chain of the a or B amino acid of the dipeptide prodrug element. In another embodiment, the dipeptide prodrug element is additionally or alternatively acylated or alkylated with a fatty acid or bile acid or salt thereof (e.g., C4-C30 fatty acid, C8-C24 fatty acid, cholic acid), C4-C30 alkyl, C8-C24 alkyl, or an alkyl group comprising a steroid moiety of a bile acid. The "A" amino acid of the dipeptide prodrug element may include, for example, d-lysine covalently bonded to an acyl or alkyl group through its side chain amino group, or d-cysteine covalently bonded to a PEG molecule through its side chain thiol group. The dipeptide prodrug element may be bound directly to the hydrophilic moiety, acyl group or alkyl group, or bound to the hydrophilic moiety, acyl group or alkyl group through a spacer, as described herein. Alternatively, the dipeptide prodrug element may be linked to a storage protein (depotprotein), such as dextran or a large PEG molecule (greater than or equal to 80,000 daltons), which serves to sequester the prodrug at the injection site until the dipeptide is cleaved to release the active bioactive peptide. Additional modifications of the dipeptide prodrug are described below in relation to the glucagon related peptide section.
The dipeptide prodrug element is designed to cleave according to an intramolecular chemical reaction that is independent of other chemical additives or enzymes. More specifically, in some embodiments, the dipeptide structure is selected to be resistant to cleavage by peptidases present in mammalian serum, including, for example, dipeptidyl peptidase IV (DPP-IV). Thus, in some embodiments, the rate of cleavage of the dipeptide prodrug element from the biologically active peptide is not significantly increased (e.g., greater than 2X) when the reaction is carried out in the presence of a serum protease using physiological conditions, as compared to a reaction carried out in the absence of the protease. Thus, under physiological conditions, the half-life of cleavage of a-B from a biologically active peptide in PBS is no more than 2, 3, 4 or 5 times the half-life of cleavage of a-B from a biologically active protein in a solution comprising DPP-IV protease. In some embodiments, the solution comprising the DPP-IV protease is serum, more particularly mammalian serum, including human serum.
According to some embodiments, the amino acid of the glucagon superfamily peptide to which a or B or a-B of the dipeptide prodrug element is attached is a non-coding amino acid. In some embodiments, an amino groupThe acid "B" is N-alkylated, but not proline. In some embodiments, the N-alkylating group of amino acid B is C1-C18Alkyl, and in some embodiments C1-C6An alkyl group.
According to some embodiments, prodrug derivatives of glucagon superfamily peptides, comprising dipeptide prodrug elements disclosed herein, may be combined with protease inhibitors (including particular DPP-IV inhibitors (e.g., such asMerck &Co, Inc)) as a means of delaying prodrug activation. In this embodiment, the amino acids of the prodrug element are selected such that the dipeptide is an acceptable substrate for DPP-IV cleavage. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. The protease inhibitor may be administered in a separate composition, or the prodrug and protease inhibitor may be formulated in a single composition. If administered as a separate composition, the protease inhibitor is typically administered within 1-5 hours, 1-2 hours, 30 minutes, or 10 minutes of administration of the prodrug. In some embodiments, the two separate compositions are administered immediately sequentially.
In some embodiments, the dipeptide prodrug element has the general structure of formula I below:
wherein
R1、R2、R4And R8Independently selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (A) (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12Cycloalkyl or aryl; or R4And R8Together with the atom to which they are attached form C3-C6A cycloalkyl group;
R3is selected from C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) NH2、(C1-C18Alkyl) SH, (C)0-C4Alkyl) (C3-C6) Cycloalkyl group, (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7And (C)1-C4Alkyl) (C3-C9Heteroaryl) or R4And R3Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring;
R5is NHR6Or OH;
R6is H, C1-C8Alkyl, or R6And R2Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from H and OH.
It will be apparent to those skilled in the art that when W is1When N, the nitrogen atom is attached to H under physiological conditions.
In other embodiments, the dipeptide prodrug element has the general structure of formula I below:
wherein
R1、R2、R4And R8Independently selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group; or R4And R8Together with the atom to which they are attached form C3-C6A cycloalkyl group;
R3is selected from C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) NH2、(C1-C18Alkyl) SH, (C)0-C4Alkyl) (C3-C6) Cycloalkyl group, (C)0-C4Alkyl) (C2-C5Heterocyclic ringsBase), (C)0-C4Alkyl) (C6-C10Aryl) R7And (C)1-C4Alkyl) (C3-C9Heteroaryl) or R4And R3Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring;
R5is NHR6Or OH;
R6is H, C1-C8Alkyl, or R6And R1Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
In some embodiments, R8Is H, R5Is NHR6。
In some embodiments, the dipeptide prodrug element has the structure of formula I, wherein
R1And R8Independently is H or C1-C8An alkyl group;
R2and R4Independently selected from H, C1-C8Alkyl radical, C2-C8Alkenyl, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7And CH2(C3-C9Heteroaryl) or R1And R2Together with the atom to which they are attached form C3-C12Cycloalkyl or aryl;
R5is NHR6(ii) a And
R6is H or C1-C8An alkyl group.
In other embodiments, the dipeptide prodrug element has the structure of formula I, wherein
R1And R8Independently is H or C1-C8An alkyl group;
R2and R4Independently selected from H, C1-C8Alkyl radical, C2-C8Alkenyl, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7And CH2(C3-C9Heteroaryl) or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group;
R3is C1-C18An alkyl group;
R5is NHR6；
R6Is H or C1-C8An alkyl group; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
The half-life of a prodrug formed in accordance with the present disclosure depends on the substituents of the dipeptide prodrug element, the position of the substituents, and the amino acid to which the substituents are attached. For example, the prodrug may comprise a glucagon superfamily peptide, wherein the dipeptide prodrug element is linked through the alpha amino group of the N-terminal amino acid of the glucagon superfamily protein. In this embodiment, t1/2The prodrug, for example, for about 1 hour, comprises a dipeptide prodrug element having the following structure:
Wherein
R1And R2Independently is C1-C18An alkyl or aryl group; or R1And R2Through- (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen; and
R5is an amine.
In other embodiments, t is1/2The prodrug, for example, for about 1 hour, comprises a dipeptide prodrug element having the following structure:
wherein
R1And R2Independently is C1-C18Alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7(ii) a Or R1And R2Through- (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen;
R5is NH2(ii) a And
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
In addition, having a dipeptide prodrug element linked to the N-terminal alpha amino acid of a glucagon superfamily peptide and t1/2Is, for example, a prodrug for between about 6 to about 24 hours, comprising a dipeptide prodrug element having the structure:
wherein R is1And R2Independently selected from hydrogen, C1-C18Alkyl and aryl, or R1And R2By (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C8Alkyl and aryl groups; and R is5Is an amine;
with the proviso that R1And R2Both are not hydrogen, with the proviso that R4Or R8One of which is hydrogen.
In some embodiments, there is a dipeptide prodrug element linked to the N-terminal alpha amino acid of a glucagon superfamily peptide and t1/2Is, for example, a prodrug for between about 12 to about 72 hours or, in some embodiments, between about 12 to about 48 hours, comprises a dipeptide prodrug element having the structure:
wherein R is1And R2Independently selected from hydrogen, C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C4Alkyl) NH2And (C)0-C4Alkyl) (C6-C10Aryl) R7Or R is1And R2By (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C8Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7；
R5Is NH2(ii) a And
R7selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen;
with the proviso that R1And R2Both are not hydrogen, with the proviso that R4Or R8At least one of which is hydrogen.
In some embodiments, there is a dipeptide prodrug element linked to the N-terminal amino acid of a glucagon superfamily peptide and t1/2A prodrug, for example, for between about 12 to about 72 hours, or in some embodiments, between about 12 to about 48 hours, comprising a dipeptide prodrug element having the structure:
wherein R is1And R2Independently selected from hydrogen, C1-C8Alkyl and (C)1-C4Alkyl) NH2Or R is1And R2By (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C8Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-6 membered heterocyclic ring;
R4selected from hydrogen and C1-C8An alkyl group; and
R5is NH2；
With the proviso that R1And R2Both are not hydrogen.
In other embodiments, there is a dipeptide prodrug element linked to the N-terminal amino acid of a glucagon superfamily peptide and t1/2A prodrug, for example, for between about 12 to about 72 hours, or in some embodiments, between about 12 to about 48 hours, comprising a dipeptide prodrug element having the structure:
wherein
R1And R2Independently selected from hydrogen, C1-C8Alkyl and (C)1-C4Alkyl) NH2；
R3Is C1-C6An alkyl group;
R4is hydrogen; and
R5is NH2；
With the proviso that R1And R2Both are not hydrogen.
In some embodiments, there is a dipeptide prodrug element linked to the N-terminal amino acid of a glucagon superfamily peptide and t1/2A prodrug, for example, for between about 12 to about 72 hours, or in some embodiments, between about 12 to about 48 hours, comprising a dipeptide prodrug element having the structure:
wherein
R1And R2Independently selected from hydrogen and C1-C8Alkyl, (C)1-C4Alkyl) NH2Or R is1And R2By (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C8An alkyl group;
R4is (C)0-C4Alkyl) (C6-C10Aryl) R7；
R5Is NH2(ii) a And
R7selected from hydrogen, C1-C8Alkyl and (C)0-C4Alkyl) OH;
with the proviso that R1And R2Both are not hydrogen.
In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. In any of these embodiments, the glucagon superfamily peptide is SEQ id no: 1-684, 701-731, 801-919, 1001-1262, 1301-1371, 1401-1518, 1701-1776 and 1801-1908.
In addition, a method of treating a glucagon superfamily peptide is provided having a dipeptide prodrug element linked to the N-terminal alpha amino acid of the peptide1/2A prodrug, for example, for about 72 to about 168 hours, wherein the dipeptide prodrug element has the following structure:
wherein R is1Selected from hydrogen, C1-C8Alkyl and aryl groups;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen; and
R5is amine or N-substituted amine or hydroxyl;
provided that if R is1Is alkyl or aryl, then R1And R5Together with the atoms to which they are attached form a 4-11 membered heterocyclic ring.
In some embodiments, the dipeptide prodrug element has the following structure:
wherein R is1Selected from hydrogen, C1-C8Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7；
R3Is C1-C18An alkyl group;
R4and R8Each is hydrogen;
R5is NHR6Or OH;
R6is H, C1-C8Alkyl, or R6And R1Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen;
provided that if R is1Is alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7Then R is1And R5Together with the atoms to which they are attached form a 4-11 membered heterocyclic ring. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide.
In some embodiments, the dipeptide prodrug element is linked to a side chain amine of an internal amino acid of a glucagon superfamily peptide. In this embodiment, t1/2A prodrug for about 1 hour, for example, has the following structure:
wherein
R1And R2Independently is C1-C8An alkyl or aryl group; or R1And R2By (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen; and R is5Is an amine.
In some embodiments, t is1/2A prodrug for about 1 hour, for example, has the following structure:
wherein
R1And R2Independently is C1-C8Alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7(ii) a Or R1And R2Through- (CH)2)p-linkage, wherein p is 2-9;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen;
R5is NH2(ii) a And
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
Furthermore, t1/2Prodrugs, for example between about 6 to about 24 hours, having a dipeptide prodrug element attached to an internal amino acid side chain, comprising a dipeptide prodrug having the structure Medicine element:
wherein R is1And R2Independently selected from hydrogen, C1-C8Alkyl and aryl, or R1And R2Through- (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently is C1-C18An alkyl or aryl group; and
R5is an amine or an N-substituted amine;
with the proviso that R1And R2Both are not hydrogen, with the proviso that R4Or R8One of which is hydrogen.
In some embodiments, t is1/2A prodrug, for example, between about 12 to about 72 hours, or in some embodiments between about 12 to about 48 hours, having a dipeptide prodrug element attached to an internal amino acid side chain, comprising a dipeptide prodrug element having the structure:
wherein R is1And R2Independently selected from hydrogen, C1-C8Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7Or R is1And R2Through- (CH)2)p-linkage, wherein p is 2-9;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently of one another is hydrogen, C1-C18Alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7；
R5Is NHR6；
R6Is H or C1-C8Alkyl, or R6And R2Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen;
with the proviso that R1And R2Both are not hydrogen, with the proviso that R4Or R8At least one of which is hydrogen. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide.
In addition, provide t1/2A prodrug, for example, for about 72 to about 168 hours, having a dipeptide prodrug element attached to an internal amino acid side chain of a glucagon superfamily peptide, wherein the dipeptide prodrug element has the structure:
wherein R is1And R2Independently selected from hydrogen, C1-C18Alkyl and aryl groups;
R3is C1-C18An alkyl group;
R4and R8Each is hydrogen; and
R5is amine or N-substituted amine or hydroxyl;
provided that if R is1And R2Both independently are alkyl or aryl, then R1Or R2One of the two passes (CH2)pAnd R5Wherein p is 2 to 9.
In some embodiments, t is provided1/2A prodrug, for example, for about 72 to about 168 hours, having a dipeptide prodrug element attached to an internal amino acid side chain of a glucagon superfamily peptide, wherein the dipeptide prodrug element has the structure:
wherein R is1Selected from hydrogen, C1-C18Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7；
R3Is C1-C18An alkyl group;
R4and R8Each is hydrogen;
R5is NHR6Or OH;
R6is H or C1-C8Alkyl, or R6And R1Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7Selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen;
provided that if R is1And R2Both independently are alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7Then R is1Or R2One of the two passes (CH2)pAnd R5Wherein p is 2 to 9. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. In any of these embodiments, the glucagon superfamily peptide is SEQ ID NO: 1-684, 701-731, 801-919, 1001-1262, 1301-1371, 1401-1518, 1701-1776 and 1801-1908.
In some embodiments, the dipeptide prodrug element is linked to a side chain amine of an internal amino acid of a glucagon superfamily peptide, wherein the internal amino acid comprises the structure of formula II:
wherein
n is an integer selected from 1 to 4. In some embodiments, n is 3 or 4, and in some embodiments, the internal amino acid is lysine. In some embodiments, the dipeptide prodrug element is linked to a primary amine of an amino acid side chain located at position 12, 16, 17, 18, 20, 28, or 29 of the glucagon superfamily peptide. In some embodiments, the amino acid at position 12, 16, 17, 18, 20, 28, or 29 comprises a structure of formula II:
Wherein n is an integer selected from 1-4, and the dipeptide prodrug element is linked to the amino acid side chain through an amide bond. In some embodiments, n is 4 and the amino acid is at position 20. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide.
In yet another embodiment, the dipeptide prodrug element is linked to the glucagon superfamily peptide through an amine present at the aromatic amino acid aryl group. In some embodiments, the aromatic amino acid is an internal amino acid of a glucagon superfamily peptide, however, the aromatic amino acid may also be an N-terminal amino acid. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. In some embodiments, the aromatic amino acid is selected from the group consisting of amino-Phe, amino-naphthylalanine, amino-tryptophan, amino-phenyl-glycine, amino-homophenylalanine, and amino tyrosine. In some embodiments, the primary amine that forms an amide bond with the dipeptide prodrug element is located para to the aryl group. In some embodiments, the aromatic amine comprises the structure of formula III:
wherein m is an integer of 1 to 3.
For embodiments in which the dipeptide prodrug element is linked to the glucagon superfamily peptide through an amine present on the aryl group of an aromatic amino acid, t1/2A prodrug for about 1 hour, for example, has the following dipeptide structure:
wherein R is1And R2Independently is C1-C18An alkyl or aryl group;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C18Alkyl radicals andan aryl group; and R is5Is amine or hydroxyl.
In some embodiments, the dipeptide prodrug element is linked to the glucagon superfamily peptide through an amine present on the aryl group of an aromatic amino acid, t1/2A prodrug for about 1 hour, for example, has the following dipeptide structure:
wherein R is1And R2Independently is C1-C18Alkyl or (C)0-C4Alkyl) (C6-C10Aryl) R7；
R3Is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-12 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C18Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7；
R5Is NH2Or OH; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen. In some embodiments, the glucagon superfamily peptide is a glucagon related peptide. In addition, a dipeptide prodrug element having a linkage through an aromatic amino acid and t1/2A prodrug, for example, for about 6 to about 24 hours, wherein the dipeptide comprises the following structure:
wherein
R1Selected from hydrogen, C1-C18Alkyl and aryl, or R1And R2Through- (CH)2)p(ii) linked, wherein p is 2-9;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-6 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C18Alkyl and aryl groups; and R is5Is an amine or an N-substituted amine.
In some embodiments, there is provided a dipeptide prodrug element having a linker connected by an aromatic amino acid and t1/2A prodrug, for example, for about 6 to about 24 hours, wherein the dipeptide comprises the following structure:
wherein
R1Selected from hydrogen, C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C4Alkyl) NH2And (C)0-C4Alkyl) (C6-C10Aryl) R7；
R3Is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-6 membered heterocyclic ring;
R4and R8Independently selected from hydrogen, C1-C18Alkyl and (C)0-C4Alkyl) (C6-C10Aryl) R7；
R5Is NHR6；
R6Is H, C1-C8Alkyl, or R6And R1Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
In addition, a dipeptide prodrug element having a linkage through an aromatic amino acid and t1/2A prodrug, for example, for about 72 to about 168 hours, wherein the dipeptide comprises the following structure:
wherein R is1And R2Independently selected from hydrogen, C1-C8Alkyl and aryl groups;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-6 membered heterocyclic ring;
R4And R8Each is hydrogen; and
R5selected from the group consisting of amines, N-substituted amines and hydroxyl groups.
In some embodiments, there is provided a dipeptide prodrug element having a linker connected by an aromatic amino acid and t1/2A prodrug, for example, for about 72 to about 168 hours, wherein the dipeptide comprises the following structure:
wherein R is1And R2Independently selected from hydrogen, C1-C8Alkyl, (C)1-C4Alkyl) COOH and (C)0-C4Alkyl) (C6-C10Aryl) R7Or R is1And R5Together with the atoms to which they are attached form a 4-11 membered heterocyclic ring;
R3is C1-C18Alkyl, or R3And R4Together with the atoms to which they are attached form a 4-6 membered heterocyclic ring;
R4is hydrogen or with R3Forming a 4-6 membered heterocyclic ring;
R8is hydrogen;
R5is NHR6Or OH;
R6is H or C1-C8Alkyl, or R6And R1Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen.
In some embodiments, the dipeptide prodrug element is linked to an aromatic amino acid through a primary amine that is present as an aryl substituent of the aromatic amino acid, wherein the aromatic amino acid is located at position 10, 13, 22, or 25 of the glucagon superfamily peptide (depending on the numbering of glucagon, see, e.g., figure 10). In some embodiments, the aromatic amino acid linking the dipeptide prodrug element is located at position 22 of the glucagon superfamily peptide.
According to some embodiments, a dipeptide prodrug element is linked at the N-terminal amine of a glucagon superfamily peptide including, for example, glucagon related peptides or osteocalcin and analogs, derivatives and conjugates thereof, wherein the dipeptide prodrug element comprises the following structure:
wherein R is1Selected from H and C1-C8An alkyl group;
R2and R4Independently selected from H, C1-C8Alkyl radical, C2-C8Alkenyl, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C6-C10Aryl) R7、CH2(C5-C9Heteroaryl) or R1And R2Together with the atom to which they are attached form C3-C6A cycloalkyl group;
R3is selected from C1-C8Alkyl, (C)3-C6) Cycloalkyl, or R4And R3Together with the atoms to which they are attached form a 5 or 6 membered heterocyclic ring;
R5is NHR6Or OH;
R6is H, or R6And R2Together with the atoms to which they are attached form a 5 or 6 membered heterocyclic ring; and
R7selected from H and OH. In some embodiments, R1Is H or C1-C8Alkyl radical, R2Selected from H, C1-C6Alkyl radical, CH2OH、(C1-C4Alkyl) NH2、(C3-C6Cycloalkyl) and CH2(C6Aryl) R7Or R is6And R2Together with the atom to which they are attached form a 5-membered heterocyclic ring, R3Is C1-C6Alkyl radical, R4Selected from H, C1-C4Alkyl, (C)3-C6) Cycloalkyl group, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH and (C)0-C4Alkyl) (C6Aryl) R7Or R is3And R4Together with the atoms to which they are attached form a 5-membered heterocyclic ring. In yet another embodiment, R3Is CH3，R5Is NHR6And in an alternative embodiment, R3And R4Together with the atom to which they are attached form a 5-membered heterocyclic ring, and R5Is NHR6。
According to another embodiment, a dipeptide prodrug element is linked at the N-terminal amine of a glucagon superfamily peptide including, for example, glucagon related peptides or osteocalcin and analogs, derivatives and conjugates thereof, wherein the dipeptide prodrug element comprises the following structure:
wherein R is1Selected from H and C1-C8An alkyl group;
R2and R4Independently selected from H, C1-C8Alkyl radical, C2-C8Alkenyl, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C6-C10Aryl) R7、CH2(C5-C9Heteroaryl) or R1And R2Together with the atom to which they are attached form C3-C6A cycloalkyl group;
R3is selected from C1-C8Alkyl, (C)3-C6) Cycloalkyl, or R4And R3Together with the atoms to which they are attached form a 5 or 6 membered heterocyclic ring;
R5is NHR6Or OH;
R6is H, or R6And R2Together with the atoms to which they are attached form a 5 or 6 membered heterocyclic ring; and
R7selected from hydrogen, C1-C18Alkyl radical, C2-C18Alkenyl, (C)0-C4Alkyl) CONH2、(C0-C4Alkyl group) COOH, (C)0-C4Alkyl) NH2、(C0-C4Alkyl) OH and halogen. In some embodiments, R1Is H or C1-C8Alkyl radical, R2Selected from H, C1-C6Alkyl radical, CH2OH、(C1-C4Alkyl) NH2、(C3-C6Cycloalkyl) and CH2(C6Aryl) R7Or R is6And R2Together with the atom to which they are attached form a 5-membered heterocyclic ring, R3Is C1-C6Alkyl radical, and R4Selected from H, C1-C4Alkyl, (C)3-C6) Cycloalkyl group, (C)1-C4Alkyl) OH, (C)1-C4Alkyl) SH and (C)0-C4Alkyl) (C6Aryl) R7Or R is3And R4Together with the atoms to which they are attached form a 5-membered hetero ringAnd (4) a ring. In yet another embodiment, R3Is CH3，R5Is NHR6And in an alternative embodiment, R3And R4Together with the atom to which they are attached form a 5-membered heterocyclic ring, and R5Is NHR6。
In some embodiments, Q is SEQ ID NO: 1-684, 701-731, 801-919, 1001-1262, 1301-1371, 1401-1518, 1701-1776 and 1801-1908.
Glucagon related peptides
In certain aspects, the disclosure relates to glucagon related peptides (as part of the indicated group "Q"). The term glucagon-related peptide refers to a peptide that is biologically active (as an agonist or antagonist) at any one or more of glucagon, GLP-1, GLP-2, and GIP receptors and comprises an amino acid sequence that has at least 40% sequence identity (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) to at least one of native glucagon, native oxyntomodulin, native exendin-4, native GLP-1, native GLP-2, or native GIP. It is to be understood that all possible subsets of the activity of glucagon-related peptides are contemplated, such as peptides that are biologically active (as agonists or antagonists) at any one or more of the glucagon or GLP-1 or GIP receptors, as well as all possible subsets of sequence identity with each listed native peptide, e.g., comprising amino acid sequences that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with native glucagon over the length of the native glucagon. In some embodiments of the invention, the glucagon related peptide is a peptide having glucagon receptor agonist activity, GIP receptor agonist activity, glucagon receptor/GLP-1 receptor co-agonist (co-agonst) activity, glucagon receptor antagonist activity, or glucagon receptor antagonist and GLP-1 receptor agonist activity. In some embodiments, the peptide maintains the alpha helical conformation of the C-terminal half of the molecule. In some embodiments, the peptide retains a position involved in receptor interaction or signaling, such as position 3 of glucagon, or position 7, 10, 12, 13, 15, or 17 of (1-37) GLP-1. Thus, the glucagon related peptide may be a peptide that may be of class 1, class 2, class 3, class 4, and/or class 5, each of which is described further herein.
According to some embodiments, the dipeptide prodrug element may be linked to any of the biologically active compounds previously disclosed in the following international application nos.: PCT/US2008/08608 (filed on 3/1/2008), PCT/US2008/053857 (filed on 13/2/2008), PCT/US2009/47437 (filed on 16/6/2009), PCT/US2009/47438 (filed on 16/6/2009), PCT/US2009/47447 (filed on 16/6/2009), PCT/US2008/080973 (filed on 23/10/2008), and PCT/US2008/081333 (filed on 27/10/2008), the disclosures of which are expressly incorporated herein by reference. In some exemplary embodiments, a dipeptide prodrug element disclosed herein may be attached to a bioactive peptide disclosed in PCT/US2008/08608, PCT/US2008/053857, PCT/US2009/47437, PCT/US2009/47438, PCT/US2009/47447, PCT/US2008/08097, and PCT/US2008/081333 through an N-terminal amine, or may be attached to a side chain amino group of a lysine at position 20 of any bioactive peptide disclosed or to an aromatic amino group of a 4-aminophenylalanine substituted for an amino acid at position 22 of any bioactive peptide disclosed. In some exemplary embodiments, the dipeptide prodrug elements disclosed herein are linked to the N-terminal amine of the bioactive peptides disclosed in PCT/US2008/08608, PCT/US2008/053857, PCT/US2009/47437, PCT/US2009/47438, and PCT/US2009/47447, PCT/US2008/08097, and PCT/US2008/081333 via an amide bond. In some embodiments, the glucagon superfamily peptide is SEQ ID NO: 1-684, 701-731, 801-919, 1001-1262, 1301-1371, 1401-1518, 1701-1776 and 1801-1908.
Decoration
Glucagon related peptides can include native glucagon amino acid sequences with modifications (SEQ ID NO; 701). In exemplary embodiments, the glucagon-related peptide may comprise a total of 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid modifications, such as conservative or non-conservative substitutions, as compared to the native glucagon sequence. In certain aspects, the modifications and substitutions described herein are made at specific positions within the glucagon related peptide, wherein the numbering of the positions is comparable to that of glucagon (SEQ ID NO: 701). In some embodiments, 1, 2, 3, 4, or 5 non-conservative substitutions are made at any of positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28, or 29, and up to 5 additional conservative substitutions are made at any of these positions. In some embodiments, 1, 2, or 3 amino acid modifications are made at amino acids 1-16, and 1, 2, or 3 amino acid modifications are made at amino acids 17-26. In some embodiments, such glucagon-related peptides retain at least 22, 23, 24, 25, 26, 27, or 28 naturally-occurring amino acids (e.g., 1-7, 1-5, or 1-3 modifications as compared to naturally-occurring glucagon) at positions corresponding to native glucagon.
DPP-IV resistance
In some embodiments, the glucagon related peptide comprises a modification at position 1 or 2 to reduce sensitivity to cleavage by dipeptidyl peptidase IV. More specifically, in some embodiments, position 1 of a glucagon related peptide (e.g., selected from the glucagon related peptides of figure 10) is substituted with an amino acid selected from the group consisting of: d-histidine, α -dimethylimidazole (imidazolium) acetic acid (DMIA), N-methylhistidine, α -methylhistidine, imidazoleacetic acid, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. More specifically, in some embodiments, position 2 of the glucagon related peptide is substituted with an amino acid selected from the group consisting of: d-serine, D-alanine, valine, glycine, N-methylserine and aminoisobutyric acid. In some embodiments, the glucagon related peptide is not D-serine at position 2.
Hydrophilic moiety
In some embodiments, a glucagon related peptide (e.g., a glucagon-like 1 peptide, a glucagon-like 2 peptide, a glucagon-like 3 peptide, a glucagon-like 4 peptide, or a glucagon-like 5 peptide) is linked (covalently bound) to a hydrophilic moiety. The hydrophilic moiety can be attached to the glucagon related peptide under any suitable conditions used to react the protein with the activated polymer molecule. Any method known in the art can be used, including other chemoselective conjugation/attachment methods by acylation, reductive alkylation, Michael addition, mercaptoalkylation, or by reaction of a reactive group on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazino) with a reactive group on the target compound (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazino). Activating groups that may be used to attach the water-soluble polymer to one or more proteins include, but are not limited to, sulfones, maleimides, sulfhydryl groups, triflate groups, aziridine (azidirine), oxirane, and 5-pyridyl. If attached to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled. See, e.g., Kinstler et al, adv. drug. delivery rev.54: 477-; roberts et al, adv. drug delivery rev.54: 459-476 (2002); and Zalipsky et al, adv. drug. delivery rev.16: 157-182(1995).
For glucagon-related peptides of classes 1-3, other activating groups that can be used to attach the water-soluble polymer to one or more proteins include alpha-halogenated acyl groups (e.g., alpha-iodoacetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid). In some embodiments wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the amino acid comprising a thiol group is modified with maleimide activated PEG in a michael addition reaction to provide a pegylated peptide comprising a thioether linkage as shown below:
in other embodiments, in the nucleophilic substitution reaction, the sulfhydryl group of an amino acid of a class 1, class 2, or class 3 glucagon related peptide is modified with a haloacetyl activated PEG to yield a pegylated peptide comprising a thioether linkage as shown below:
suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyalkylene oxide, polyethylene glycol propionaldehyde, ethylene glycol/propylene glycol copolymers, polyethylene glycol monomethyl ether, mono- (C1-C10) alkoxy-or aryloxy-polyethylene glycol, carboxymethylcellulose, polyacetal, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trisubstituted-polyethylene glycol, poly (oxyethylene) glycol, poly ( Alkanes, ethylene/maleic anhydride copolymers, poly (. beta. -amino acids) (homo-or random copolymers), poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, colinic acid (colonic acid) or other polysaccharide polymers, Ficoll or dextran and mixtures thereof. Dextran is a polysaccharide polymer of glucose subunits, primarily linked by α 1-6 linkages. A number of molecular weight ranges of dextran are available, for example from about 1kD to about 100kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD.
In some embodiments, the hydrophilic moiety is a polyethylene glycol (PEG) chain or other water soluble polymer covalently attached on the side chain of an amino acid residue at one or more of positions 16, 17, 21, 24, 29, 40 of said glucagon related peptide, within the C-terminal overhang or on the C-terminal amino acid. In some embodiments, the natural amino acid at this position is substituted with an amino acid having a side chain suitable for crosslinking with the hydrophilic moiety to facilitate bonding of the hydrophilic moiety to the peptide. Exemplary amino acids include Cys, Lys, Orn, homocysteine, or acetylphenylalanine (Ac-Phe). In other embodiments, an amino acid modified to include a hydrophilic group is added to the C-terminus of the peptide.
According to some embodiments, the molecular weight of the hydrophilic moiety (e.g., polyethylene glycol chain) is selected from about 500 to about 40,000 daltons. In some embodiments, the molecular weight of the polyethylene glycol chain is selected from about 500 to about 5,000 daltons or about 1,000 to about 5,000 daltons. In another embodiment, the hydrophilic moiety (e.g., polyethylene glycol chain) has a molecular weight of about 10,000 to about 20,000 daltons. In other exemplary embodiments, the hydrophilic moiety (e.g., polyethylene glycol chain) has a molecular weight of about 20,000 to about 40,000 daltons.
Linear or branched hydrophilic polymers are contemplated. The resulting preparation of the conjugate can be substantially monodisperse or polydisperse, and can have about 0.5, 0.7, 1, 1.2, 1.5, or 2 polymer moieties per peptide.
Acylation
In some embodiments, the glucagon related peptide (e.g., glucagon-like 1 peptide, glucagon-like 2 peptide, glucagon-like 3 peptide, glucagon-like 4 peptide, or glucagon-like 5 peptide) is modified to comprise an acyl group. For example, the glucagon related peptide may be one of class 1, class 2, or class 3, and may comprise an acyl group that is non-natural to a naturally occurring amino acid. Acylation may be carried out at any position within the glucagon related peptide (including any of positions 1-29), at a position within the C-terminal overhang, or at the C-terminal amino acid, provided that the activity possessed by the non-acylated glucagon related peptide is retained upon acylation. For example, if the non-acylated peptide has glucagon agonist activity, the acylated peptide retains glucagon agonist activity. Likewise, if the non-acylated peptide has glucagon antagonist activity, for example, the acylated peptide retains glucagon antagonist activity. For example, if the non-acylated peptide has GLP-1 agonist activity, the acylated peptide retains GLP-1 agonist activity. Non-limiting examples include acylation at position 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29 (according to the amino acid number of wild-type glucagon). For class 1, class 2 and class 3 glucagon related peptides, acylation may occur at any of positions 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 40, 41, 42 or 43 (according to the amino acid numbering of wild-type glucagon). The acyl group may be covalently linked directly to an amino acid of the glucagon related peptide or indirectly via a spacer, wherein the spacer is located between the amino acid of the glucagon related peptide and the acyl group. The glucagon related peptide may be acylated at the same amino acid position to which the hydrophilic moiety is attached or at a different amino acid position. Non-limiting examples include acylation at position 10 (according to the amino acid numbering of wild-type glucagon), and pegylation at one or more positions (e.g., positions 24, 28, or 29) in the C-terminal portion of the glucagon peptide (according to the amino acid numbering of wild-type glucagon), within the C-terminal overhang, or at the C-terminus (e.g., by addition of a C-terminal Cys).
In a particular aspect of the invention, the glucagon related peptide is modified to comprise an acyl group by direct acylation of an amine, hydroxyl or thiol group of an amino acid side chain of the glucagon related peptide. In some embodiments, the glucagon related peptide is directly acylated by a side chain amine, hydroxyl, or sulfhydryl group of an amino acid. In some embodiments, the acylation is at position 10, 20, 24 or 29 (according to the amino acid numbering of wild-type glucagon). In this aspect, the acylated glucagon related peptide may comprise SEQ ID NO: 701, or a modified amino acid sequence of such a sequence comprising one or more of the amino acid modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) is modified to any amino acid comprising a side chain amine, hydroxyl or sulfhydryl group. In some embodiments of the invention, the glucagon related peptide is acylated directly by an amino acid side chain amine, hydroxyl or sulfhydryl group at position 10 (numbered according to the amino acids of wild-type glucagon).
In some embodiments, the amino acid comprising a side chain amine is an amino acid of formula I:
Wherein n is 1-4
[ formula I ]
In some exemplary embodiments, the amino acid of formula I is an amino acid wherein n is 4(Lys) or n is 3 (Orn).
In other embodiments, the amino acid comprising a side chain hydroxyl group is an amino acid of formula II:
wherein n is 1-4
[ formula II ]
In some exemplary embodiments, the amino acid of formula II is an amino acid (Ser) wherein n is 1.
In still other embodiments, the amino acid comprising a side chain thiol group is an amino acid of formula III below:
wherein n is 1-4
[ formula III ]
In some exemplary embodiments, the amino acid of formula III is an amino acid (Cys) wherein n is 1.
In still other embodiments, wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the amino acid comprising a side chain amine, hydroxyl or thiol group is a disubstituted amino acid comprising the same structure of formula I, formula II or formula III except that the hydrogen bound to the alpha carbon of the amino acid of formula I, formula II or formula III is replaced by a second side chain.
In some embodiments of the invention, the acylated glucagon related peptide comprises a spacer between the peptide and the acyl group. In some embodiments, the glucagon related peptide is covalently bound to a spacer, which is covalently bound to an acyl group. In some exemplary embodiments, the glucagon related peptide is modified to comprise an acyl group by acylation of an amine, hydroxyl, or sulfhydryl group of a spacer attached to the side chain or C-terminal amino acid of amino acid position 10, 20, 24, or 29 (numbered according to amino acids of wild-type glucagon) of the glucagon related peptide. The amino acid to which the spacer is attached may be any amino acid comprising a moiety that allows attachment of the spacer. For example, containing side chain-NH2Amino acids of-OH or-COOH (e.g., Lys, Orn, Ser, Asp or Glu) are suitable. Similarly, for glucagon related peptides of class 1, class 2 and class 3, the side chain-NH is included2Amino acids (e.g., single or double α -substituted amino acids) of-OH or-COOH (e.g., Lys, Orn, Ser, Asp, or Glu) are suitable. In this aspect, the acylated glucagon related peptide may comprise SEQ id no: 701, or a modified amino acid sequence of such a sequence comprising one or more of the amino acid modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) is modified to any amino acid comprising a side chain amine, hydroxyl or carboxyl (carboxylate).
In some embodiments, the spacer is an amino acid comprising a side chain amine, hydroxyl, or thiol group, or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol group. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
When acylation is performed via the amine group of a spacer amino acid, acylation may be performed via the alpha amine or side chain amine of the amino acid. In the case where the alpha amine is acylated, the spacer amino acid may be any amino acid. For example, the spacer amino acid can be a hydrophobic amino acid, such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer amino acid may be, for example, a hydrophobic amino acid, such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr, 6-aminocaproic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid. Alternatively, the spacer amino acid may be an acidic residue, such as Asp and Glu. In the case where the side chain amine of the spacer amino acid is acylated, the spacer amino acid is an amino acid comprising a side chain amine, such as an amino acid of formula I (e.g., Lys or Orn). In this case, it is possible that both the alpha amine and the side chain amine of the spacer amino acid are acylated, so that the glucagon peptide is diacylated. Embodiments of the present invention include such diacylated molecules.
When the acylation is carried out via the hydroxyl group of the spacer amino acid, the amino acid or one of the amino acids of the di-or tripeptide may be an amino acid of formula II. In a specific exemplary embodiment, the amino acid is Ser.
When the acylation is carried out via the thiol group of a spacer amino acid, one of the amino acids, or the amino acids of the di-or tripeptide, may be an amino acid of formula III. In a specific exemplary embodiment, the amino acid is Cys.
In some embodiments, the spacer comprises a hydrophilic bifunctional spacer. In a particular embodiment, the spacer comprises an aminopoly (alkoxy) carboxylic acid. In this aspect, the spacer may comprise, for example, NH2(CH2CH2O)n(CH2)mCOOH, where m is any integer from 1 to 6 and n is any integer from 2 to 12, such as 8-amino-3, 6-dioxaoctanoic acid available from Peptides International, Inc. (Louisville, KY).
In some embodiments related only to class 1, class 2 and class 3 glucagon related peptides, the spacer comprises a hydrophilic bifunctional spacer. In certain embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises two or more reactive groups, such as an amine, a hydroxyl, a thiol, and a carboxyl group, or any combination thereof. In certain embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a hydroxyl group and a carboxyl group. In other embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises an amine group and a carboxyl group. In other embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a thiol group and a carboxyl group.
In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer is a hydrophobic bifunctional spacer. Hydrophobic bifunctional spacers are known in the art. See, e.g., Bioconjugate Techniques, g.t. hermanson (Academic Press, San Diego, CA, 1996), incorporated herein by reference in its entirety. In certain embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises two or more reactive groups, such as an amine, a hydroxyl, a thiol, and a carboxyl group, or any combination thereof. In certain embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a hydroxyl group and a carboxyl group. In other embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises an amine group and a carboxyl group. In other embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a thiol group and a carboxyl group. Suitable hydrophobic bifunctional spacers comprising a carboxyl group and a hydroxyl or thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.
In some embodiments, the bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide is not a dicarboxylic acid comprising an unbranched methylene group of 1 to 7 carbon atoms between carboxylic acid groups. In some embodiments, the bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide is a dicarboxylic acid comprising an unbranched methylene group of 1 to 7 carbon atoms between carboxylic acid groups.
In particular embodiments where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the spacer (e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer or hydrophobic bifunctional spacer) is 3-10 atoms, such as 6-10 atoms (e.g., 6, 7, 8, 9 or 10 atoms) in length. In more specific embodiments where the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer is about 3-10 atoms (e.g., 6-10 atoms) in length and the acyl group is a C12-C18 fatty acyl group, e.g., a C14 fatty acyl group, a C16 fatty acyl group, such that the total length of the spacer and acyl groups is 14-28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 atoms. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer and acyl groups are 17-28 (e.g., 19-26, 19-21) atoms in length.
According to certain embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the bifunctional spacer may be a synthetic or naturally occurring amino acid (including but not limited to any of the amino acids described herein) comprising an amino acid backbone of 3-10 atoms in length (e.g., 6-aminocaproic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, and 8-aminocaprylic acid). Alternatively, the spacer attached to the class 1, class 2 or class 3 glucagon related peptide may be of a peptide backbone length ofA dipeptide or tripeptide spacer of 3-10 atoms (e.g., 6-10 atoms). Each amino acid of the dipeptide or tripeptide spacer linked to the class 1, class 2 or class 3 glucagon related peptide may be the same or different from the other amino acids of the dipeptide or tripeptide and may be independently selected from: naturally occurring and/or non-naturally occurring amino acids, including, for example, any D or L isomer of a naturally occurring amino acid (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or L isomer of a non-naturally occurring amino acid selected from the group consisting of: beta-alanine (. beta. -Ala.), N-alpha-methyl-alanine (Me-Ala), aminobutyric acid (Abu), gamma-aminobutyric acid (gamma-Abu), aminocaproic acid (. epsilon. -Ahx), aminoisobutyric acid (Aib), aminomethylpyrrolecarboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, beta-aspartic acid (. beta. -Asp), azetidinecarboxylic acid, 3- (2-benzothiazolyl) alanine, alpha-tert-butylglycine, 2-amino-5-ureido-N-pentanoic acid (citrulline, Cit), beta-cyclohexylalanine (Cha), acetamidomethyl-cysteine, diaminobutyric acid (Dab), Diaminopropionic acid (Dpr), Dihydroxyphenylalanine (DOPA), Dimethylthiazolidine (DMTA), gamma-glutamic acid (gamma-Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N-methylamide, methyl-isoleucine (MeIle), 4-piperidinecarboxylic acid (Isn), methyl-leucine (melleu), methyl-lysine, dimethyl-lysine, trimethyl-lysine, methanoproline (methanoprolamine), methionine-sulfoxide (Met (O)), methionine-sulfone (Met (O)))2) Norleucine (Nle), methyl-norleucine (Me-Nle), norvaline (Nva), ornithine (Orn), p-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-chlorophenylalanine (Phe (4-Cl)), 4-fluorophenylalanine (Phe (4-F)), 4-nitrophenylalanine (Phe (4-NO))2) 4-cyanophenylalanine ((Phe (4-CN)), phenylglycine (Phg), piperidinylalanine, piperidinylglycine, 3, 4-dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), O-benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta), 4-amino-5-Cyclohexyl-3-hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), 1, 2, 3, 4, -tetrahydro-isoquinoline-3-carboxylic acid (Tic), tetrahydropyran glycine, thienylalanine (Thi), O-benzyl-phosphotyrosine, O-phosphotyrosine, methoxytyrosine, ethoxytyrosine, O- (bis-dimethylamino-phosphono) -tyrosine, tetrabutyl-amine tyrosine sulphate, methyl-valine (MeVal) and alkylated 3-mercaptopropionic acid.
In some embodiments wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the dipeptide is not any dipeptide of the general structure a-B, wherein a is selected from Gly, gin, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe and Pro, wherein B is selected from Lys, His, Trp. In some embodiments wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the dipeptide spacer is selected from: Ala-Ala, beta-Ala-beta-Ala, Leu-Leu, Pro-Pro, gamma-aminobutyric acid-gamma-aminobutyric acid, and gamma-Glu-gamma-Glu.
In some embodiments of the invention wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the glucagon related peptide is modified to comprise an acyl group by acylation of a long alkane by the glucagon related peptide. In particular aspects where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the long chain alkane comprises an amine, hydroxyl or sulfhydryl group (e.g., octadecylamine, tetradecanol, and octadecanethiol) that reacts with the carboxyl group or activated form of the glucagon related peptide. The carboxyl group or activated form of the glucagon related peptide class 1, class 2 or class 3 may be part of the side chain of an amino acid of the glucagon related peptide (e.g., glutamic acid, aspartic acid) or may be part of the peptide backbone.
In certain embodiments, the class 1, class 2 or class 3 glucagon-related peptide is modified to comprise an acyl group by acylation of the long chain alkane by a spacer linked to the glucagon peptide. In particular aspects, the long chain alkane comprises an amine, hydroxyl, or sulfhydryl group that reacts with the carboxyl group of the spacer or an activated form thereof. Suitable spacers comprising a carboxyl group or an activated form thereof are described herein and include, for example, bifunctional spacers such as amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers, and hydrophobic bifunctional spacers.
The term "activated form of a carboxyl group" as used herein refers to a carboxyl group having the general formula R (C ═ O) X, wherein X is a leaving group and R is a glucagon related peptide or spacer. For example, activated forms of carboxyl groups may include, but are not limited to, acid chlorides, anhydrides, and esters. In some embodiments, the activated carboxyl group is an ester with an N-hydroxysuccinimide (NHS) leaving group.
With respect to these aspects of the invention wherein the long chain alkane is acylated with a glucagon related peptide of class 1, class 2 or class 3 or a spacer, the long chain alkane may be of any size and may comprise any carbon chain length. The long chain alkane may be straight chain or branched. In certain aspects wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the long chain alkane is a C4-C30 alkane. For example, the long-chain alkane may be any one of a C4 alkane, a C6 alkane, a C8 alkane, a C10 alkane, a C12 alkane, a C14 alkane, a C16 alkane, a C18 alkane, a C20 alkane, a C22 alkane, a C24 alkane, a C26 alkane, a C28 alkane, or a C30 alkane. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the long chain alkane comprises a C8-C20 alkane, e.g., a C14 alkane, a C16 alkane, or a C18 alkane.
Also, in some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the amine, hydroxyl or sulfhydryl group of the glucagon related peptide is acylated with a cholesterol acid. In a specific embodiment, the class 1, class 2 or class 3 glucagon related peptide is linked to the cholesterol acid via an alkylated deaminated Cys spacer (i.e., an alkylated 3-mercaptopropionic acid spacer).
Suitable methods for peptide acylation via amine, hydroxyl and thiol groups are known in the art. See, e.g., Miller, Biochem Biophys Res Commun 218: 377-382 (1996); shimohigashi and Stammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al, Biochim biophysis Acta 263: 7-13(1972) (for the method by acylation of hydroxyl group); and San and Silvius, J Pept Res 66: 169-180(2005) (method for acylation by thiol); bioconjugate chem, "Chemical Modifications of proteins: history and Applications "page 1, 2-12 (1990); hashimoto et al, pharmaceutical Res. "Synthesis of Palmitoyl Derivatives of Insulin and dtheir Biological Activity" Vol.6, 2 nd p.171-.
The acyl group of the acylated glucagon related peptide may be of any size, e.g., a carbon chain of any length, and may be straight or branched. In some embodiments of the invention, the acyl group is a C4-C30 fatty acid. For example, the acyl group may be any one of a C4 fatty acid, a C6 fatty acid, a C8 fatty acid, a C10 fatty acid, a C12 fatty acid, a C14 fatty acid, a C16 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, or a C30 fatty acid. In some embodiments, the acyl group is a C8-C20 fatty acid, e.g., a C14 fatty acid or a C16 fatty acid.
In an alternative embodiment, the acyl group is a bile acid. The bile acid can be any suitable bile acid including, but not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholic acid.
The acylated glucagon related peptides described herein may also be further modified to include a hydrophilic moiety. In some embodiments, the hydrophilic moiety may comprise a polyethylene glycol (PEG) chain. Incorporation of the hydrophilic moiety can be accomplished by any suitable method, such as any of the methods described herein. In this aspect, the acylated glucagon related peptide may comprise SEQ ID NO: 701, including any of the modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) comprises an acyl group and at least one of the amino acids at positions 16, 17, 21, 24 or 29 (numbered according to the amino acids of wild-type glucagon), a position within the C-terminal overhang or the C-terminal amino acid is modified to Cys, Lys, Orn, homocysteine or Ac-Phe, and the side chain of the amino acid is covalently bound to a hydrophilic moiety (e.g., PEG). In some embodiments, the acyl group is optionally linked to position 10 (numbered according to amino acids of wild-type glucagon) through a spacer comprising Cys, Lys, Orn, homocysteine, or Ac-Phe, and the hydrophilic moiety is incorporated into the Cys residue at position 24.
Alternatively, the acylated glucagon related peptide may comprise a spacer, wherein the spacer is both acylated and modified to comprise a hydrophilic moiety. Non-limiting examples of suitable spacers include spacers comprising one or more amino acids selected from Cys, Lys, Orn, homocysteine, and Ac-Phe.
Alkylation
According to some embodiments, the glucagon related peptide, e.g., glucagon-like 1 peptide, glucagon-like 2 peptide, glucagon-like 3 peptide, glucagon-like 4 peptide, or glucagon-like 5 peptide, is modified to comprise an alkyl group linked to the glucagon related peptide by an ether, thioether, or amino bond to extend the circulatory half-life and/or delay onset and/or extend duration of action and/or improve resistance to a protease (e.g., DPP-IV). In exemplary embodiments where the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the glucagon related peptide comprises an alkyl group that is non-natural to the naturally occurring amino acid.
The alkylation may be performed at any position within the glucagon related peptide, including any of positions 1-29, a position within the C-terminal overhang, or a C-terminal amino acid, provided that agonist or antagonist activity of the glucagon related peptide on the glucagon, GLP-1, or other glucagon related peptide receptor is maintained. In some embodiments, the alkylated peptide retains glucagon agonist activity if the non-alkylated peptide has glucagon agonist activity. In other embodiments, the alkylated peptide retains glucagon antagonist activity if the non-alkylated peptide has glucagon antagonist activity. In some embodiments, the alkylated peptide retains GLP-1 agonist activity if the non-alkylated peptide has GLP-1 agonist activity. Non-limiting examples include alkylation at position 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29 (according to the amino acid numbering of wild-type glucagon). For class 1, class 2 and class 3 glucagon-related peptides, alkylation may occur at position 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 40, 41, 42 or 43 (according to the amino acid numbering of wild-type glucagon). The alkyl group may be covalently linked directly to an amino acid of the glucagon related peptide or indirectly via a spacer, wherein the spacer is located between the amino acid of the glucagon related peptide and the alkyl group. The glucagon related peptide may be alkylated at the same amino acid position to which the hydrophilic moiety is attached, or alkylated at a different amino acid position. Non-limiting examples include alkylation at position 10 (according to amino acid numbering of wild-type glucagon) and pegylation at one or more positions of the C-terminal portion of the glucagon related peptide, such as position 24, 28 or 29 (according to amino acid numbering of wild-type glucagon), within the C-terminal overhang or at the C-terminus (e.g., by addition of a C-terminal Cys).
In a particular aspect of the invention, the glucagon related peptide is modified to comprise an alkyl group by direct alkylation of an amine, hydroxyl or thiol group of an amino acid side chain of the glucagon related peptide. In some embodiments, the glucagon related peptide is directly alkylated through a side chain amine, hydroxyl, or sulfhydryl group of an amino acid. In some embodiments, the alkylation is carried out at position 10, 20, 24 or 29 (according to amino acid numbering of wild-type glucagon). In this aspect, the alkylated glucagon related peptide may comprise SEQ ID NO: 701, or a modified amino acid sequence of such a sequence comprising one or more of the amino acid modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) is modified to any amino acid comprising a side chain amine, hydroxyl or sulfhydryl group. In some embodiments of the invention, direct alkylation of the glucagon related peptide is performed via a side chain amine, hydroxyl or sulfhydryl group of an amino acid at position 10 (according to the amino acid numbering of wild-type glucagon).
In some embodiments, the amino acid comprising a side chain amine is an amino acid of formula I. In some exemplary embodiments, the amino acid of formula I is an amino acid wherein n is 4(Lys) or n is 3 (Orn).
In other embodiments, the amino acid comprising a side chain hydroxyl group is an amino acid of formula II. In some exemplary embodiments, the amino acid of formula II is an amino acid (Ser) wherein n is 1.
In still other embodiments, the amino acid comprising a side chain thiol group is an amino acid of formula III. In some exemplary embodiments, the amino acid of formula II is an amino acid (Cys) wherein n is 1.
In still other embodiments, wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the amino acid comprising a side chain amine, hydroxyl or thiol group is a disubstituted amino acid comprising the same structure of formula I, formula II or formula III except that the hydrogen bound to the alpha carbon of the amino acid of formula I, formula II or formula III is replaced by a second side chain.
In some embodiments of the invention, the alkylated glucagon related peptide comprises a spacer between the peptide and the alkyl group. In some embodiments, the glucagon related peptide is covalently bound to a spacer, which is covalently bound to an alkyl group. In some exemplary embodiments, the glucagon related peptide is modified to comprise an alkyl group by alkylation of an amine, hydroxyl or sulfhydryl group of a spacer group that is complementary to that at 10, 20, 24 or 29 of the glucagon related peptide The side chain of the amino acid at position (numbered according to amino acids of wild-type glucagon). The amino acid to which the spacer is attached may be any amino acid comprising a moiety that allows attachment to the spacer. For class 1, class 2 and class 3 glucagon-related peptides, the amino acid to which the spacer is attached can be any amino acid that comprises a moiety that allows attachment to the spacer (e.g., a single alpha-substituted amino acid or an alpha, alpha-disubstituted amino acid). For example, containing side chain-NH2Amino acids of-OH or-COOH (e.g., Lys, Orn, Ser, Asp or Glu) are suitable. In this aspect, the alkylated glucagon related peptide may comprise SEQ ID NO: 701, or a modified amino acid sequence of such a sequence comprising one or more of the amino acid modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) is modified to any amino acid comprising a side chain amine, hydroxyl or carboxyl group.
In some embodiments, the spacer is an amino acid comprising a side chain amine, hydroxyl, or thiol group or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl, or thiol group. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
When alkylation is performed by an amine group of an amino acid of the spacer group, alkylation may be performed by an alpha amine or a side chain amine of the amino acid. In the case where the alpha amine is alkylated, the spacer amino acid may be any amino acid. For example, the spacer amino acid can be a hydrophobic amino acid, such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr. Alternatively, the spacer amino acid may be an acidic residue, such as Asp and Glu. In exemplary embodiments where the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer amino acid may be a hydrophobic amino acid, such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr, 6-aminocaproic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid. Alternatively, the spacer amino acid attached to the class 1, class 2 or class 3 glucagon-related peptide may be an acidic residue, such as Asp and Glu, provided that the alkylation is performed on the alpha amine of the acidic residue. In the case where the side chain amine of the spacer amino acid is alkylated, the spacer amino acid is an amino acid comprising a side chain amine, such as an amino acid of formula I (e.g., Lys or Orn). In this case, it is possible that both the alpha and side chain amines of the spacer amino acid are alkylated such that the glucagon peptide is dialkylated. Embodiments of the present invention include such dialkylated molecules.
When alkylation is carried out via the hydroxyl group of a spacer amino acid, the amino acid or one of the amino acids of the spacer may be an amino acid of formula II. In a specific exemplary embodiment, the amino acid is Ser.
When alkylation is by a sulfhydryl group of a spacer amino acid, the amino acid or one of the amino acids of the spacer may be an amino acid of formula III. In a specific exemplary embodiment, the amino acid is Cys.
In some embodiments, the spacer comprises a hydrophilic bifunctional spacer. In a particular embodiment, the spacer comprises an aminopoly (alkoxy) carboxylic acid. In this aspect, the spacer may comprise, for example, NH2(CH2CH2O)n(CH2)mCOOH, where m is any integer from 1 to 6 and n is any integer from 2 to 12, such as 8-amino-3, 6-dioxaoctanoic acid available from Peptides International, Inc. (Louisville, KY).
In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer is a hydrophilic bifunctional spacer. In certain embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises two or more reactive groups, such as an amine, a hydroxyl, a thiol, and a carboxyl group, or any combination thereof. In certain embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a hydroxyl group and a carboxyl group. In other embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises an amine group and a carboxyl group. In other embodiments, the hydrophilic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a thiol group and a carboxyl group.
In some embodiments, the spacer linked to the class 1, class 2 or class 3 glucagon-related peptide is a hydrophobic bifunctional spacer. In certain embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises two or more reactive groups, such as an amine, a hydroxyl, a thiol, and a carboxyl group, or any combination thereof. In certain embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a hydroxyl group and a carboxyl group. In other embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises an amine group and a carboxyl group. In other embodiments, the hydrophobic bifunctional spacer attached to the class 1, class 2 or class 3 glucagon-related peptide comprises a thiol group and a carboxyl group. Suitable hydrophobic bifunctional spacers comprising a carboxyl group and a hydroxyl or thiol group are known in the art and include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoic acid.
In particular embodiments where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the spacer (e.g., an amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer or hydrophobic bifunctional spacer) is 3-10 atoms (e.g., 6, 7, 8, 9, or 10 atoms)) in length. In more specific embodiments, the spacer attached to the class 1, class 2 or class 3 glucagon related peptide is about 3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl group is a C12 to C18 alkyl group, e.g., C14 alkyl, C16 alkyl, such that the total length of the spacer and alkyl group is 14 to 28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 atoms. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the spacer and alkyl groups are 17-28 (e.g., 19-26, 19-21) atoms in length.
According to certain of the foregoing embodiments in which the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the bifunctional spacer may be a synthetic or non-naturally occurring amino acid comprising an amino acid backbone of 3-10 atoms in length (e.g., 6-aminocaproic acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, and 8-aminocaprylic acid). Alternatively, the spacer attached to the class 1, class 2 or class 3 glucagon related peptide may be a dipeptide or tripeptide spacer having a peptide backbone of 3-10 atoms in length (e.g., 6-10 atoms). The dipeptide or tripeptide spacer linked to the class 1, class 2 or class 3 glucagon-related peptide may be comprised of naturally occurring and/or non-naturally occurring amino acids including, for example, any of the amino acids taught herein. In some embodiments wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the spacer comprises an overall negative charge, e.g., comprises one or two negatively charged amino acids. In some embodiments wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the dipeptide spacer is selected from: Ala-Ala, beta-Ala-beta-Ala, Leu-Leu, Pro-Pro, gamma-aminobutyric acid-gamma-aminobutyric acid, and gamma-Glu-gamma-Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
Suitable methods for alkylating peptides by amine, hydroxyl and thiol groups are known in the art. For example, Williamson ether synthesis (Williamson ether synthesis) can be used to form an ether linkage between a glucagon related peptide and an alkyl group. Likewise, nucleophilic substitution reactions of peptides with alkyl halides can produce any of ether, thioether, or amino linkages.
The alkyl group of the alkylated glucagon related peptide may be of any size, e.g., a carbon chain of any length, and may be straight or branched. In some embodiments of the invention, the alkyl group is a C4-C30 alkyl group. For example, the alkyl group may be any of a C4 alkyl group, a C6 alkyl group, a C8 alkyl group, a C10 alkyl group, a C12 alkyl group, a C14 alkyl group, a C16 alkyl group, a C18 alkyl group, a C20 alkyl group, a C22 alkyl group, a C24 alkyl group, a C26 alkyl group, a C28 alkyl group, or a C30 alkyl group. In some embodiments, the alkyl is a C8-C20 alkyl, e.g., C14 alkyl or C16 alkyl.
In some embodiments, the alkyl group comprises a steroid moiety of a bile acid, such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholecolic acid.
In some embodiments of the invention wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the glucagon related peptide is modified to comprise an alkyl group by reacting a nucleophilic long chain alkane with the glucagon related peptide, wherein the glucagon related peptide comprises a leaving group suitable for nucleophilic substitution. In particular aspects where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the nucleophilic group of the long chain alkane comprises an amine, a hydroxyl group, or a sulfhydryl group (e.g., octadecylamine, tetradecanol, and hexadecanethiol). The leaving group of a glucagon related peptide of class 1, class 2 or class 3 may be part of the amino acid side chain or may be part of the peptide backbone. Suitable leaving groups include, for example, N-hydroxysuccinimide, halogen, and sulfonate esters.
In certain embodiments, the class 1, class 2 or class 3 glucagon related peptide is modified to comprise an alkyl group by reacting a nucleophilic long-chain alkane with a spacer linked to the glucagon related peptide, wherein the spacer comprises a leaving group. In particular aspects wherein the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the long alkane comprises an amine, a hydroxyl, or a thiol group. In certain embodiments where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, the spacer comprising a leaving group may be any of the spacers discussed herein, such as amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacers, and hydrophobic bifunctional spacers that further comprise a suitable leaving group.
For those aspects of the invention in which the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3 and the long chain alkane is alkylated with the glucagon related peptide or spacer, the long chain alkane may be of any size and may comprise any carbon chain length. The long chain alkane may be straight chain or branched. In certain aspects, the long chain alkane is a C4-C30 alkane. For example, the long-chain alkane may be any of a C4 alkane, a C6 alkane, a C8 alkane, a C10 alkane, a C12 alkane, a C14 alkane, a C16 alkane, a C18 alkane, a C20 alkane, a C22 alkane, a C24 alkane, a C26 alkane, a C28 alkane, or a C30 alkane. In some embodiments wherein the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, the long chain alkane comprises a C8-C20 alkane, e.g., a C14 alkane, a C16 alkane, or a C18 alkane.
Also, in some embodiments where the glucagon related peptide is a class 1, class 2 or class 3 glucagon related peptide, alkylation may occur between the glucagon related peptide and the cholesterol moiety. For example, the hydroxyl group of cholesterol may replace a leaving group on a long chain alkane to form a cholesterol-glucagon peptide product.
The alkylated glucagon related peptides described herein may be further modified to include a hydrophilic moiety. In some embodiments, the hydrophilic moiety may comprise a polyethylene glycol (PEG) chain. Incorporation of the hydrophilic moiety can be accomplished by any suitable method, such as any of the methods described herein. In this aspect, the alkylated glucagon related peptide may comprise seq id NO: 701, or a modified amino acid sequence of such a sequence comprising one or more of the amino acid modifications described herein, wherein at least one of the amino acids at positions 10, 20, 24 and 29 (numbered according to the amino acids of wild-type glucagon) comprises an alkyl group, at least one of the amino acids at positions 16, 17, 21, 24 and 29, a position within the C-terminal overhang or the C-terminal amino acid is modified to Cys, Lys, Orn, homocysteine or Ac-Phe, and the side chain of the amino acid is covalently bound to a hydrophilic moiety (e.g., PEG). In some embodiments, the alkyl group is optionally linked to position 10 (numbered according to amino acids of wild-type glucagon) through a spacer comprising Cys, Lys, Orn, homocysteine, or Ac-Phe, and the hydrophilic moiety is incorporated into the Cys residue at position 24.
Alternatively, the alkylated glucagon related peptide may comprise a spacer, wherein the spacer is both alkylated and modified to comprise a hydrophilic moiety. Non-limiting examples of suitable spacers include spacers comprising one or more amino acids selected from Cys, Lys, Orn, homocysteine, and Ac-Phe.
Stabilization of alpha helical structures
In some embodiments, an intramolecular bridge is formed between the 2 amino acid side chains to stabilize the three-dimensional structure of the carboxy-terminal portion of the glucagon-related peptide, e.g., amino acids 12-29 (numbered according to amino acids of wild-type glucagon). The 2 amino acid side chains may be linked to each other by hydrogen bonding, ionic interactions (e.g., formation of salt bridges), or by covalent bonds.
In some embodiments, an intramolecular bridge is formed between 2 amino acids that are 3 amino acids apart, e.g., the amino acids at positions i and i +4, where i is any integer between 12 and 25 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) numbered according to the amino acids of wild-type glucagon. More specifically, the side chains of amino acid pairs of 12 and 16, 16 and 20, 20 and 24 or 24 and 28 (wherein i ═ amino acid pair of 12, 16, 20 or 24) are linked to each other according to the amino acid numbering of wild-type glucagon, thereby stabilizing the glucagon alpha helix. Or i may be 17.
In some embodiments in which the amino acids at positions i and i +4 are connected by an intramolecular bridge, the linker is about 8 atoms in size or about 7-9 atoms in size.
In other embodiments, an intramolecular bridge is formed between 2 amino acids that are 2 amino acids apart, e.g., the amino acids at positions j and j +3, where j is any integer between 12 and 26 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26) according to the amino acid numbering of wild-type glucagon. In some embodiments, j is 17.
In some embodiments in which the amino acids at positions j and j +3 are connected by an intramolecular bridge, the linker is about 6 atoms in size, or about 5-7 atoms in size.
In still other embodiments, an intramolecular bridge is formed between 2 amino acids that are 6 amino acids apart, e.g., the amino acids at positions k and k +7, where k is any integer between 12 and 22 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22) according to the amino acid numbering of wild-type glucagon. In some embodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.
Examples of pairs of amino acids capable of covalent bonding to form a 6-atom bridge include Orn and Asp, Glu and amino acids of formula I (where n is 2), homoglutamic acid and amino acids of formula I (where n is 1), wherein formula I is:
wherein n is 1-4
[ formula I ]
Examples of amino acid pairs capable of covalent bonding to form a 7-atom connecting bridge include Orn-Glu (lactam ring), Lys-Asp (lactam) or homoserine-homoglutamic acid (lactone). Examples of amino acid pairs that can form an 8-atom linker include Lys-Glu (lactam), homolysine-Asp (lactam), Orn-homoglutamic acid (lactam), 4-aminophe-Asp (lactam), or Tyr-Asp (lactone). Examples of amino acid pairs that can form a 9-atom linker include homolysine-Glu (lactam), Lys-homoglutamic acid (lactam), 4-aminophe-Glu (lactam), or Tyr-Glu (lactone). Any side chains of these amino acids may also be substituted with other chemical groups, as long as the three-dimensional structure of the alpha helix is not disrupted. One of ordinary skill in the art can expect alternative pairs or alternative amino acid analogs, including chemically modified derivatives, that can produce stable structures of similar size and desired effect. For example, homocysteine-homocysteine disulfide bridges are 6 atoms long and may be further modified to provide the desired effect. Even without covalent bonds, the above-described amino acid pairings, or similar pairings as would be expected by one of ordinary skill in the art, may provide additional stability to the alpha helix through non-covalent bonds (e.g., formation of salt bridges or hydrogen bonding interactions).
The size of the lactam ring may vary with the length of the amino acid side chain, and in some embodiments, the lactam is formed by the attachment of the side chain of a lysine amino acid to a glutamic acid side chain. Further exemplary embodiments (by amino acid numbering of wild-type glucagon) include the following pairs optionally having a lactam bridge: glu at position 12 and Lys at position 16, natural Lys at position 12 and Glu at position 16, Glu at position 16 and Lys at position 20, Lys at position 16 and Glu at position 20, Glu at position 20 and Lys at position 24, Lys at position 20 and Glu at position 24, Glu at position 24 and Lys at position 28, and Lys at position 24 and Glu at position 28. Alternatively, the order of the amide bonds in the lactam ring may be reversed (e.g., a lactam ring may be formed between the side chains of Lys12 and Glu16 or between Glu12 and Lys 16).
Intramolecular bridges other than lactam bridges can be used to stabilize the alpha helix of glucagon related peptides. In some embodiments, the intramolecular bridge is a hydrophobic bridge. In this case, the intramolecular bridge is optionally interposed between the side chains of 2 amino acids which are part of the hydrophobic face of the alpha helix of the glucagon related peptide. For example, one of the amino acids linked by a hydrophobic bridge may be the amino acids at positions 10, 14 and 18 (numbered according to amino acids of wild-type glucagon).
In a particular aspect, one or both turns of the alpha helix of a glucagon related peptide are crosslinked using olefin metathesis using an all hydrocarbon crosslinking system. In this case, the glucagon related peptide may comprise alpha-methylated amino acids carrying olefinic side chains of different lengths and arranged in R or S stereochemistry at positions i and i +4 or i + 7. For example, the olefin side may Comprise (CH)2) n, wherein n is any integer between 1 and 6. In some embodiments, for 8A cross-link of one atom length, n is 3. Suitable methods for forming such intramolecular bridges are described in the art. See, e.g., schaffeister et al, j.am.chem.soc.122: 5891-: 1466-1470(2004). Alternatively, the glucagon peptide may comprise O-allyl Ser residues adjacent to the spiro turn angle, which are bridged together by ruthenium-catalyzed ring-closing metathesis. Such crosslinking methods are described, for example, by Blackwell et al, angelw, chem., int.ed.37: 3281-3284(1998).
In another specific aspect, the use of an unnatural thiodipropionine amino acid, lanthionine (which is widely used as a peptidomimetic of cystine), is used to crosslink one turn of an alpha helix. Suitable methods based on lanthionine cyclization are known in the art. See, e.g., Matteucci et al, Tetrahedron Letters 45: 1399-1401 (2004); mayer et al, J.peptide Res.51: 432-436 (1998); polinsky et al, j.med.chem.35: 4185-4194 (1992); osapay et al, j.med.chem.40: 2241-2251 (1997); fukase et al, Bull. chem. Soc. Jpn.65: 2227-; harpp et al, J.org.chem.36: 73-80 (1971); goodman and Shao, Pure appl. chem.68: 1303, 1308 (1996); and Osapay and Goodman, j.chem.soc.chem.Commun.1599-1600 (1993).
In some embodiments, an alpha, omega-diaminoalkane linker (tether) between the 2 Glu residues at positions i and i +7 (e.g., 1, 4-diaminopropane and 1, 5-diaminopentane) is used to stabilize the alpha helix of the glucagon peptide. Such linkers result in the formation of bridges that are 9 atoms or more in length, depending on the length of the diaminoalkane linker. Suitable methods for generating peptides that crosslink with such linkers are described in the art. See, e.g., Phelan et al, j.am.chem.soc.119: 455-460(1997).
In yet another embodiment of the invention, one or both turns of the alpha helix of the glucagon related peptide are cross-linked using a disulfide bridge. Alternatively, modified disulfide bridges, in which one or both sulfur atoms are replaced by a methylene group resulting in isosteric cyclization, are used to stabilize the alpha helix of glucagon related peptides. Suitable methods for modifying peptides with disulfide bridges or sulfur-based cyclization are described, for example, in Jackson et al, j.am.chem.soc.113: 9391-9392(1991) and Rudinger and Jost, Experientia 20: 570-571(1964).
In yet another embodiment, the alpha helix of the glucagon related peptide is stabilized by binding of a metal atom to 2 His residues or His and Cys pairs located at positions i and i + 4. The metal atom may be, for example, Ru (III), Cu (II), Zn (II), or Cd (II). Such methods for stabilizing alpha helices based on metal binding are known in the art. See, e.g., Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); ghadiri et al, j.am.chem.soc.112: 1630-1632 (1990); and Ghadiri et al, j.am.chem.soc.119: 9063-9064(1997).
The alpha helix of glucagon related peptides or other methods by which peptide cyclization can be stabilized are reviewed by Davies, j.peptide.sci.9: 471-501(2003). The alpha helix may be stabilized by the formation of amide bridges, thioether bridges, thioester bridges, urea bridges, carbamate bridges, sulfonamide bridges, and the like. For example, a thioester bridge may be formed between the C-terminus and the side chain of a Cys residue. Alternatively, thioesters may be formed through the side chain of an amino acid having a sulfhydryl group (Cys) and a carboxylic acid (e.g., Asp, Glu). In another approach, a crosslinking agent (e.g., a dicarboxylic acid such as suberic acid) can introduce a linkage between 2 functional groups (e.g., free amino groups, hydroxyl groups, thiol groups, and combinations thereof) of the amino acid side chain.
According to some embodiments, the alpha helix of the glucagon related peptide is stabilized by incorporating hydrophobic amino acids in the i and i +4 positions. For example, i may be Tyr, i +4 may be Val or Leu; i may be Phe, i +4 may be Cys or Met; i may be Cys, i +4 may be Met; or i can be Phe and i +4 can be Ile. It will be appreciated that for purposes herein, the above amino acid pairing can be reversed such that the amino acid given at position i can alternatively be located at position i +4, and the i +4 amino acid can be located at position i.
According to other embodiments of the present invention, wherein the glucagon related peptide is a peptide having glucagon agonist activity, GIP agonist activity, glucagon antagonist and GLP-1 activity, the alpha helix is stabilized by incorporating (by amino acid substitution or insertion) one or more alpha helix stabilizing amino acids in the C-terminal portion of the glucagon related peptide (in the vicinity of amino acids 12-29, depending on the numbering of the wild-type glucagon amino acid). In a particular embodiment, the amino acid that stabilizes the alpha helix is an alpha, alpha-disubstituted amino acid, including, but not limited to, any of the following: aminoisobutyric Acid (AIB), amino acids substituted with the same or different groups selected from methyl, ethyl, propyl and n-butyl or disubstituted with cyclooctane or cycloheptane (e.g. 1-aminocyclooctane-1-carboxylic acid). In some embodiments, the glucagon related peptide is substituted with an α, α -disubstituted amino acid at 1, 2, 3, 4 or more of positions 16, 17, 18, 19, 20, 21, 24 or 29. In a particular embodiment, 1, 2, 3 or all of positions 16, 20, 21 and 24 are substituted with AIB.
Conjugates
The present disclosure also includes conjugates in which a glucagon related peptide (e.g., a glucagon-like 1 peptide, a glucagon-like 2 peptide, a glucagon-like 3 peptide, a glucagon-like 4 peptide, or a glucagon-like 5 peptide) is optionally covalently bound and optionally linked to a conjugate moiety by a linker. Attachment may be achieved by covalent chemical bonds, physical forces such as electrostatic, hydrogen, ionic, van der waals, or hydrophobic or hydrophilic interactions. Various non-covalent coupling systems may also be employed, including biotin-avidin, ligands/receptors, enzymes/substrates, nucleic acids/nucleic acid binding proteins, lipids/lipid binding proteins, cell adhesion molecule partners; or any binding partners or fragments thereof having affinity for each other.
The glucagon-related peptides can be attached to the conjugate moiety by direct covalent attachment via reaction of targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or N-or C-terminal residues of these targeted amino acids. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, or other derivatizing agents known in the art. Alternatively, the conjugate moiety may be indirectly linked to the peptide through an intermediate carrier, such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these and other amino acids (e.g., serine) to provide the desired solubility properties on the resulting load carrier.
Most commonly, the cysteinyl residue is reacted with an α -haloacetate (and the corresponding amine), such as chloroacetic acid or chloroacetamide, to give a carboxymethyl derivative or a carboxyamidomethyl derivative. Cysteinyl residues are also derivatized by reaction with: bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetylphosphoric acid (chloroacetyl phosphate), N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri4-nitrophenol or chloro-7-nitrobenzo-2- -1, 3-diazole.
Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this reagent is relatively specific for histidyl side chains. P-bromobenzoylmethyl bromide may also be used; the reaction is preferably carried out at pH 6.0 in 0.1M sodium cacodylate.
Lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing the α -amino group-containing residue include imidoesters, such as methyl picoliniminate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, orthomethylisourea, 2, 4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.
Arginyl residues are modified by reaction with one or several common reagents, among which phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions, since the pK of the guanidine functionaHigh. In addition, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.
Specific modifications can be made to tyrosyl residues, of which particular interest is the introduction of spectroscopic tags into tyrosyl residues by reaction with aromatic diazo compounds or tetranitromethane. N-acetylimidazole and tetranitromethane are most commonly used to form O-acetyltyrosyl and 3-nitro derivatives, respectively.
The pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N ═ C ═ N-R '), where R and R' are different alkyl groups, e.g. 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-nitrogen) carbodiimide-4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of the lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N-terminal amine and/or amidation or esterification of the C-terminal carboxylic acid group.
Another type of covalent modification involves chemical or enzymatic coupling of the glycoside to the peptide. The saccharide may be linked to (a) arginine and histidine, (b) a free carboxyl group, (c) a free thiol group (e.g., of cysteine), (d) a free hydroxyl group (e.g., of serine, threonine, or hydroxyproline), (e) an aromatic residue (e.g., of tyrosine or tryptophan) or (f) an amide group of glutamine. These methods are described in WO87/05330, published 11.9.1987, and Aplin and Wriston, CRC Crit. Rev. biochem., 259-306 (1981).
Exemplary conjugate moieties that can be linked to any of the glucagon related peptides described herein include, but are not limited to, heterologous peptides or polypeptides (including, e.g., plasma proteins), targeting agents, immunoglobulins or portions thereof (e.g., variable regions, CDR or Fc regions), diagnostic labels (e.g., radioisotopes, fluorophores, or enzyme labels), polymers (including water-soluble polymers), or other therapeutic or diagnostic agents. In some embodiments, conjugates are provided comprising a glucagon related peptide of the invention and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferrin, fibrinogen and globulin. In some embodiments, the plasma protein portion of the conjugate is albumin or transferrin. In some embodiments, the linker comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms of the linker backbone are selected from C, O, N and S. The chain atoms and linker may be selected according to their intended solubility (hydrophilicity) so as to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is cleaved/cleaved by an enzyme or other catalyst or hydrolysis conditions present in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the likelihood of steric hindrance. If the linker is a covalent bond or a peptide bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such peptidyl linkers may be of any length. Exemplary linkers are about 1-50 amino acids, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length. Alternatively, such fusion proteins can be produced by recombinant genetic engineering methods known to those of ordinary skill in the art.
As described above, in some embodiments, the glucagon-related peptide is conjugated, e.g., fused to an immunoglobulin or portion thereof (e.g., a variable region, CDR, or Fc region). Known immunoglobulin (Ig) classes include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain, which is responsible for binding to Fc receptors that effect activity (e.g., cyclization, which results in extended half-life, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).
For example, according to certain definitions, the human IgG heavy chain Fc region extends from Cys226 to the C-terminus of the heavy chain. The "hinge region" typically extends from Glu216 to Pro230 of human IgG1 (the hinge region of other IgG isotypes can be aligned to the IgG1 sequence by aligning the cysteines involved in cysteine binding). The Fc region of IgG comprises 2 constant domains CH2 and CH 3. The CH2 domain of the human IgGFc region typically extends from amino acid 231 to amino acid 341. The CH3 domain of the human IgG Fc region typically extends from amino acids 342 to 447. The amino acid numbering of the immunoglobulins or immunoglobulin fragments or regions mentioned is based on Kabat et al, 1991, Sequences of Proteins of Immunological Interest, U.S. department of public Health, Bethesda, Md. In a related embodiment, the Fc region may comprise one or more native or modified constant regions of an immunoglobulin heavy chain other than CH1, such as the CH2 and CH3 regions of IgG and IgA or the CH3 and CH4 regions of IgE.
Suitable conjugate moieties include those portions of an immunoglobulin sequence that comprise an FcRn binding site. FcRn, a salvage receptor (salvaging receptor), is responsible for cyclizing the immunoglobulin and reinfusing it back into the blood circulation. The region of the IgG Fc part that binds the FcRn receptor was described according to X-ray crystallography (Burmeister et al, 1994, Nature 372: 379). The primary contact region of Fc to FcRn is near the junction of the CH2 and CH3 domains. The Fc-FcRn contact points are all within a single Ig heavy chain. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain.
Some conjugate moieties may or may not include an fcyr binding site. Fc γ R is responsible for ADCC and CDC. Examples of positions within the Fc region which are in direct contact with Fc γ R are the amino acids 234-. The lower hinge region of IgE is also involved in FcRI binding (Henry et al, Biochemistry36, 15568-15578, 1997). Residues involved in IgA receptor binding are described by Lewis et al (JImmunol.175: 6694-701, 2005). Amino acid residues involved in IgE receptor binding are described in Sayers et al (J Biol chem.279 (34): 35320-5, 2004).
Amino acid modifications may be made to the Fc region of an immunoglobulin. Such variant Fc regions comprise at least one amino acid modification in the CH3 domain (residues 342-447) and/or at least one amino acid modification in the CH2 domain (residues 231-341) of the Fc region. Mutations believed to confer increased affinity to FcRn include T256A, T307A, E380A and N434A (Shields et al, 2001, j.biol.chem.276: 6591). Other mutations may reduce the binding of the Fc region to Fc γ RI, Fc γ RIIA, Fc γ RIIB, and/or Fc γ RIIIA without significantly reducing the affinity for FcRn. For example, substitution of Asn at position 297 of the Fc region by Ala or other amino acid eliminates a well-conserved N-glycosylation site and can result in reduced immunogenicity with increased half-life of the Fc region and reduced binding to Fc γ R (Routledge et al, 1995, Transplantation 60: 847; Friend et al, 1999, Transplantation 68: 1632; Shields et al, 1995, J.biol.chem.276: 6591). Amino acid modifications at position 233-236 of IgG1 that reduce binding to Fc γ R can be performed (Ward and Ghetie, 1995, Therapeutic Immunology 2: 77 and Armour et al, 1999, Eur. J. immunol. 29: 2613). Some exemplary amino acid substitutions can be found in U.S. patents 7,355,008 and 7,381,408, each of which is incorporated by reference herein in its entirety.
rPEG
In some embodiments, the conjugates of the present invention comprise glucagon superfamily peptides, including glucagon-related peptides, osteocalcin, and analogs, derivatives and conjugates of the aforementioned biologically active peptides fused to helper peptides capable of forming extended conformations similar to chemical PEG (e.g., recombinant PEG (rpeg) molecules), such as the helper peptides described in international patent application publication No. WO2009/023270 and U.S. patent application publication No. US 2008/0286808. The rPEG molecule is not polyethylene glycol. In some aspects, the rPEG molecule is a polypeptide comprising one or more of glycine, serine, glutamic acid, aspartic acid, alanine, or proline. In some aspects, the rPEG is a homopolymer, such as polyglycine, polyserine, polyglutamic acid, polyaspartic acid, polyalanine, or polyproline. In other embodiments, rPEG comprises repeating two types of amino acids, such as poly (Gly-Ser), poly (Gly-Glu), poly (Gly-Ala), poly (Gly-Asp), poly (Gly-Pro), poly (Ser-Glu), and the like. In some aspects, rPEG comprises three different types of amino acids, such as poly (Gly-Ser-Glu). In particular aspects, the rPEG extends the half-life of the glucagon superfamily peptide or osteocalcin. In some aspects, the rPEG comprises a net positive charge or a net negative charge. In some aspects, the rPEG lacks secondary structure. In some embodiments, the rPEG is greater than or equal to 10 amino acids in length, and in some embodiments, from about 40 to about 50 amino acids in length. In some aspects, the helper peptide is fused to the N-terminus or C-terminus of the peptide of the invention via a peptide bond or protease cleavage site, or inserted into the loop of the peptide of the invention. In some aspects, the rPEG comprises an affinity tag or is linked to a PEG of greater than 5 kDa. In some embodiments, rPEG confers increased hydrodynamic radius, serum half-life, protease resistance or solubility to a peptide of the invention, and in some aspects, reduced immunogenicity to the peptide.
Fusion peptide-C terminal overhang
In certain embodiments, the glucagon related peptide may comprise a C-terminal or C-terminal amino acid sequence including, but not limited to: COOH, CONH2、GPSSGAPPPS(SEQID NO：710)、GPSSGAPPPS-CONH2(SEQ ID NO: 711), an oxyntomodulin carboxy-terminal overhang, KRNRNNIA (SEQ ID NO: 714) or KGKKNDWKHNITQ (SEQ ID NO: 713). For example, the terminal 10 amino acids of Exendin-4 (i.e., the sequence of SEQ ID NO: 710 (GPSSGAPPPS)), with the present disclosureThe carboxy-terminal linkage of a class 1 glucagon-related peptide, a class 2 glucagon-related peptide, a class 3 glucagon-related peptide, a class 4 glucagon-related peptide, or a class 5 glucagon-related peptide of the disclosure.
Another compound that causes weight loss is oxyntomodulin, a naturally occurring digestive hormone present in the small intestine (see Diabetes 2005; 54: 2390-. Oxyntomodulin is a 37 amino acid peptide (SEQ ID NO: 706) comprising the 29 amino acid sequence of glucagon followed by the amino acid sequence of SEQ ID NO: 714 (KRNRNNIA). Thus, in some embodiments, there is provided a polypeptide further comprising SEQ ID NO: 714 sequence or a prodrug derivative of a glucagon related peptide having a 4-amino acid extension of the sequence KRNR.
Glucagon modification at position 3
The 3-position (according to amino acid numbering of wild-type glucagon) of the glucagon related peptides of classes 1-3 described herein may be modified to maintain or increase activity at the glucagon receptor.
In some embodiments wherein the glucagon related peptide is a glucagon type 1, 2 or 3 peptide, maintenance or enhancement of glucagon receptor activity may be achieved by modifying the Gln at position 3 with a glutamine analog. For example, the activity of a glucagon related peptide class 1, class 2 or class 3 comprising a glutamine analog at position 3 at the glucagon receptor can be shown to be about 5%, about 10%, about 20%, about 50% or about 85% or more of the activity of native glucagon (SEQ ID NO: 701). In some embodiments, the activity of a glucagon related peptide of class 1, class 2 or class 3 comprising a glutamine analog at position 3 at the glucagon receptor may be manifested as about 20%, about 50%, about 75%, about 100%, about 200% or about 500% or more of the activity of the corresponding glucagon peptide having the same amino acid sequence as the peptide comprising the glutamine analog except for the modified amino acid at position 3. In some embodiments, the glucagon related peptide class 1, class 2 or class 3 comprising a glutamine analog at position 3 has increased activity at the glucagon receptor, but the increased activity is less than 1000%, 10,000%, 100,000% or 1,000,000% of the activity of native glucagon or a corresponding glucagon related peptide having the same amino acid sequence as the peptide comprising the glutamine analog except for the modified amino acid at position 3.
In some embodiments, the glutamine analog is a naturally occurring or non-naturally occurring amino acid comprising a side chain of the following structure I, II or III:
structure I
Structure II
Structure III
Wherein R is1Is C0-3Alkyl or C0-3A heteroalkyl group; r2Is NHR4Or C1-3An alkyl group; r3Is C1-3An alkyl group; r4Is H or C1-3An alkyl group; x is NH, O or S; and Y is NHR4、SR3OR OR3. In some embodiments, X is NH or Y is NHR4. In some embodiments, R1Is C0-2Alkyl or C1A heteroalkyl group. In some embodiments, R2Is NHR4Or C1An alkyl group. In some implementationsIn the scheme, R4Is H or C1An alkyl group. In exemplary embodiments where the glucagon related peptide is a glucagon related peptide of class 1, class 2 or class 3, amino acids comprising a side chain of structure I are provided, wherein R is1Is CH2-S, X is NH, R2Is CH3(acetamidomethyl-cysteine, c (acm)); r1Is CH2X is NH, R2Is CH3(acetyldiaminobutyric acid, dab (ac)); r1Is C0Alkyl, X is NH, R2Is NHR4，R4Is H (carbamoyldiaminopropionic acid, Dap (urea)); or R1Is CH2-CH2X is NH, R2Is CH3(acetylornithine, Orn (Ac)). In an exemplary embodiment, an amino acid comprising a side chain of structure II is provided, wherein R1Is CH2Y is NHR4，R4Is CH3(methyl glutamine, q (me)); in an exemplary embodiment, an amino acid comprising a side chain of structure IIII is provided, wherein R1Is CH2，R4Is H (methionine-sulfoxide, M (O)); in a specific embodiment, the amino acid at position 3 is substituted with dab (ac).
Dimer
For class 1, class 2 and class 3 glucagon related peptides, the glucagon related peptide may be a component of a dimer, trimer or higher order multimer comprising at least 2, 3 or more peptides bound by a linker, wherein at least one or both peptides are glucagon related peptides. The dimer may be a homodimer or a heterodimer. In some embodiments, the linker is selected from the group consisting of difunctional thiol crosslinkers and difunctional amine crosslinkers. In certain embodiments, the linker is a PEG, e.g., a 5kDa PEG, a 20kDa PEG. In some embodiments, the linker is a disulfide bond. For example, each monomer of the dimer may comprise a Cys residue (e.g., a Cys located terminally or internally), and the sulfur atom of each Cys residue is involved in the formation of a disulfide bond. In some aspects of the invention, the monomers are linked by a terminal amino acid (e.g., N-or C-terminus), by an internal amino acid, or by a terminal amino acid of at least one monomer and an internal amino acid of at least one other monomer. In particular aspects, the monomers are not linked by an N-terminal amino acid. In some aspects, monomers of a multimer are linked together in a "tail-to-tail" orientation, wherein the C-terminal amino acids of each monomer are linked together. The conjugate moiety can be covalently linked to any of the glucagon related peptides described herein, including dimers, trimers or higher order multimers.
Process for preparing glucagon related peptide
The glucagon related peptides (and prodrugs) disclosed herein may be prepared by standard synthetic methods, recombinant DNA techniques, or any other method of preparing peptides and fusion proteins. Although certain unnatural amino acids are not expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. In addition to standard peptide chemistry, where applicable, the compounds of the invention comprising a non-peptide moiety can be synthesized by standard organic chemistry.
The classes of glucagon related peptides are described in detail below. With respect to the various portions of the disclosure relating to class 1, 2, 3, 4 and 5 glucagon-like peptides, modifications of the glucagon-like peptide portion (Q) with respect to the prodrug compounds detailed above are described. Thus, the structural element described according to the glucagon related peptide class is that of Q, which is then further modified to produce the prodrug compound as described above.
Class 1 glucagon related peptides
In certain embodiments, the glucagon related peptide is a class 1 glucagon related peptide described herein and described in international patent application No. PCT US2009/47437 (filed 6/16/2009), international patent application publication No. WO 2008/086086 (published 7/17/2008), and U.S. provisional application No. 61/090,415, the contents of which are incorporated herein by reference in their entirety.
The biological sequences mentioned in the following sections in relation to the class 1 glucagon-like related peptide (SEQ ID NO: 801-915) correspond to the sequence of SEQ ID NO of International patent application No. PCT US 2009/47437: 1-115.
Activity of
Glucagon type 1 peptides retain glucagon receptor activity relative to native glucagon peptide (SEQ ID NO: 801). For example, the glucagon peptide can retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% activity, 80% activity, 85% activity, or 90% of the native glucagon activity (calculated as an inverse ratio of EC50 for glucagon peptide versus glucagon, e.g., measured by cAMP production using the assay outlined in example 5). In some embodiments, the glucagon-related peptide class 1 has the same and greater activity (used in the same sense as the term "potency" herein) as compared to glucagon. In some embodiments, the activity of the glucagon peptides described herein is no more than about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of the native glucagon peptide.
Any of the glucagon-like peptide class 1 described herein can exhibit an EC50 for the human glucagon receptor of about 100nM, 75nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, or less when cAMP induced in HEK293 cells overexpressing the glucagon receptor is determined, e.g., using the assay of example 5. Typically, pegylated peptides may have a higher EC50 than non-pegylated peptides. For example, a glucagon-like peptide class 1 described herein, when not pegylated, can exhibit an activity at the glucagon receptor that is at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, 150%, 200%, 400%, 500%, or more) of the activity of native glucagon (SEQ ID NO: 801) at the glucagon receptor. In certain embodiments, the glucagon related peptide class 1 described herein exhibits a labeled activity% of native glucagon at the glucagon receptor in the absence of the hydrophilic moiety, but exhibits a decreased activity% of native glucagon at the glucagon receptor when the hydrophilic moiety is included. For example, a class 1 glucagon-related peptide described herein, when pegylated, exhibits an activity at the glucagon receptor that can be at least 2% (e.g., at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the activity of native glucagon.
In some embodiments, the glucagon-like peptide class 1 exhibits less than about 5%, 4%, 3%, 2%, or 1% activity at the GLP-1 receptor than native GLP-1 and/or about 5-fold, 10-fold, or 15-fold selectivity at the glucagon receptor over the GLP-1 receptor. For example, in some embodiments, the glucagon-like peptide class 1 exhibits less than 5% activity at the GLP-1 receptor as compared to native GLP-1, and is 5-fold selective for the glucagon receptor as compared to the GLP-1 receptor.
Improved solubility
Native glucagon is poorly soluble in aqueous solutions, particularly at physiological pH, and tends to aggregate and precipitate over time. In contrast, after 24 hours at 25 ℃ between pH 6 and 8 or between 6 and 9 (e.g., at pH 7), the glucagon-related peptide class 1 has in some embodiments at least 2-fold, 5-fold, or even higher solubility compared to native glucagon.
Thus, in some embodiments, the class 1 glucagon-related peptide is modified to improve the solubility of the peptide in aqueous solution, particularly at a pH in the range of about 5.5 to about 8.0, while maintaining the biological activity of the native peptide, as compared to the wild-type peptide of His-Ser-Gln-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 801).
For example, the solubility of any of the class 1 glucagon-related peptides described herein can be further improved by linking the hydrophilic moiety to the peptide. The introduction of such groups also extends the duration of action, for example as measured by an extended circulating half-life. Hydrophilic moieties are further described herein.
Modification with charged residues
In some embodiments, the solubility is increased by adding a charge to the class 1 glucagon-related peptide by substituting a charged amino acid selected from the group consisting of lysine, arginine, histidine, aspartic acid, and glutamic acid for a naturally uncharged amino acid, or by adding a charged amino acid to the amino-or carboxy-terminus of the peptide.
According to some embodiments, the glucagon related peptide class 1 has improved solubility due to the fact that by introducing a charged amino acid into the C-terminal portion of the peptide and in some embodiments into the sequence of SEQ ID NO: 801 at the C-terminal position 27, to modify the peptide. Optionally, 1, 2 or 3 charged amino acids may be introduced into the C-terminal portion, in some embodiments from the C-terminus to position 27. According to some embodiments, the natural amino acid at position 28 and/or 29 is substituted with a charged amino acid, and/or 1-3 charged amino acids are added to the C-terminus of the peptide, e.g. after position 27, 28 or 29. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. In other embodiments, 1, 2, 3, or all of the charged amino acids have a positive charge.
In specific exemplary embodiments, the glucagon-like peptide class 1 may comprise modifications of either or both of: n28 substituted with E; n28 substituted with D; t29 substituted by D; t29 substituted with E; insert E after position 27, 28 or 29; d was inserted after position 27, 28 or 29. For example, D28E29, E28E29, E29E30, E28E30, D28E 30.
According to an exemplary embodiment, the glucagon-like related peptide class 1 comprises SEQ ID NO: 811 or an analog thereof comprising 1-3 additional amino acid modifications (described herein for glucagon agonists) as compared to native glucagon or a glucagon agonist analog thereof. SEQ ID NO: 811 represents a modified glucagon-like peptide of class 1 wherein the asparagine residue at position 28 of the native protein is replaced with aspartic acid. In another exemplary embodiment, the glucagon-like related peptide class 1 comprises SEQ ID NO: 838 in which the asparagine residue at position 28 of the native protein is replaced with glutamic acid. Other exemplary embodiments include SEQ id nos: 824. 825, 826, 833, 835, 836 and 837.
Substitution of the amino acids normally present at positions 28 and/or 29 with charged amino acids, and/or addition of 1-2 charged amino acids at the carboxy terminus of the class 1 glucagon-related peptide, increases the solubility and stability of the glucagon peptide in aqueous solution at physiologically relevant pH (i.e., pH of about 6.5 to about 7.5) by at least a factor of 5 and as much as a factor of 30. Thus, at a given pH (e.g., pH 7) between about 5.5 and 8, the glucagon-like peptide class 1 of some embodiments retains glucagon activity and exhibits at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-fold or greater solubility relative to native glucagon when measured after 24 hours at 25 ℃.
Other modifications, such as conservative substitutions, which will be further described herein, can be made to the glucagon-like peptide class 1 that still maintain glucagon activity.
Improved stability
Any of the glucagon-like peptide class 1 may additionally exhibit improved stability and/or reduced degradation, e.g., retention of at least 95% of the starting peptide after 24 hours at 25 ℃. Any of the glucagon related peptide class 1 peptides disclosed herein can additionally exhibit improved stability, e.g., at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the starting peptide is retained after 24 hours at 25 ℃, in the ph range of 5.5-8. In some embodiments, the glucagon-related peptide class 1 of the present invention exhibits improved stability such that after about one or more weeks (e.g., about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months) in solution at least a 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, greater than 95%, up to 100%) peptide concentration or less than about 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%, 4%) is detectable by an Ultraviolet (UV) detector at 280nm after about one or more weeks (e.g., about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months) at least 20 ℃ (e.g., 21 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, at least 27.g., 5%, 4%), and at a temperature of less than 100 ℃, less than 85 ℃, 3%, 2%, 1%, down to 0%) of a degrading peptide. The class 1 glucagon-related peptide may include other modifications that alter its pharmaceutical properties, such as increasing potency, increasing circulating half-life, increasing shelf life, reducing precipitation or aggregation, and/or reducing degradation, such as reduced occurrence of post-storage cleavage or chemical modification.
In further exemplary embodiments, the expression of the polypeptide of SEQ ID NO: the amino acid at position 15 of 801 additionally modifies any of the aforementioned glucagon-like peptide class 1 to improve stability, thereby reducing degradation of the peptide over time, particularly in acidic or basic buffers. In exemplary embodiments, Asp at position 15 is substituted with Glu, homoglutamic acid, cysteic acid, or homocysteic acid.
Alternatively, the expression of the polypeptide of SEQ ID NO: 801 the amino acid at position 16 makes other modifications to any of the glucagon related class 1 peptides described herein to improve stability. In exemplary embodiments, the Ser at position 16 is substituted with Thr or AIB, or any of the amino acid substitutions described herein for class 1 glucagon related peptides that are associated with increased potency at the glucagon receptor. Such modifications reduce cleavage of the peptide bond between Asp15-Ser 16.
In some embodiments, any of the glucagon related peptide class 1 peptides described herein can be further modified by modifying any of the 1, 2, 3, or all 4 amino acids at positions 20, 21, 24, or 27 to reduce degradation at different amino acid positions. Exemplary embodiments include substitutions of Gln at position 20 with Ser, Thr, Ala or AIB, substitution of Asp with Glu at position 21, substitution of Gln at position 24 with Ala or AIB, and substitution of Met with Leu or Nle at position 27. Removal or substitution of methionine reduces degradation caused by oxidation of methionine. Removal or substitution of Gln or Asn reduces degradation due to deamidation of Gln or Asn. Removal or substitution of the Asp reduces degradation by dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid.
Increased potency
According to another embodiment, there is provided a class 1 glucagon-related peptide with increased potency at the glucagon receptor, wherein the peptide comprises an amino acid modification at position 16 of native glucagon (SEQ ID NO: 801). By way of non-limiting example, such increased potency may be provided as follows: the naturally occurring serine at position 16 is replaced by glutamic acid or by another negatively charged amino acid having a side chain of 4 atoms in length, or any of glutamine, homoglutamic acid, or homocysteine acid, or a charged amino acid having a side chain containing at least one heteroatom (e.g., N, O, S, P) and having a side chain length of about 4 (or 3-5) atoms. Substitution of the serine at position 16 with glutamic acid increases glucagon activity at the glucagon receptor by at least 2-fold, 4-fold, 5-fold, and up to more than 10-fold. In some embodiments, the glucagon-like peptide class 1 remains selective for the glucagon receptor relative to the GLP-1 receptor, e.g., at least 5-fold, 10-fold, or 15-fold selective.
DPP-IV resistance
In some embodiments, the glucagon-like peptide class 1 disclosed herein is further modified at position 1 or 2 to reduce sensitivity to cleavage by dipeptidyl peptidase IV. More specifically, in some embodiments, position 1 and/or position 2 of the glucagon-like peptide class 1 is substituted with a DPP-IV resistant amino acid as described herein. In some embodiments, the 2-position of the analog peptide is substituted with aminoisobutyric acid. In some embodiments, the 2-position of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, D-alanine, glycine, N-methylserine, and epsilon-aminobutyric acid. In another embodiment, position 2 of the glucagon-like related peptide type 1 is substituted with an amino acid selected from the group consisting of D-serine, glycine, and aminoisobutyric acid. In some embodiments, the amino acid at position 2 is not D-serine.
Reduced glucagon activity upon modification of the amino acids at position 1 and/or position 2 of the glucagon peptide can be restored by stabilizing the alpha helical structure (around amino acids 12-29) in the C-terminal portion of the glucagon peptide. As further described herein, the alpha helix structure can be stabilized, for example, by forming covalent or non-covalent intramolecular bridges (e.g., lactam bridges between the flanking chains of amino acids at positions "i" and "i + 4", where i is an integer from 12 to 25), substitution and/or insertion of amino acids near positions 12-29 with amino acids that stabilize the alpha helix (e.g., alpha-disubstituted amino acids).
Modification at position 3
Glucagon receptor activity can be decreased by modification at amino acid position 3 (according to the amino acid numbering of wild-type glucagon), e.g., substitution of the naturally occurring glutamine at position 3 with an acidic, basic or hydrophobic amino acid. For example, substitution of glutamic acid, ornithine or norleucine at position 3 greatly reduces or abolishes glucagon receptor activity.
As described herein, maintenance or enhancement of glucagon receptor activity can be achieved by modifying the Gln at position 3 with a glutamine analog. For example, the glucagon agonist can comprise SEQ ID NO: 863. SEQ ID NO: 869. SEQ ID NO: 870. SEQ ID NO: 871. SEQ ID NO: 872. SEQ ID NO: 873 and SEQ ID NO: 874 amino acid sequence.
Increasing GLP-1 activity with C-terminal amides and esters
Replacement of the carboxylic acid of the C-terminal amino acid with a charge neutral group (e.g., amide or ester) provides increased activity at the GLP-1 receptor. Conversely, the native carboxylic acid that retains the C-terminus of the peptide retains the relative high selectivity of the class 1 glucagon-related peptide for the glucagon receptor as compared to the GLP-1 receptor (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fold for the former as compared to the latter).
Other modifications and combinations
Other modifications can be made to the glucagon-related peptide class 1 which can further enhance solubility and/or stability and/or glucagon activity. The glucagon-like peptide class 1 may also comprise other modifications that do not substantially affect solubility or stability and do not substantially decrease glucagon activity. In exemplary embodiments, the glucagon-related peptide class 1 may comprise a total of up to 11, or up to 12, or up to 13, or up to 14 amino acid modifications as compared to the native glucagon sequence. For example, conservative or non-conservative substitutions, additions or deletions may be made at any of positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29.
Exemplary modifications of class 1 glucagon related peptides include, but are not limited to:
(a) non-conservative substitutions, additions or deletions while retaining at least part of the glucagon agonist activity, e.g., conservative substitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; tyr at position 10 is substituted with Val or Phe; lys at position 12 is substituted with Arg; one or more of these positions is substituted with Ala;
(b) amino acid deletions at positions 29 and/or 28 and optionally 27, while retaining at least a portion of glucagon agonist activity;
(c) modification of the aspartic acid at position 15, for example by substitution with glutamic acid, homoglutamic acid, cysteic acid, or homocysteic acid, which can reduce degradation; or serine at position 16, such as by threonine, AIB, glutamic acid, or by another negatively charged amino acid with a side chain of 4 atoms in length, or by any of glutamine, homoglutamic acid, or homocysteine, again to reduce degradation due to cleavage of the Asp15-Ser16 bond;
(d) as described herein, for example, adding a hydrophilic moiety (e.g., a water-soluble polymer polyethylene glycol) at position 16, 17, 20, 21, 24, 29, 40 or at the C-terminal amino acid may improve solubility and/or extend half-life;
(e) Modification of the methionine at position 27, e.g., by substitution with leucine or norleucine, to reduce oxidative degradation;
(f) modification of the Gln at positions 20 or 24, for example by substitution with Ser, Thr, Ala or AIB to reduce degradation by deamidation of the Gln;
(g) modification of the Asp at position 21, for example by substitution with Glu, to reduce degradation by dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid;
(h) a modification at position 1 or 2 that improves resistance to DPP-IV cleavage as described herein, optionally in combination with an intramolecular bridge, e.g., a lactam bridge between the "i" and "i + 4" positions, wherein i is an integer from 12 to 25, e.g., 12, 16, 20, 24;
(i) acylating or alkylating a glucagon peptide as described herein, which may increase activity at the glucagon receptor and/or the GLP-1 receptor, extend half-life in circulation and/or extend duration of action and/or delay onset, optionally in combination with the addition of a hydrophilic moiety, additionally or alternatively, optionally in combination with a modification that selectively reduces activity at the GLP-1 peptide, e.g. modification of the Thr at position 7, e.g. substitution of the Thr at position 7 with an amino acid lacking a hydroxyl group (e.g. Abu or Ile); deletion of the amino acids from the C-terminus to position 27 (e.g., deletion of one or both of the amino acids at positions 28 and 29, resulting in a peptide of 27 or 28 amino acids);
(j) A C-terminal overhang as described herein;
(k) homo-or heterodimerization as described herein; and
(a) a combination of (a) - (k).
In some embodiments, exemplary modifications of the glucagon-like peptide class 1 include at least one amino acid modification selected from group A and one or more amino acid modifications selected from group B and/or group C,
wherein group A is:
(ii) Asn at position 28 is substituted with a charged amino acid;
asn at position 28 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine;
(xiii) substitution at position 28 with Asn, Asp or Glu;
asp at position 28;
substituted at position 28 with Glu;
thr at position 29 is substituted with a charged amino acid;
thr at position 29 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine;
asp, Glu or Lys at position 29;
substituted at position 29 with Glu;
1-3 charged amino acids are inserted after position 29;
glu or Lys inserted after position 29;
insertion of Gly-Lys or Lys-Lys after position 29;
or a combination thereof;
wherein group B is:
asp at position 15 substituted with Glu;
ser at position 16 substituted with Thr or AIB;
and wherein group C is:
His at position 1 is substituted with an unnatural amino acid that reduces the susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
substitution of the Ser at position 2 with an unnatural amino acid that reduces the susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV),
lys at position 12 is substituted with Arg;
gln at position 20 substituted with Ser, Thr, Ala or AIB;
asp at position 21 substituted with Glu;
gln at position 24 substituted with Ser, Thr, Ala or AIB;
met at position 27 is substituted by Leu or Nle;
a deletion of amino acids at positions 27-29;
deletion of amino acids at positions 28-29;
a deletion of the amino acid at position 29;
or a combination thereof.
In an exemplary embodiment, Lys at position 12 is substituted with Arg. In other exemplary embodiments, the amino acids at positions 29 and/or 28, and optionally 27, are deleted.
In some embodiments, the glucagon peptides comprise (a) an amino acid modification at positions 1 and/or 2 that confers DPP-IV resistance, e.g., a substitution at position 1 with DMIA, or a substitution at position 2 with AIB, (b) an intramolecular bridge within positions 12-29 (e.g., positions 16 and 20), or one or more substitutions of amino acids at positions 16, 20, 21, and 24 with an α, α -disubstituted amino acid, optionally (C) a linkage to a hydrophilic moiety (e.g., PEG), e.g., via a Cys or C-terminal amino acid at positions 24, 29, optionally (d) an amino acid modification at position 27, e.g., to replace Met with Nle, optionally (e) an amino acid modification at positions 20, 21, and 24 that reduces degradation, and optionally (f) an amino acid modification with seq id NO: 820 are connected. In certain embodiments, if the glucagon peptide is not identical to SEQ id no: 820 linkage, the amino acid at position 29 is Thr or Gly. In other specific embodiments, the glucagon peptides comprise (a) Asp28Glu29, or Glu28Glu29, or Glu29Glu30, or Glu28Glu30, or Asp28Glu30, and optionally (b) an amino acid modification at position 16 to replace Ser with, for example, Thr or AIB, and optionally (c) an amino acid modification at position 27 to replace Met with, for example, Nle, and optionally (d) amino acid modifications at positions 20, 21, and 24 to reduce degradation. In a particular embodiment, the glucagon peptide is T16, a20, E21, a24, Nle27, D28, E29.
In some embodiments, the glucagon-like peptide class 1 comprises the amino acid sequence:
X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z (SEQ ID NO: 839) with 1-3 amino acid modifications,
wherein X1 and/or X2 are unnatural amino acids that decrease the sensitivity of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV) (or increase resistance to dipeptidyl peptidase IV),
wherein Z is selected from the group consisting of-COOH (naturally occurring C-terminal carboxyl), -Asn-COOH, Asn-Thr-COOH and Y-COOH, wherein Y is 1-2 amino acids, and
wherein an intramolecular bridge (preferably a covalent bond) links the side chain of the amino acid in position i and the amino acid in position i +4, wherein i is 12, 16, 20 or 24.
In some embodiments, the intramolecular bridge is a lactam bridge. In some embodiments, the nucleic acid sequence of SEQ ID NO: 839 are the amino acids at positions i and i +4 Lys and Glu, such as Glu16 and Lys 20. In some embodiments, X1 is selected from: D-His, N-methyl-His, α -methyl-His, imidazoleacetic acid, deamination-His, hydroxy-His, acetyl-His, homohistidine and α, α -dimethylimidazoleacetic acid (DMIA). In other embodiments, X2 is selected from: D-Ser, D-Ala, Gly, N-methyl-Ser, Val and alpha-aminoisobutyric Acid (AIB). In some embodiments, the glucagon peptide is covalently linked to the hydrophilic moiety at any amino acid at position 16, 17, 20, 21, 24, 29, 40, within the C-terminal overhang, or at the C-terminal amino acid. In exemplary embodiments, the hydrophilic moiety is covalently attached to a Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine residue at any of these positions. Exemplary hydrophilic moieties include, for example, polyethylene glycol (PEG) having a molecular weight of from about 1,000 daltons to about 40,000 daltons or from about 20,000 daltons to about 40,000 daltons.
In other embodiments, the glucagon-like peptide class 1 comprises the amino acid sequence:
X1-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Z(SEQ ID NO：839)，
wherein X1 and/or X2 are unnatural amino acids that decrease the sensitivity of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV) (or increase resistance to dipeptidyl peptidase IV),
wherein 1, 2, 3, 4 or more of the glucagon peptides 16, 20, 21 and 24 are substituted with an alpha, alpha-disubstituted amino acid, and
wherein Z is selected from the group consisting of-COOH (naturally occurring C-terminal carboxyl), -Asn-COOH, Asn-Thr-COOH and Y-COOH, wherein Y is 1-2 amino acids.
Exemplary additional amino acid modifications of the foregoing glucagon-like peptide class 1 or analogs include substitution of Thr at position 7 with an amino acid lacking a hydroxyl group (e.g., aminobutyric acid (Abu), Ile), optionally in combination with substitution or addition of an amino acid comprising a side chain covalently linked (optionally through a spacer) to an acyl group or an alkyl group that is non-natural to the naturally occurring amino acid, with substitution of Lys at position 12 with Arg; asp at position 15 substituted with Glu; ser at position 16 substituted with Thr or AIB; gln at position 20 substituted with Ser, Thr, Ala or AIB; asp at position 21 substituted with Glu; gln at position 24 substituted with Ser, Thr, Ala or AIB; met at position 27 is substituted by Leu or Nle; (ii) Asn at position 28 is substituted with a charged amino acid; asn at position 28 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine; (xiii) substitution at position 28 with Asn, Asp or Glu; asp at position 28; substituted at position 28 with Glu; thr at position 29 is substituted with a charged amino acid; thr at position 29 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine; asp, Glu or Lys at position 29; substituted at position 29 with Glu; 1-3 charged amino acids are inserted after position 29; insertion of Glu or Lys at position 30 (i.e. after position 29); optionally with an insertion of Lys at position 31; SEQ ID NO: 820 to the C-terminus, optionally wherein the amino acid at position 29 is Thr or Gly; substitution or addition of amino acids covalently linked to a hydrophilic moiety; or a combination thereof.
Any of the above modifications mentioned with reference to class 1 glucagon agonists that increase glucagon receptor activity, maintain partial glucagon receptor activity, increase solubility, increase stability or decrease degradation, alone or in combination, may be used for the class 1 glucagon peptide. Thus, a class 1 glucagon-related peptide can be prepared that retains at least 20% of the activity of native glucagon at the glucagon receptor and is soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 or between 6 and 9 (e.g., pH 7), and optionally retains at least 95% of the starting peptide (e.g., 5% or less of the starting peptide is degraded or cleaved) after 24 hours at 25 ℃. Alternatively, high potency glucagon-like peptide class 1 can be prepared that exhibits at least about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or more than 10 times the activity of native glucagon at the glucagon receptor, and is optionally soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 or between 6 and 9 (e.g., pH 7), and optionally retains at least 95% of the starting peptide (e.g., 5% or less of the starting peptide is degraded or cleaved) after 24 hours at 25 ℃. In some embodiments, a class 1 glucagon peptide described herein can exhibit at least any of the indicated relative levels of activity at the glucagon receptor but no more than 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon at the glucagon receptor.
Examples of class 1 glucagon-like related peptide embodiments
According to some embodiments, the peptide of SEQ ID NO: 801 native glucagon peptide. In an alternative embodiment, the amino acid sequence of SEQ ID NO: 801 native glucagon peptide. According to some embodiments, there is provided a glucagon analog having improved solubility and stability, wherein the analog comprises SEQ ID NO: 834 with the proviso that at least one amino acid at position 28 or 29 is substituted with an acidic amino acid and/or that a further acidic amino acid is added to the amino acid sequence of SEQ ID NO: 834 on the carboxy terminus. In some embodiments, the acidic amino acids are independently selected from Asp, Glu, cysteic acid, and homocysteine.
According to some embodiments, a glucagon agonist with improved solubility and stability is provided, wherein the agonist comprises the sequence of SEQ ID NO: 833 in which at least one of the amino acids at position 27, 28 or 29 is substituted with a non-natural amino acid residue (i.e. at least one of the amino acids present at position 27, 28 or 29 of said analogue is an acidic amino acid different from the amino acid present at the corresponding position of SEQ ID NO: 801). According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 833, provided that if the amino acid at position 28 is asparagine and the amino acid at position 29 is threonine, then the peptide further comprises 1-2 amino acids independently selected from Lys, Arg, His, Asp, or Glu added to the carboxy terminus of the glucagon peptide.
It is reported that certain positions of the native glucagon peptide can be modified while maintaining the activity of at least some of the parent peptide. Thus, the applicant expects that the nucleic acid sequence located in SEQ ID NO: one or more of the amino acids at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, or 29 of the 811 peptide may be substituted with an amino acid other than that present in the native glucagon peptide and still maintain the increased potency, physiological pH stability, and biological activity of the parent glucagon peptide. For example, according to some embodiments, the methionine residue present at position 27 of the native peptide is replaced with leucine or norleucine to prevent oxidative degradation of the peptide.
In some embodiments, provided is SEQ ID NO: 833 wherein 1-6 amino acids at positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog are different from the amino acid sequence of SEQ ID NO: 801 corresponding amino acids. According to another embodiment, there is provided SEQ ID NO: 833 wherein 1-3 of the amino acids at positions 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21 or 24 of the analog are different from the amino acid sequence of SEQ ID NO: 801 corresponding amino acids. In another embodiment, provided is seq id NO: 807. SEQ ID NO: 808 or SEQ ID NO: 834, wherein 1-2 amino acids selected from position 1, 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 24 of the analog differ from SEQ ID NO: 801 and in yet another embodiment, the 1-2 different amino acids represent conservative amino acid substitutions as compared to the amino acid present in the native sequence (SEQ ID NO: 801). In some embodiments, provided is SEQ ID NO: 811 or SEQ ID NO: 813, wherein the glucagon peptide further comprises 1, 2, or 3 amino acid substitutions at a position selected from the group consisting of 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, or 29. In some embodiments, the substitution at position 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27, or 29 is a conservative amino acid substitution.
In some embodiments, there is provided a nucleic acid comprising SEQ ID NO: 801, wherein the analog is a glucagon agonist wherein the analog has an amino acid other than serine at position 2 and a natural amino acid at position 28 or 29 replaced with an acidic amino acid or an acidic amino acid added to the glucagon peptide of SEQ ID NO: 801, and is different from SEQ ID NO: 801. in some embodiments, the acidic amino acid is aspartic acid or glutamic acid. In some embodiments, provided is SEQ ID NO: 809. SEQ ID NO: 812. SEQ ID NO: 813 or SEQ ID NO: 832, wherein said analog differs from the parent molecule by a substitution at position 2. More specifically, the 2-position of the analog peptide is substituted with an amino acid selected from the group consisting of D-serine, alanine, D-alanine, glycine, n-methylserine and aminoisobutyric acid.
In another embodiment, there is provided a polypeptide comprising SEQ ID NO: 801, wherein the analog is a glucagon agonist wherein the analog is a peptide of SEQ ID NO: 801, and is different from SEQ ID NO: 801. in some embodiments, the acidic amino acid is aspartic acid or glutamic acid. In some embodiments, provided is SEQ ID NO: 809. SEQ ID NO: 812. SEQ ID NO: 813 or SEQ ID NO: 832, wherein the analog differs from the parent molecule by a substitution at position 1. More specifically, the analog peptide is substituted at position 1 with an amino acid selected from the group consisting of DMIA, D-histidine, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine.
According to some embodiments, the modified glucagon peptide comprises a sequence selected from the group consisting of SEQ ID NO: 809. SEQ ID NO: 812. SEQ ID NO: 813 and SEQ ID NO: 832 sequence of the sequence. In yet another embodiment, there is provided a polypeptide comprising SEQ ID NO: 809. SEQ ID NO: 812. SEQ ID NO: 813 or SEQ ID NO: 832, which sequence further comprises 1-2 glucagon peptides added to the sequence of SEQ ID NO: 809. SEQ ID NO: 812. SEQ ID NO: 813 or SEQ ID NO: 832, wherein the additional amino acid is independently selected from Lys, Arg, His, Asp, Glu, cysteic acid or homocysteine. In some embodiments, the additional amino acid added to the carboxy terminus is selected from Lys, Arg, His, Asp, or Glu, or in yet another embodiment, the additional amino acid is Asp or Glu.
In another embodiment, the glucagon peptide comprises SEQ ID NO: 7 or a glucagon agonist analog thereof. In some embodiments, the peptide comprises a sequence selected from SEQ ID NOs: 808. SEQ ID NO: 810. SEQ ID NO: 811. SEQ ID NO: 812 and SEQ ID NO: 813. In another embodiment, the peptide comprises a sequence selected from SEQ ID NOs: 808. SEQ ID NO: 810 and SEQ ID NO: 811, respectively. In some embodiments, the glucagon peptide comprises SEQ ID NO: 808. SEQ ID NO: 810 and SEQ ID NO: 811 further comprising an additional amino acid selected from Asp and Glu added to the C-terminus of the glucagon peptide. In some embodiments, the glucagon peptide comprises SEQ ID NO: 811 or SEQ ID NO: 813, and in yet another embodiment, the glucagon peptide comprises the sequence of SEQ ID NO: 811, respectively.
According to some embodiments, there is provided a glucagon agonist comprising a modified glucagon peptide selected from the group consisting of:
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R(SEQ ID NO：834)、
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R (SEQ ID NO: 811) and
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Glu-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-R(SEQ ID NO：813)
wherein Xaa at position 15 isAsp, Glu, cysteic acid, homoglutamic acid or homocysteic acid, Xaa at position 28 is Asn or an acidic amino acid, Xaa at position 29 is Thr or an acidic amino acid, and R is an acidic amino acid, COOH or CONH2With the proviso that an acidic amino acid residue is present at one of positions 28, 29 or 30. In some embodiments, R is COOH, while in another embodiment, R is CONH2。
The present disclosure also includes glucagon fusion peptides wherein a second peptide is fused to the C-terminus of the glucagon peptide to increase the stability and solubility of the glucagon peptide. More specifically, the fused glucagon peptide may comprise a glucagon agonist analog comprising glucagon peptide NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Xaa-Xaa-R (SEQ ID NO: 834), wherein R is an acidic amino acid or a chemical bond and SEQ ID NO: 820(GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822 (KRNR). In some embodiments, the glucagon peptide is selected from the group consisting of SEQ ID NO: 833. SEQ ID NO: 807 or SEQ ID NO: 808, further comprising SEQ ID NO: 820(GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822(KRNR) amino acid sequence. In some embodiments, the glucagon fusion peptide comprises SEQ ID NO: 802. SEQ ID NO: 803. SEQ ID NO: 804. SEQ ID NO: 805 and SEQ ID NO: 806 or a glucagon agonist analog thereof, further comprising the sequence of SEQ ID NO: 820(GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822 (KRNR). According to some embodiments, the fusion peptide further comprises a PEG chain linked to the amino acid at position 16, 17, 21, 24, 29, within the C-terminal overhang or at the C-terminal amino acid, wherein said PEG chain is selected from the range of 500-40,000 daltons. In some embodiments, the nucleic acid sequence of SEQ ID NO: 820(GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822(KRNR) with glucagon peptide via a peptide bond Amino acid 29. In some embodiments, the glucagon peptide portion of the glucagon fusion peptide comprises a sequence selected from the group consisting of SEQ ID NO: 810. SEQ ID NO: 811 and SEQ ID NO: 813. In some embodiments, the glucagon peptide portion of the glucagon fusion peptide comprises SEQ ID NO: 811 or SEQ ID NO: 813 wherein the PEG chains are linked at positions 21, 24, 29, within the C-terminal overhang or at the C-terminal amino acid, respectively.
In another embodiment, the glucagon peptide sequence of the fusion peptide comprises SEQ id no: 811, further comprising the sequence of SEQ id no: 820(GPSSGAPPPS), SEQ ID NO: 821(KRNRNNIA) or SEQ ID NO: 822 (KRNR). In some embodiments, the glucagon fusion peptide comprises a sequence selected from the group consisting of SEQ ID NO: 824. SEQ ID NO: 825 and SEQ ID NO: 826, respectively. In general, the fusion peptides of the invention may have a C-terminal amino acid with a standard carboxylic acid group. However, analogs of those sequences in which the C-terminal amino acid has an amide substituted for a carboxylic acid are also included as embodiments. According to some embodiments, the fusion glucagon peptide comprises a sequence selected from the group consisting of SEQ ID NO: 810. SEQ ID NO: 811 and SEQ ID NO: 813 further comprising the glucagon agonist analog of seq id NO: 823(GPSSGAPPPS-CONH2) The amino acid sequence of (a).
The glucagon agonists of the present invention may be further modified to enhance the solubility and stability of the peptide in aqueous solution while maintaining the biological activity of the glucagon peptide. According to some embodiments, it is contemplated that the sequence of one or more of SEQ ID NOs: 811 or a glucagon agonist analog thereof, introduces hydrophilic groups at positions 16, 17, 20, 21, 24, and 29 to increase the solubility and stability of the pH stable glucagon analog. More specifically, in some embodiments, the sequence set forth for SEQ ID NO: 810. SEQ ID NO: 811. SEQ ID NO: 813 or SEQ ID NO: 832 is modified to comprise one or more hydrophilic groups covalently attached to the side chains of the amino acids present at positions 21 and 24 of the glucagon peptide.
According to some embodiments, the sequence of SEQ ID NO: the glucagon peptide of 811 is modified to contain one or more amino acids substituted at positions 16, 17, 20, 21, 24 and/or 29, wherein the natural amino acid is substituted with an amino acid having a side chain suitable for crosslinking with a hydrophilic moiety, including, for example, PEG. The natural peptide may be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non-naturally occurring amino acids refer to amino acids that are not naturally occurring in the body but can nevertheless be incorporated into the peptide structures described herein.
In some embodiments, provided is SEQ ID NO: 810, SEQ ID NO: 811 or SEQ ID NO: 813 wherein the native glucagon peptide sequence is modified to contain a naturally occurring or synthetic amino acid at least one of positions 16, 17, 21, 24, 29 of the native sequence, within the C-terminal overhang or at the C-terminal amino acid, wherein the amino acid substitution further comprises a hydrophilic moiety. In some embodiments, the substitution is at position 21 or 24, while in yet another embodiment, the hydrophilic moiety is a PEG chain. In some embodiments, the nucleic acid sequence of SEQ ID NO: the glucagon peptide of 811 is substituted with at least one cysteine residue wherein the side chain of the cysteine residue is further modified with a sulfhydryl-reactive reagent including, for example, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl and haloacyl. These thiol-reactive reagents may contain carboxyl, keto, hydroxyl and ether groups as well as other hydrophilic moieties such as polyethylene glycol units. In an alternative embodiment, the native glucagon peptide is substituted with lysine and the side chain of the substituted lysine residue is further modified using an amine reactive reagent such as the aldehyde of an active ester of a carboxylic acid (succinimidyl, anhydride, etc.) or a hydrophilic moiety (e.g., polyethylene glycol). In some embodiments, the glucagon peptide is selected from the group consisting of SEQ ID NO: 814. SEQ ID NO: 815. SEQ ID NO: 816. SEQ ID NO: 817. SEQ ID NO: 818 and SEQ ID NO: 819.
According to some embodiments, the pegylated glucagon peptide comprises two or more polyethylene glycol chains covalently bound to the glucagon peptide, wherein the total molecular weight of the glucagon chain is from about 1,000 to about 5,000 daltons. In some embodiments, the pegylated glucagon agonist comprises SEQ ID NO: 806, wherein the PEG chains are covalently attached to the amino acid residues at positions 21 and 24, and wherein the 2 PEG chains have a total molecular weight of about 1,000 to about 5,000 daltons. In another embodiment, the pegylated glucagon agonist comprises SEQ ID NO: 806, wherein the PEG chains are covalently attached to the amino acid residues at positions 21 and 24, and wherein the 2 PEG chains have a total molecular weight of about 5,000 to about 20,000 daltons.
The polyethylene glycol chain may be in the form of a straight chain or may be branched. According to some embodiments, the polyethylene glycol chains have an average molecular weight selected from about 500 to about 40,000 daltons. In some embodiments, the polyethylene glycol chain has a molecular weight selected from about 500 to about 5,000 daltons. In another embodiment, the polyethylene glycol chain has a molecular weight of about 20,000 to about 40,000 daltons.
Any of the above glucagon peptides may be further modified to include within the C-terminal portion of the glucagon peptide (amino acid positions 12-29) amino acids that are covalently or non-covalently intra-molecular bridges or that stabilize the alpha helix. According to some embodiments, the glucagon peptide comprises any one or more of the above modifications in addition to the amino acid substitution at position 16, 20, 21, or 24 (or a combination thereof) with an α, α -disubstituted amino acid (e.g., AIB). According to another embodiment, the glucagon peptide comprises any one or more of the above modifications in addition to an intramolecular bridge (e.g. lactam) between the side chains of the amino acids at positions 16 and 20 of the glucagon peptide.
According to some embodiments, the glucagon peptide comprises SEQ ID NO: 877, wherein Xaa at position 3 is an amino acid comprising a side chain of the following structure I, II or III:
structure I
Structure II
Structure III
Wherein R is1Is C0-3Alkyl or C0-3A heteroalkyl group; r2Is NHR4Or C1-3An alkyl group; r3Is C1-3An alkyl group; r4Is H or C1-3An alkyl group; x is NH, O or S; and Y is NHR4、SR3OR OR3. In some embodiments, X is NH or Y is NHR4. In some embodiments, R1Is C0-2Alkyl or C1A heteroalkyl group. In some embodiments, R2Is NHR4Or C1An alkyl group. In some embodiments, R4Is H or C1An alkyl group. In an exemplary embodiment, amino acids comprising a side chain of structure I are provided, wherein R1Is CH2-S, X is NH, R2Is CH3(acetamidomethyl-cysteine, c (acm)); r1Is CH2X is NH, R2Is CH3(acetyldiaminobutyric acid, dab (ac)); r1Is C0Alkyl, X is NH, R2Is NHR4，R4Is H (carbamoyldiaminopropionic acid, Dap (urea)); or R1Is CH2-CH2X is NH, R2Is CH3(acetylornithine, Orn (Ac)). In an exemplary embodiment, an amino acid comprising a side chain of structure II is provided, wherein R1Is CH2Y is NHR4，R4Is CH3(methyl glutamine, q (me)); in an exemplary embodiment, an amino acid comprising a side chain of structure III is provided, wherein R1Is CH2，R4Is H (methionine-sulfoxide, M (O)); in a specific embodiment, the amino acid at position 3 is substituted with dab (ac). For example, the glucagon agonist can comprise SEQ ID NO: 863. SEQ ID NO: 869. SEQ ID NO: 871. SEQ ID NO: 872. SEQ ID NO: 873 and SEQ ID NO: 874 amino acid sequence.
In certain embodiments, the glucagon peptide is SEQ ID NO: 877 analog of a glucagon peptide. In particular aspects, the analog comprises any of the above amino acid modifications, including but not limited to: (ii) Asn at position 28 is substituted with a charged amino acid; asn at position 28 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine; (xiii) substitution at position 28 with Asn, Asp or Glu; asp at position 28; substituted at position 28 with Glu; thr at position 29 is substituted with a charged amino acid; thr at position 29 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine; asp, Glu or Lys at position 29; substituted at position 29 with Glu; 1-3 charged amino acids are inserted after position 29; glu or Lys inserted after position 29; insertion of Gly-Lys or Lys-Lys after position 29; and combinations thereof.
In certain embodiments, SEQ ID NO: 877 comprises an alpha, alpha-disubstituted amino acid, such as AIB, at 1, 2, 3 or all of positions 16, 20, 21 and 24.
In certain embodiments, SEQ ID NO: 877 comprises one or more of the following modifications: the His at position 1 is substituted with an unnatural amino acid that decreases the susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), the Ser at position 2 is substituted with an unnatural amino acid that decreases the susceptibility of the glucagon peptide to cleavage by dipeptidyl peptidase IV (DPP-IV), the Thr at position 7 is substituted with an amino acid that lacks a hydroxyl group (e.g., Abu or Ile); tyr at position 10 is substituted with Phe or Val; lys at position 12 is substituted with Arg; asp at position 15 substituted by Glu, Ser at position 16 substituted by Thr or AIB, Gln at position 20 substituted by Ala or AIB, Asp at position 21 substituted by Glu, Gln at position 24 substituted by Ala or AIB, Met at position 27 substituted by Leu or Nle, amino acid deletion at positions 27-29, amino acid deletion at positions 28-29, amino acid deletion at position 29; SEQ ID NO: 820 to the C-terminus, wherein the amino acid at position 29 is Thr or Gly; or a combination thereof.
According to a particular embodiment, the glucagon peptide comprises SEQ ID NO: 862-867 and 869-874.
In certain embodiments, the polypeptide comprising SEQ ID NO: 877 comprises a hydrophilic moiety, such as PEG, covalently attached to the amino acid at any of positions 16, 17, 20, 21, 24 and 29 or at the C-terminal amino acid.
In certain embodiments, the polypeptide comprising SEQ ID NO: 877 comprises an amino acid having a side chain covalently attached, optionally through a spacer, to an acyl group or an alkyl group that is non-natural to the naturally occurring amino acid. In some embodiments, the acyl group is a C4-C30 fatty acyl group. In other embodiments, the alkyl group is a C4-C30 alkyl group. In a particular aspect, the acyl group or alkyl group is covalently attached to the side chain of the amino acid at position 10. In some embodiments, the amino acid at position 7 is Ile or Abu.
The glucagon agonist may be a glucagon peptide comprising SEQ ID NO: peptides of any of amino acid sequences 801-919, optionally with up to 1, 2, 3, 4, or 5 other modifications that maintain glucagon agonist activity. In certain embodiments, the glucagon agonist comprises SEQ ID NO: 859-919.
Glucagon-like 2 peptide
In certain embodiments, the glucagon related peptide is a glucagon-like peptide class 2, described herein and international patent application No. PCT US2009/47447 (filed 6/16/2009), U.S. provisional application No. 61/090,448, and U.S. application No. 61/151,349, the contents of which are incorporated by reference in their entirety.
The biological sequences referred to in the following sections in relation to glucagon-like peptide type 2 (SEQ ID NO: 1001-1262) correspond to the SEQ ID NO of International patent application No. PCT US 2009/47447: 1-262.
Activity of
Native glucagon does not activate the GIP receptor and typically has about 1% of the activity of native-GLP-1 at the GLP-1 receptor. Modifications of the native glucagon sequences described herein produce glucagon-like peptide class 2 that can exhibit potent glucagon activity equal to or better than that of native glucagon (SEQ ID NO: 1001), potent GIP activity equal to or better than that of native GIP (SEQ ID NO: 1004), and/or potent GLP-1 activity equal to or better than that of native GLP-1. In this aspect, the glucagon-like peptide type 2 can be one of a glucagon/GIP co-agonist, a glucagon/GIP/GLP-1 triple agonist (tri-aginst), a GIP/GLP-1 co-agonist, or a GIP agonist glucagon peptide, as further described herein.
In some embodiments, the glucagon-like peptide type 2 described herein has an EC50 for GIP receptor activating activity of about 100nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2, or 1nM or less. In some embodiments, the glucagon receptor activation by the glucagon-like peptide type 2 has an EC50 of about 100nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2, or 1nM or less. In some embodiments, the glucagon-like peptide type 2 has an EC50 for GLP-1 receptor activation of about 100nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2, or 1nM or less. Receptor activation can be determined by in vitro assays that measure cAMP induced in HEK293 cells overexpressing the receptor, e.g., HEK293 cells co-transfected with DNA encoding the receptor and a luciferase gene linked to a cAMP-reactive component, as described in example 5 herein.
In some embodiments, the glucagon-like peptide type 2 exhibits at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, or 200% or more activity at the GIP receptor as compared to native GIP (GIP potency). In some embodiments, the glucagon peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GIP receptor as compared to native GIP. In some embodiments, the glucagon related peptide type 2 exhibits at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more activity at the glucagon receptor as compared to native glucagon (glucagon potency). In some embodiments, the glucagon peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the glucagon receptor as compared to native glucagon. In some embodiments, the glucagon-like peptide type 2 exhibits at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, or 200% or more activity at the GLP-1 receptor as compared to native GLP-1 (GLP-1 potency). In some embodiments, the glucagon peptides described herein exhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity at the GLP-1 receptor as compared to native GLP-1. The activity of class 2 glucagon related peptides on the receptor relative to the natural ligand of the receptor was calculated based on the inverse ratio of EC50 for class 2 glucagon related peptides relative to the natural ligand.
In some embodiments, the glucagon-like peptide class 2 is active at both the glucagon receptor and the GIP receptor ("glucagon/GIP co-agonist"). These class 2 glucagon-related peptides lose the selectivity of native glucagon for the glucagon receptor compared to the GIP receptor. In some embodiments, the class 2 glucagon-related peptide has an EC50 for the GIP receptor that differs from (is higher or lower than) its EC50 for the glucagon receptor by less than about 50-fold, 40-fold, 30-fold, or 20-fold. In some embodiments, the GIP potency of the glucagon-like peptide class 2 differs from its glucagon potency by less than about 500-fold, 450-fold, 400-fold, 350-fold, 300-fold, 250-fold, 200-fold, 150-fold, 100-fold, 75-fold, 50-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold. In some embodiments, the ratio of EC50 of class 2 glucagon related peptide to GIP receptor divided by EC50 of class 2 glucagon related peptide to glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of EC50 for the GIP receptor divided by EC50 for the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the glucagon related peptide type 2 compared to the glucagon potency of the glucagon related peptide type 2 is less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the potency at the GIP receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, GLP-1 activity is significantly reduced or disrupted, for example by an amino acid modification at position 7, deletion of an amino acid from the C-terminus of the amino acid to position 27 or 28 to give a 27-amino acid peptide or a 28-amino acid peptide, or a combination thereof.
In another aspect, the class 2 glucagon-related peptide has activity at glucagon, GIP and GLP-1 receptors ("glucagon/GIP/GLP-1 triple agonist"). These class 2 glucagon-related peptides lose the selectivity of native glucagon for the glucagon receptor, compared to both GLP-1 and GIP receptors. In some embodiments, the class 2 glucagon-related peptide has an EC50 at the GIP receptor that differs (is higher or lower) from its corresponding EC50 at the glucagon and GLP-1 receptors by less than about 50-fold, 40-fold, 30-fold, or 20-fold. In some embodiments, the GIP potency of the glucagon-like-peptide class 2 differs from (is higher or lower than) its glucagon and GLP-1 potency by less than about 500-fold, 450-fold, 400-fold, 350-fold, 300-fold, 250-fold, 200-fold, 150-fold, 100-fold, 75-fold, 50-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold. In some embodiments, the ratio of EC50 for the triple agonist to the GIP receptor divided by EC50 for the triple agonist to the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of EC50 for the GIP receptor divided by EC50 for the GLP-1 receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the triple agonist compared to the GLP-1 potency of the triple agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the potency at the GIP receptor divided by the potency at the GLP-1 receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In related embodiments, the ratio of EC50 for the triple agonist to the GIP receptor divided by EC50 for the triple agonist to the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of EC50 for the GIP receptor divided by EC50 for the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency of the triple agonist compared to the glucagon potency of the triple agonist is less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the potency at the GIP receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of EC50 of the triple agonist to the GLP-1 receptor divided by EC50 of the triple agonist to the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of EC50 for the GLP-1 receptor divided by EC50 for the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of GLP-1 potency of the triple agonist compared to the glucagon potency of the triple agonist is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the potency at the GLP-1 receptor divided by the potency at the glucagon receptor is about 1 or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2).
In another aspect, the class 2 glucagon-related peptide is active at the GLP-1 and GIP receptors, but wherein glucagon activity is significantly reduced or abolished, e.g., by modification of the amino acid at position 3 ("GIP/GLP-1 co-agonist"). For example, substitution at this position with an acidic, basic or hydrophobic amino acid (glutamic acid, ornithine, norleucine) decreases glucagon activity. In some embodiments, the glucagon peptide has an EC50 for the GIP receptor that differs from (is higher or lower than) its EC50 for the GLP-1 receptor by less than about 50-fold, 40-fold, 30-fold, or 20-fold. In some embodiments, the GIP potency of the glucagon-like peptide class 2 differs from (is higher or lower than) its GLP-1 potency by less than about 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold. In some embodiments, these class 2 glucagon-related peptides have about 10% or less, for example about 1-10% or about 0.1-10% or greater than about 0.1% but less than about 10% of the activity of native glucagon at the glucagon receptor. In some embodiments, the ratio of EC50 of the glucagon related peptide type 2 to the GIP receptor divided by EC50 of the glucagon related peptide type 2 to the GLP-1 receptor is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, but not less than 1. In some embodiments, the ratio of the GIP potency of the glucagon related peptide type 2 compared to the GLP-1 potency of the glucagon related peptide type 2 is less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, but not less than 1.
In yet another aspect, the class 2 glucagon-related peptide has activity at the GIP receptor, wherein glucagon and GLP-1 activity is significantly reduced or disrupted, for example, by modification of the amino acid at position 3 with Glu and modification at position 7 with Ile ("GIP agonist glucagon peptide"). In some embodiments, these class 2 glucagon-related peptides have less than about 10% of the activity of native glucagon at the glucagon receptor, e.g., about 1-10% or about 0.1-10% or greater than about 0.1%, 0.5% or 1% but less than about 1%, 5% or 10%. In some embodiments, these glucagon-like peptide class 2 also have less than about 10% of the activity of native GLP-1 at the GLP-1 receptor, e.g., about 1-10% or about 0.1-10% or greater than about 0.1%, 0.5% or 1% but less than about 1%, 5% or 10%.
In some embodiments, if the glucagon-like peptide type 2 is not pegylated, the glucagon-like peptide type 2 has an EC50 for GIP receptor activation of about 4, 2, 1nM, or less, or the analog has at least about 1%, 2%, 3%, 4%, or 5% of the activity of native GIP at the GIP receptor. In related embodiments, the non-pegylated glucagon-like-2-like peptide has an EC50 for GLP-1 receptor activation of about 4, 2, 1nM or less, or at least about 1%, 2%, 3%, 4% or 5% of the activity of native GLP-1 at the GLP-1 receptor. In other related embodiments, the PEGylated glucagon 2-like peptide has an EC50 for glucagon receptor activation of about 4, 2, 1nM or less, or at least about 5%, 10%, 15% or 20% of the activity of native glucagon at the glucagon receptor. In some embodiments, the non-pegylated type 2 glucagon-related peptide has less than about 1% of the activity of native glucagon at the glucagon receptor. In other embodiments, the non-pegylated glucagon-like peptide type 2 has less than about 10%, 5% or 1% of the activity of native GLP-1 at the GLP-1 receptor.
In embodiments in which the glucagon-like peptide class 2 is linked to a hydrophilic moiety (e.g., PEG), the relative EC50 for one or more receptors may be higher, e.g., about 10-fold higher. For example, the pegylated analogs have an EC50 for GIP receptor activation of about 10nM or less, or the glucagon related peptide type 2 has at least about 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the activity of native GIP at the GIP receptor. In related embodiments, the PEGylated glucagon-like peptide type 2 has an EC50 for GLP-1 receptor activation of about 10nM or less, or at least about 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% of the activity of native GLP-1 at the GLP-1 receptor. In other related embodiments, the pegylated type 2 glucagon-related peptide has an EC50 for glucagon receptor activation of about 10nM or less, or at least about 0.5%, 1%, 1.5%, or 2% of the activity of native glucagon at the glucagon receptor. In some embodiments, the glucagon-like peptide type 2 has less than about 1% of the activity of native glucagon at the glucagon receptor. In other embodiments, the glucagon-like peptide type 2 has less than about 10%, 5% or 1% of the activity of native GLP-1 at the GLP-1 receptor.
Decoration
The modifications disclosed herein with respect to the class 2 glucagon-related peptide allow manipulation of glucagon (SEQ ID NO: 1001) to produce glucagon peptides with increased GIP activity, glucagon activity, and/or GLP-1 activity. Other modifications disclosed herein with respect to the class 2 glucagon-related peptides extend the half-life, increase solubility or increase stability of the resulting peptide. Further modifications disclosed herein with respect to the glucagon related peptide class 2 have no effect on activity or can be made without disruption of the desired activity or activities. Any combination of related class 2 glucagon related peptides used for the same purpose (e.g., to increase GIP activity) can be used alone or in combination. Single combinations or combinations of related class 2 glucagon related peptides endowed with improved properties may be used alone or in combination, e.g. increased GIP and/or GLP-1 activity may be combined with increased half-life. In related embodiments, 1, 2, 3, 4, 5, 6, or more amino acid modifications may be non-conservative substitutions, additions, or deletions. In some embodiments, 1, 2, 3, 4, 5, 6, or more amino acid modifications can be conservative substitutions.
Modifications affecting GIP activity
The activity on the GIP receptor is enhanced by amino acid modification at position 1. For example, His at position 1 is substituted with a large aromatic amino acid that is optionally Tyr, Phe, Trp, amino-Phe, nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3-amino Tyr. The combination of Tyr at position 1 with stabilization of the alpha helix within the region corresponding to amino acids 12-29 provides glucagon related peptide class 2 that activates the GIP receptor as well as the GLP-1 receptor and the glucagon receptor. The alpha helix structure may be stabilized, for example, by forming covalent or non-covalent intramolecular bridges, or by substitution and/or insertion of amino acids near positions 12-29 with alpha helix-stabilizing amino acids (e.g., alpha-disubstituted amino acids).
The activity towards the GIP receptor is also increased by amino acid modifications at positions 27 and/or 28 and optionally at position 29. For example, Met at position 27 is substituted with a large aliphatic amino acid (optionally Leu), Asn at position 28 is substituted with a small aliphatic amino acid (optionally Ala), and Thr at position 29 is substituted with a small aliphatic amino acid (optionally Gly). Substitution of LAG at positions 27-29 results in increased GIP activity compared to the native MNT sequence at those positions.
The activity on the GIP receptor is also enhanced by the amino acid modification at position 12. For example, position 12 is substituted with a large aliphatic nonpolar amino acid (optionally Ile).
The activity on the GIP receptor is also increased by amino acid modifications at positions 17 and/or 18. For example, position 17 is substituted with a polar residue (optionally Gln) and position 18 with a small aliphatic amino acid (optionally Ala). Substitution at positions 17 and 18 with QA results in increased GIP activity compared to the native RR sequence at those positions.
The activity towards the GIP receptor is enhanced by modifications that allow the formation of intramolecular bridges between the amino acid side chains at positions 12-29. For example, intramolecular bridges may be formed by covalent bonds between the side chains of 2 amino acids at positions i and i +4, or between positions j and j +3, or between positions k and k + 7. In exemplary embodiments, the intramolecular bridge is between positions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20. In other embodiments, non-covalent interactions, such as salt bridges, may be formed between positively and negatively charged amino acids at these positions.
Any of the modifications described herein that increase GIP receptor activity may be used alone or in combination. Combinations of modifications that increase GIP receptor activity generally provide higher GIP activity than if only any one of such modifications were employed.
Modifications affecting glucagon activity
In some embodiments, glucagon potency is increased by an amino acid modification at position 16 of native glucagon (SEQ ID NO: 1001). By way of non-limiting example, such an increase in potency may be obtained by substituting the naturally occurring serine at position 16 with: glutamic acid or another negatively charged amino acid having a side chain of 4 atoms in length; or any one of glutamine, homoglutamic acid, or homocysteic acid; or charged amino acids having a side chain containing at least one heteroatom (e.g., N, O, S, P) and a pendant chain length of about 4 (or 3-5) atoms. In some embodiments, the glucagon peptide retains its original selectivity for the glucagon receptor over the GLP-1 receptor.
Glucagon receptor activity can be decreased by amino acid modification at position 3, for example, by substituting the naturally occurring glutamine at position 3 with an acidic, basic, or hydrophobic amino acid. For example, substitution at position 3 with glutamic acid, ornithine or norleucine greatly reduces or abolishes glucagon receptor activity.
As described herein, modification of Gln at position 3 with glutamine analogs can achieve maintenance or enhancement of activity at the glucagon receptor. For example, the glucagon agonist can comprise SEQ ID NO: the amino acid sequence of any one of 1243-1248, 1250, 1251 and 1253-1256.
Glucagon activity, reduced by amino acid modifications at positions 1 and 2, is restored by modification of the alpha helical structure (amino acids 12-29) of the C-terminal portion of a stabilized glucagon peptide or analog thereof. For example, intramolecular bridges may be formed by covalent bonds between the side chains of 2 amino acids at positions i and i +4, or between positions j and j +3, or between positions k and k + 7. In other embodiments, non-covalent interactions, such as salt bridges, may be formed between positively and negatively charged amino acids at these positions. In yet other embodiments, one or more α, α -disubstituted amino acids are inserted or substituted into the C-terminal moiety (amino acids 12-29) at positions that retain the desired activity. For example, 1, 2, 3 or all 4 positions at positions 16, 20, 21 or 24 are substituted with an α, α -disubstituted amino acid (e.g., AIB).
Modifications affecting GLP-1 activity
By replacing the carboxylic acid of the C-terminal amino acid with an electrically neutral group (e.g. amide or ester), the activity towards the GLP-1 receptor is increased.
The activity at the GLP-1 receptor is also enhanced by stabilizing the alpha helical structure of the C-terminal portion of glucagon (near amino acids 12-29), as further described herein, for example by forming an intramolecular bridge between the side chains of the 2 amino acids, or by substitution and/or insertion of amino acids near positions 12-29 with amino acids that stabilize the alpha helix (e.g., alpha-disubstituted amino acids). In exemplary embodiments, the side chains of the amino acid pairs of 12 and 16, 13 and 17, 16 and 20, 17 and 21, 20 and 24, or 24 and 28 (where i-the amino acid pair of 12, 16, 20, or 24) are linked to each other to stabilize the glucagon alpha helix. In some embodiments, the bridge or linker is about 8 (or about 7-9) atoms in length, particularly when the bridge is between the i and i +4 positions. In some embodiments, the bridge or linker is about 6 (or about 5 to 7) atoms in length, particularly when the bridge is located between the j and j +3 positions.
In some embodiments, the intramolecular bridge is formed as follows: (a) substituting the naturally occurring serine at position 16 with glutamic acid or with another negatively charged amino acid having a side chain that is 4 atoms in length, or with any of glutamine, homoglutamic acid, or homocysteic acid, or with a charged amino acid having a side chain that contains at least one heteroatom (e.g., N, O, S, P) and has a side chain length of about 4 (or 3-5) atoms, and (b) substituting the naturally occurring glutamine at position 20 with another hydrophilic amino acid (e.g., lysine, citrulline, arginine, or ornithine) that contains a side chain that is charged or has hydrogen bonding capability and is at least about 5 (or about 4-6) atoms in length. The side chains of such amino acids at positions 16 and 20 may form salt bridges or may be covalently linked. In some embodiments, 2 amino acids are combined with each other to form a lactam ring.
In some embodiments, stabilization of the alpha helical structure of the C-terminal portion of the glucagon peptide is achieved by forming an intramolecular bridge that is not a lactam bridge. For example, suitable covalent bonding methods include any one or more of the following: olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, use of α, ω -diaminoalkane linkers, formation of metal-atom bridges, and other methods of peptide cyclization are used to stabilize the α helix.
In yet other embodiments, one or more α, α -disubstituted amino acids are inserted or substituted into the C-terminal moiety (amino acids 12-29) at positions that retain the desired activity. For example, 1, 2, 3 or all 4 positions at positions 16, 20, 21 or 24 are substituted with an α, α -disubstituted amino acid (e.g., AIB).
The activity at the GLP-1 receptor is increased by an amino acid modification at position 20 as described herein.
The activity on the GLP-1 receptor was increased by adding GPSSGAPPPS (SEQ ID NO: 1095) or XGPSGAPPPS (SEQ ID NO: 1096) to the C-terminus. GLP-1 activity of such analogs can be further enhanced by modifying the amino acids at positions 18, 28, or 29, or at positions 18 and 29, as described herein.
By modifying the amino acid in position 10 to a large aromatic amino acid residue (optionally Trp), GLP-1 potency is further moderately increased.
The activity at the GLP-1 receptor is reduced, for example, by an amino acid modification at position 7, as described herein.
Potency at the GLP-1 receptor can be further improved by substituting alanine for the native arginine at position 18.
Any of the modifications described above in relation to the class 2 glucagon-related peptide that increase GLP-1 receptor activity may be used alone or in combination. Combinations of modifications that increase GLP-1 receptor activity generally provide higher GLP-1 activity than if only any one of such modifications were used. For example, the present invention provides glucagon peptides comprising modifications at positions 16, 20 and the C-terminal carboxylic acid group, optionally with a covalent bond between the amino acids at positions 16 and 20; a modified glucagon peptide comprising a modification at the 16-position and the C-terminal carboxylic acid group; a glucagon peptide comprising a modification at positions 16 and 20, optionally with a covalent bond between the amino acids at positions 16 and 20; and a modified glucagon peptide comprising a modification at the 20-position and at the C-terminal carboxylic acid group.
Modifications to improve DPP-IV resistance
Modifications at position 1 and/or 2 may increase the resistance of the peptide to cleavage by dipeptidyl peptidase iv (dpp iv). For example, position 1 and/or position 2 may be substituted with a DPP-IV resistant amino acid as described herein. In some embodiments, the amino acid at position 2 is substituted with N-methylalanine.
It was observed that modifications at position 2 (e.g., AIB at position 2) and in some cases at position 1 (e.g., DMIA at position 1) reduced glucagon activity, sometimes significantly; unexpectedly, this reduction in glucagon activity can be restored by stabilizing the alpha helical structure of the C-terminal portion of glucagon (near amino acids 12-29) (e.g., by forming covalent bonds between side chains of 2 amino acids, as described herein). In some embodiments, the covalent bond is between the amino acids at positions "i" and "i + 4", or "j" and "j + 3", such as between positions 12 and 16, 16 and 20, 20 and 24, 24 and 28, or 17 and 20. In an exemplary embodiment, this covalent bond is a lactam bridge between the glutamic acid at position 16 and the lysine at position 20. In some embodiments, such covalent bond is an intramolecular bridge other than the lactam bridge described herein.
Modification to reduce degradation
In further exemplary embodiments, the expression of the polypeptide of SEQ ID NO: any glucagon 2-like peptide is further modified at position 15 and/or 16 of 1001 to reduce degradation of the peptide, particularly in acidic or basic buffers, over time to improve stability. Such modifications reduce cleavage of the peptide bond Asp15-Ser 16. In exemplary embodiments, the amino acid modification at position 15 is a deletion or substitution of Asp with glutamic acid, homoglutamic acid, cysteic acid, or homocysteic acid. In other exemplary embodiments, the amino acid modification at position 16 is a deletion or a substitution of Ser with Thr or AIB. In other exemplary embodiments, the Ser at position 16 is substituted with glutamic acid, or with another negatively charged amino acid having a side chain that is 4 atoms long, or with any of glutamine, homoglutamic acid, or homocysteine.
In some embodiments, the methionine residue present at position 27 of the native peptide is modified, e.g., by deletion or substitution. Such modifications may prevent oxidative degradation of the peptide. In some embodiments, the Met at position 27 is substituted with leucine, isoleucine, or norleucine. In some embodiments, the Met at position 27 is substituted with leucine or norleucine.
In some embodiments, the Gln at positions 20 and/or 24 is modified, e.g., by deletion or substitution. Such modifications can reduce degradation by deamidation of Gln. In some embodiments, the Gln at positions 20 and/or 24 is substituted with Ser, Thr, Ala or AIB. In some embodiments, the Gln at positions 20 and/or 24 is substituted with Lys, Arg, Orn, or citrulline.
In some embodiments, the Asp at position 21 is modified, e.g., by deletion or substitution. Such modifications may reduce degradation that occurs through dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid. In some embodiments, position 21 is substituted with Glu, homoglutamic acid, or homocysteic acid. In some embodiments, position 21 is substituted with Glu.
Stabilization of alpha helical structures
Stabilization of the alpha helical structure (around amino acids 12-29) of the C-terminal portion of the glucagon-like-2-related peptide results in increased GLP-1 and/or GIP activity and restoration of glucagon activity that is decreased by amino acid modifications at positions 1 and/or 2. The alpha helix structure may be stabilized, for example, by forming covalent or non-covalent intramolecular bridges or by substituting and/or inserting amino acids near positions 12-29 with alpha helix stabilizing amino acids (e.g., alpha-disubstituted amino acids). Stabilization of the alpha helical structure of GIP agonists can be achieved as described herein.
Acylation and alkylation
As described herein, according to some embodiments, the glucagon peptides disclosed herein are modified to comprise an acyl group or an alkyl group, e.g., an acyl group or an alkyl group that is non-natural to naturally occurring amino acids. Acylation or alkylation may extend the half-life of the glucagon peptide in circulation. Acylation or alkylation may advantageously delay onset and/or prolong duration of action at the glucagon and/or GLP-1 receptor and/or improve resistance to proteases (e.g. DPP-IV) and/or improve solubility. The activity of the glucagon peptide on glucagon and/or GLP-1 and/or GIP receptors is maintained after acylation. In some embodiments, the potency of the acylated glucagon peptide is comparable to the unenylated form of the glucagon peptide. As described herein, the glucagon-like peptide class 2 can be acylated or alkylated at the same amino acid position or at a different amino acid position attached to the hydrophilic moiety.
In some embodiments, the present invention provides glucagon peptides modified to comprise an acyl or alkyl group covalently attached to the amino acid at position 10 of the glucagon peptide. The glucagon peptide may further comprise a peptideA spacer between the amino acid at position 10 of the glucagon peptide and the acyl or alkyl group. In some embodiments, the acyl group is a fatty acid or bile acid or salt thereof, for example, a C4-C30 fatty acid, a C8-C24 fatty acid, a cholic acid, a C4-C30 alkyl group, a C8-C24 alkyl group, or an alkyl group comprising the steroid moiety of a bile acid. The spacer is any moiety having a suitable reactive group to which an acyl or alkyl group is attached. In exemplary embodiments, the spacer comprises an amino acid, dipeptide, tripeptide, hydrophilic bifunctional or hydrophobic bifunctional spacer. In some embodiments, the spacer is selected from: trp, Glu, Asp, Cys and NH-containing2(CH2CH2O)n(CH2) Spacer groups of mCOOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12. Such acylated or alkylated glucagon peptides may also comprise a hydrophilic moiety, optionally a polyethylene glycol. Any of the foregoing glucagon peptides may comprise 2 acyl groups or 2 alkyl groups or a combination thereof.
Conjugates and fusions
The GIP agonist can be linked to the conjugate moiety described herein, optionally by covalent bonding and optionally by a linker.
In other embodiments, the second peptide is XGPSGAPPPS (SEQ ID NO: 1096), wherein X is selected from one of the 20 common amino acids, such as glutamic acid, aspartic acid, or glycine. In some embodiments, X represents an amino acid (e.g., Cys) that further comprises a hydrophilic moiety covalently attached to the side chain of the amino acid. Such C-terminal overhangs enhance solubility and may improve GIP or GLP-1 activity. In some embodiments in which the glucagon peptide further comprises a carboxy-terminal extension, the carboxy-terminal amino acid of the extension terminates in an amide group or an ester group rather than a carboxylic acid.
In some embodiments, for example, in a glucagon peptide comprising a C-terminal overhang, the threonine at position 29 of the native glucagon peptide is replaced with glycine. For example, a glucagon peptide having a glycine substituted for the threonine at position 29 and comprising the C-terminal overhang of GPSSGAPPPS (SEQ ID NO: 1095) would have a potency at the GLP-1 receptor that is 4-fold higher than native glucagon modified to comprise the same C-terminal overhang. This T29G substitution can be used in combination with other modifications disclosed herein to increase the affinity of glucagon peptide for the GLP-1 receptor. For example, the T29G substitution may be combined with S16E and N20K amino acid substitutions, optionally with a lactam bridge between the 16 and 20 amino acids and optionally with the addition of a PEG chain as described herein.
In some embodiments, an amino acid is added to the C-terminus, the added amino acid being selected from the group consisting of glutamic acid, aspartic acid, and glycine.
Modifications to improve solubility
In another embodiment, the solubility of any glucagon peptide can be improved by amino acid substitutions and/or additions that introduce a charged amino acid into the C-terminal portion of the peptide (preferably at the position C-terminal at position 27 of SEQ ID NO: 1001). Optionally, 1, 2 or 3 charged amino acids may be introduced into the C-terminal portion, preferably C-terminal to position 27. In some embodiments, the natural amino acid at position 28 and/or 29 is substituted with one or two charged amino acids, and/or in yet another embodiment, 1-3 charged amino acids are added to the C-terminus of the peptide. In exemplary embodiments, 1, 2, or all of the charged amino acids carry a negative charge. In some embodiments, the negatively charged (acidic amino acid) is aspartic acid or glutamic acid.
Other modifications, such as conservative substitutions, can be made to the glucagon peptide that still maintain GIP activity (and optionally GLP-1 activity and/or glucagon activity).
Other modifications
Any of the modifications described above with respect to class 2 peptides that increase or decrease GIP activity, increase or decrease glucagon receptor activity, and increase GLP-1 receptor activity may be used alone or in combination. Any of the modifications described above in relation to the glucagon related peptide class 2 may also be combined with other modifications described herein in relation to the glucagon related peptide class 2 to confer other desirable properties, such as increased solubility and/or stability and/or duration of action. Alternatively, any of the modifications described above in relation to the glucagon related peptide class 2 may be combined with other modifications described herein in relation to the glucagon related peptide class 2 which do not substantially affect solubility or stability or activity. Exemplary modifications include, but are not limited to:
(A) Solubility is improved, for example, by introducing 1, 2, 3, or more charged amino acids into the C-terminal portion of native glucagon, preferably at positions from the C-terminus to position 27. Such charged amino acids can be introduced by, for example, substituting the natural amino acid with a charged amino acid at position 28 or 29, or by, for example, adding a charged amino acid after position 27, 28, or 29. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. In other embodiments, 1, 2, 3, or all of the charged amino acids have a positive charge. Such modifications increase solubility, e.g., provide at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-fold or greater solubility compared to native glucagon, when measured after 24 hours at 25 ℃, at a given pH (e.g., pH 7) between about 5.5 and 8.
(B) Solubility is enhanced and the duration of action or half-life in the circulation is extended by, for example, adding a hydrophilic moiety as described herein (e.g., a polyethylene glycol chain) at position 16, 17, 20, 21, 24 or 29 of the peptide, within the C-terminal overhang or at the C-terminal amino acid.
(C) Increasing solubility and/or extending duration of action or half-life in circulation and/or delaying onset of action by acylation or alkylation of a glucagon peptide as described herein;
(D) By introducing resistance to cleavage by dipeptidyl peptidase IV (DPP IV) via amino acid modification at position 1 or 2 as described herein, the duration of action or half-life in circulation is extended.
(E) Stability is improved by modifying the Asp at position 15, for example by deletion or substitution with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid. Such modifications can reduce degradation or cleavage in the pH range of 5.5-8, e.g., at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, up to 100% of the starting peptide is retained after 24 hours at 25 ℃. Such modifications reduce cleavage of the peptide bond between Asp15-Ser 16.
(F) Stability is improved by modifying the Ser at position 16, for example by substitution with Thr or AIB. Such modifications also reduce cleavage of the peptide bond between Asp15-Ser 16.
(G) Stability is improved by modifying the methionine at position 27, for example by substitution with leucine or norleucine. Such modifications can reduce oxidative degradation. Stability may also be improved by modifying the Gln at positions 20 or 24, for example by substitution with Ser, Thr, Ala or AIB. Such modifications can reduce degradation by deamidation of Gln. Stability can be improved by modifying the Asp at position 21, for example by substitution with Glu. Such modifications may reduce degradation that occurs through dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid.
(H) Non-conservative or conservative substitutions, additions or deletions that do not substantially affect activity, for example conservative substitutions at one or more of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; one or more of these positions is substituted with Ala; a deletion of one or more amino acids in positions 27, 28 or 29; or the deletion of amino acid 29, optionally in combination with a C-terminal amide or ester replacement for the C-terminal carboxylic acid group; lys at position 12 is substituted with Arg; tyr at position 10 is substituted with Val or Phe;
maintenance of activity after PEGylation was provided by adding GPSSGAPPPS (SEQ ID NO: 1095) to the C-terminus.
Some positions of the native glucagon peptide may be modified while maintaining at least some activity of the parent peptide. Thus, applicants contemplate that one or more of the amino acids at positions 2, 5, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28, or 29 may be substituted with an amino acid other than that present in the native glucagon peptide and still retain activity at the glucagon receptor.
In some embodiments, position 18 is substituted with an amino acid selected from Ala, Ser, or Thr. In some embodiments, the amino acid at position 20 is substituted with Ser, Thr, Lys, Arg, Orn, citrulline, or AIB. In some embodiments, position 21 is substituted with Glu, homoglutamic acid, or homocysteic acid. In some embodiments, the glucagon peptide comprises 1-10 amino acid modifications selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28, and 29. In exemplary embodiments, the modification is one or more amino acid substitutions selected from Gln17, Ala18, Glu21, Ile23, Ala24, Val27, and Gly 29. In some embodiments, 1-2 amino acids selected from positions 17-26 are different from the parent peptide. In other embodiments, 1-2 amino acids selected from positions 17-22 are different from the parent peptide. In still other embodiments, the modifications are Gln17, Ala18, Glu21, Ile23, and Ala 24.
In some embodiments, one or more amino acids are added to the carboxy terminus of the glucagon peptide. The amino acid is typically selected from one of the 20 common amino acids, and in some embodiments, the amino acid has an amide group of a carboxylic acid that replaces the natural amino acid. In exemplary embodiments, the added amino acids are selected from glutamic acid and aspartic acid and glycine.
Other modifications that do not destroy activity include W10 or R20.
In some embodiments, the class 2 glucagon-related peptides disclosed herein are modified by truncation of the C-terminus by 1 or 2 amino acid residues, while still maintaining similar activity and potency at the glucagon, GLP-1 and/or GIP receptors. In this aspect, the amino acids at positions 29 and/or 28 may be deleted.
Exemplary embodiments
According to some embodiments of the invention, the analog of glucagon having GIP agonist activity (SEQ ID NO: 1001) comprises SEQ ID NO: 1001 having: (a) an amino acid modification at position 1 that confers GIP agonist activity, (b) a modification that stabilizes the alpha helical structure of the C-terminal portion of the analog (amino acids 12-29), and (C) optionally 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) additional amino acid modifications. In some embodiments, the analogs have at least about 1% of the activity of native GIP at the GIP receptor or any other level of activity described herein at the GIP receptor.
In certain embodiments, the modification that stabilizes the alpha helical structure is a modification that provides or introduces an intramolecular bridge, including, for example, a covalent intramolecular bridge, such as any of the covalent intramolecular bridges described herein. In some embodiments, the covalent intramolecular bridge is a lactam bridge. The lactam bridge of the analogs of these embodiments can be a lactam bridge as described herein. See, for example, the teaching of lactam bridges in the "stabilization of the alpha helix" section. For example, the lactam bridge may be a lactam bridge between the amino acid side chains at positions i and i +4, where i is 12, 13, 16, 17, 20 or 24, or between the amino acid side chains at positions j and j +3, where j is 17. In certain embodiments, the lactam bridge may be between the amino acids at positions 16 and 20, wherein one of the amino acids at positions 16 and 20 is substituted with Glu and the other amino acid at positions 16 and 20 is substituted with Lys.
In alternative embodiments, the modification to stabilize the alpha helical structure is the introduction of 1, 2, 3 or 4 alpha, alpha-disubstituted amino acids at positions 16, 20, 21 and 24 of the analog. In some embodiments, the α, α -disubstituted amino acid is AIB. In certain aspects, an α, α -disubstituted amino acid (e.g., AIB) is located at position 20 and the amino acid at position 16 is substituted with a positively charged amino acid (e.g., an amino acid of formula IV described herein). The amino acid of formula IV can be homolysine, Lys, Orn, or 2, 4-diaminobutyric acid (Dab).
In a particular aspect of the invention, the amino acid modification at position 1 is a substitution of His with an amino acid without an imidazole side chain, such as a large aromatic amino acid (e.g., Tyr).
In certain aspects, analogs of glucagon comprise amino acid modifications at 1, 2, or all of positions 27, 28, and 29. For example, Met at position 27 can be substituted with a large aliphatic amino acid (optionally Leu), Asn at position 28 can be substituted with a small aliphatic amino acid (optionally Ala), Thr at position 29 can be substituted with a small aliphatic amino acid (optionally Gly), or a combination of 2 or 3 of the foregoing modifications. In particular embodiments, the analog of glucagon comprises Leu at position 27, Ala at position 28, and Gly or Thr at position 29.
In certain embodiments of the invention, the analog of glucagon comprises an overhang of 1-21 amino acids C-terminal to amino acid 29. For example, the overhang may comprise seq id NO: 1095 or 1096. Additionally or alternatively, the analog of glucagon may comprise an extension in which 1-6 amino acids of the extension are positively charged amino acids. The positively charged amino acid can be an amino acid of formula IV, including but not limited to Lys, homolysine, Orn, and Dab.
In some embodiments, the analog of glucagon is acylated or alkylated as described herein. For example, an acyl or alkyl group may be attached to an analog of glucagon at the 10 or 40 position of the analog, with or without a spacer, as further described herein. Additionally or alternatively, analogs can be modified to include hydrophilic moieties as further described herein. Further, in some embodiments, the analogs comprise any one or combination of the following modifications:
(a) ser at position 2 substituted by D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val or alpha-amino-N-butanoic acid;
(b) tyr at position 10 is substituted with Trp, Lys, Orn, Glu, Phe, or Val;
(c) acyl is linked to Lys at position 10;
(d) lys at position 12 is substituted with Arg or Ile;
(e) ser at position 16 substituted with Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly or AIB;
(f) arg at position 17 substituted with Gln;
(g) arg at position 18 substituted with Ala, Ser, Thr, or Gly;
(h) gln at position 20 is substituted with Ser, Thr, Ala, Lys, citrulline, Arg, Orn, or AIB;
(i) asp at position 21 is substituted by Glu, homoglutamic acid, homocysteic acid;
(j) val at position 23 is substituted with Ile;
(k) Gln at position 24 is substituted with Asn, Ser, Thr, Ala or AIB;
(l) And conservative substitutions at any of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 8, 19, 20, 21, 24, 27, 28 and 29.
In an exemplary embodiment, an analog of glucagon with GIP agonist activity (SEQ ID NO: 1001) comprises the following modifications:
(a) an amino acid modification at position 1 that confers GIP agonist activity,
(b) a lactam bridge between the amino acid side chains in positions i and i +4 or between the amino acid side chains in positions j and j +3, wherein i is 12, 13, 16, 17, 20 or 24, wherein j is 17,
(c) amino acid modifications at 1, 2 or all of positions 27, 28 and 29, e.g.at positions 27 and/or 28, and
(d)1-9 or 1-6 additional amino acid modifications, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional amino acid modifications, and the analogs have an EC50 for GIP receptor activation of about 10nM or less.
The lactam bridge of the analogs of these embodiments can be a lactam bridge as described herein. See, for example, the teaching of lactam bridges under the "stabilization of the alpha helix structure" section. For example, the lactam bridge may be between the amino acids in positions 16 and 20, wherein one of the amino acids in positions 16 and 20 is substituted with Glu and the other amino acid in positions 16 and 20 is substituted with Lys.
According to these embodiments, the analog may comprise, for example, SEQ ID NO: 1005-1094.
In other exemplary embodiments, analogs of glucagon having GIP agonist activity (SEQ ID NO: 1001) comprise the following modifications:
(a) an amino acid modification at position 1 that confers GIP agonist activity,
(b) analogs 16, 20, 21 and 24 position 1, 2, 3 or all amino acids are substituted with alpha, alpha-disubstituted amino acids,
(c) amino acid modifications at 1, 2 or all of positions 27, 28 and 29, e.g.at positions 27 and/or 28, and
(d)1-9 or 1-6 further amino acid modifications, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications,
and the analogs have an EC50 for GIP receptor activation of about 10nM or less.
The α, α -disubstituted amino acid of the analogs of these embodiments may be any α, α -disubstituted amino acid, including but not limited to amino isobutyric Acid (AIB), amino acids disubstituted with the same or different groups selected from methyl, ethyl, propyl and n-butyl, or with cyclooctane or cycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In certain embodiments, the α, α -disubstituted amino acid is AIB. In certain embodiments, the amino acid at position 20 is substituted with an α, α -disubstituted amino acid (e.g., AIB).
According to these embodiments, the analog may comprise, for example, SEQ ID NO: 1099-.
In still other exemplary embodiments, the analog of glucagon having GIP agonist activity (SEQ ID NO: 1001) comprises the following modifications:
(a) an amino acid modification at position 1 that confers GIP agonist activity,
(b) amino acid substitution of Ser at position 16 with an amino acid of formula IV:
[ formula IV ],
wherein n is 1-16, or 1-10, or 1-7, or 1-6, or 2-6, R1And R2Each independently selected from H, C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) NH2、(C1-C18Alkyl) SH, (C)0-C4Alkyl) (C3-C6) Cycloalkyl group, (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7And (C)1-C4Alkyl) (C3-C9Heteroaryl) in which R is7Is H or OH, the side chain of the amino acid of the formula IV comprises a free amino group,
(c) amino acid substitution of Gln at position 20 with an alpha, alpha-disubstituted amino acid,
(d) amino acid modifications at 1, 2 or all of positions 27, 28 and 29, e.g.at positions 27 and/or 28, and
(e)1-9 or 1-6 further amino acid modifications, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 further amino acid modifications,
And the analogs have an EC50 for GIP receptor activation of about 10nM or less.
The amino acid of formula IV of the analogs of these embodiments can be any amino acid, for example, an amino acid of formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4 or 5, in which case the amino acid is Dab, Orn, Lys or homolysine, respectively.
The α, α -disubstituted amino acid of the analogs of these embodiments may be any α, α -disubstituted amino acid, including but not limited to amino isobutyric Acid (AIB), amino acids disubstituted with the same or different groups selected from methyl, ethyl, propyl and n-butyl, or with cyclooctane or cycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In certain embodiments, the α, α -disubstituted amino acid is AIB.
According to these embodiments, the analog may comprise, for example, SEQ ID NO: 1099-1165.
In still other exemplary embodiments, the analog of glucagon having GIP agonist activity (SEQ ID NO: 1001) comprises:
(a) amino acid modification at position 1 that confers GIP agonist activity, and
(b) an extension of about 1 to about 21 amino acids C-terminal to amino acid 29, wherein at least one amino acid of the extension is acylated or alkylated,
Wherein the analog has an EC50 for GIP receptor activation of about 10nM or less.
In some embodiments, the acylated or alkylated amino acid is an amino acid of formula I, formula II, or formula III. In more specific embodiments, the amino acid of formula I is Dab, Orn, Lys, or homolysine. Furthermore, in some embodiments, the about 1 to about 21 amino acid overhang contains the following amino acid sequence: GPSSGAPPPS (SEQ ID NO: 1095) or XGPPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid; or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSGAPPPK (SEQ ID NO: 1171) or XGPSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small aliphatic or apolar or slightly polar amino acid. In some embodiments, about 1 to about 21 amino acids may comprise a sequence that is identical to SEQ ID NO: 1095. 1096, 1170, 1171, or 1172 contain one or more conservative substitutions. In some embodiments, the acylated or alkylated amino acid is at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminal overhang analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminal overhang analog.
In some embodiments, the analog having GIP agonist activity further comprises amino acid modifications at 1, 2, or all of positions 27, 28, and 29, e.g., amino acid modifications at positions 27 and/or 28.
In any of the above exemplary embodiments, the amino acid modification at position 1 that confers GIP agonist activity may be a substitution of His with an amino acid without an imidazole side chain. The amino acid modification at position 1 may be, for example, a substitution of His with a large aromatic amino acid. In some embodiments, a large aromatic amino acid is any large aromatic amino acid described herein, including, for example, Tyr.
Further, with respect to the exemplary embodiments described above, the amino acid modifications at 1, 2, or all of positions 27, 28, and 29 can be any of those positions described herein. For example, Met at position 27 can be substituted with a large aliphatic amino acid (optionally Leu), Asn at position 28 can be substituted with a small aliphatic amino acid (optionally Ala), and/or Thr at position 29 can be substituted with a small aliphatic amino acid (optionally Gly). Alternatively, the analog may comprise such amino acid modifications at positions 27 and/or 28.
The analogs of the above exemplary embodiments can further comprise 1-9 or 1-6 additional other amino acid modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional amino acid modifications, such as any of the modifications described herein that increase or decrease activity at any of GIP, GLP-1, and glucagon receptors, increase solubility, improve duration of action or half-life in circulation, delay onset, or increase stability. The analog may further comprise, for example, an amino acid modification at position 12, optionally substituted with Ile; and/or amino acid modifications at positions 17 and 18, optionally substituted with Q at position 17 and A at position 18; and/or the use of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSGAPPPS (SEQ ID NO: 1096), or a combination thereof with the amino acid sequence of SEQ ID NO: 1095 or 1096 is added to the C-terminus as compared to a sequence containing one or more conservative substitutions. The analog may comprise one or more of the following modifications:
(i) Ser at position 2 substituted by D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val or alpha-amino-N-butanoic acid;
(ii) tyr at position 10 is substituted with Trp, Lys, Orn, Glu, Phe, or Val;
(iii) acyl is linked to Lys at position 10;
(iv) lys at position 12 is substituted with Arg;
(v) ser at position 16 substituted with Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly or AIB;
(vi) arg at position 17 substituted with Gln;
(vii) arg at position 18 substituted with Ala, Ser, Thr, or Gly;
(viii) gln at position 20 is substituted with Ala, Ser, Thr, Lys, citrulline, Arg, Orn, or AIB;
(ix) asp at position 21 is substituted by Glu, homoglutamic acid, homocysteic acid;
(x) Val at position 23 is substituted with Ile;
(xi) Gln at position 24 is substituted with Asn, Ala, Ser, Thr or AIB; and
(xii) Conservative substitutions at any one of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 and 29.
In some embodiments, the analog comprises a combination of modifications (i) - (xii). Additionally or alternatively, the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gln with Glu), wherein the analog has less than 1% glucagon activity at the glucagon receptor. Additionally or alternatively, the analog can comprise an amino acid modification at position 7 (e.g., an amino acid substitution of Thr with an amino acid without a hydroxyl group (e.g., Abu or Ile)), wherein the analog has less than about 10% GLP-1 activity at the GLP-1 receptor.
For the exemplary embodiments, the analog can be covalently linked to a hydrophilic moiety. In some embodiments, the analog is covalently attached to a hydrophilic moiety at any amino acid at position 16, 17, 20, 21, 24, 29, 40 or the C-terminus. In certain embodiments, the analog comprises a C-terminal overhang (e.g., the amino acid sequence of SEQ ID NO: 1095) and an amino acid comprising a hydrophilic moiety is added such that the hydrophilic moiety is covalently attached to the analog at position 40.
In some embodiments, the hydrophilic moiety is covalently attached to Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. Lys, Cys, Orn, homocysteine or acetyl-phenylalanine may be an amino acid that is native to the glucagon sequence (SEQ ID NO: 1001) or may be a substitution of the amino acid sequence of SEQ ID NO: 1001, and a natural amino acid. In some embodiments, wherein the hydrophilic moiety is linked to Cys, the linkage to the hydrophilic moiety may comprise the following structure:
for analogs comprising a hydrophilic moiety, the hydrophilic moiety can be any of the hydrophilic moieties described herein. See, e.g., the teaching under the section "attachment of hydrophilic moieties". In some embodiments, the hydrophilic moiety is polyethylene glycol (PEG). In certain embodiments, the PEG has a molecular weight of about 1,000 daltons to about 40,000 daltons, e.g., about 20,000 daltons to about 40,000 daltons.
For exemplary embodiments, analogs can comprise modified amino acids in which the side chain is covalently attached to an acyl group or an alkyl group (e.g., an acyl or alkyl group that is not natural to a naturally occurring amino acid). The acylated or alkylated analogs can be identical to the acylated or alkylated peptides described in the "acylated and alkylated" section. In some embodiments, the acyl group is a C4-C30 fatty acyl group, e.g., a C10 fatty acyl group or alkyl group, a C12 fatty acyl group or alkyl group, a C14 fatty acyl group or alkyl group, a C16 fatty acyl group or alkyl group, a C18 fatty acyl group or alkyl group, a C20 acyl group or alkyl group, or a C22 acyl group or alkyl group. The acyl or alkyl group may be covalently attached to any amino acid of the analog, including but not limited to the amino acid at position 10 or 40 or the C-terminal amino acid. In certain embodiments, the analog comprises a C-terminal overhang (e.g., the amino acid sequence of SEQ ID NO: 1095) and an amino acid comprising an acyl group or an alkyl group is added such that the acyl group or alkyl group is covalently attached to the analog at position 40. In some embodiments, the acyl group or alkyl group is covalently attached to a side chain of an amino acid of formula I, formula II, or formula III (e.g., a Lys residue). The acyl or alkyl group may be covalently linked to an amino acid that is native to the glucagon sequence (SEQ ID NO: 1001), or may be covalently linked to an amino acid added to the sequence of SEQ ID NO: 1001 sequence or to a sequence followed by SEQ ID NO: 1095 SEQ ID NO: 1001 (at the N-or C-terminus) or may be linked to an amino acid replacing the natural amino acid (e.g.Tyr in position 10 of SEQ ID NO: 1001).
In the above exemplary embodiments, where the analog comprises an acyl group or an alkyl group, the analog can be attached to the acyl group or alkyl group through a spacer as described herein. The spacer can be, for example, 3-10 atoms in length, and can be, for example, an amino acid (e.g., 6-aminocaproic acid, any of the amino acids described herein), a dipeptide (e.g., Ala-Ala, β Ala- β Ala, Leu-Leu, Pro-Pro, γ Glu- γ Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects, the total length of the spacer and acyl or alkyl group is from about 14 to about 28 atoms. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
In further exemplary embodiments, the analog of glucagon having GIP agonist activity comprises SEQ ID NO: 1227. an amino acid sequence of any one of 1228, 1229 or 1230, further comprising the following modifications:
(a) optionally an amino acid modification at position 1 conferring GIP agonist activity,
(b) an overhang of about 1 to about 21 amino acids C-terminal to amino acid 29, wherein at least one amino acid of the overhang is acylated or alkylated, and
(d) up to 6 other amino acid modifications,
Wherein the analog has an EC50 for GIP receptor activation of about 10nM or less.
In some aspects, the acylated or alkylated amino acid is an amino acid of formula I, formula II, or formula III. In more specific embodiments, the amino acid of formula I is Dab, Orn, Lys, or homolysine. Also, in some embodiments, about 1 to about 21 amino acids comprise the following amino acid sequence: GPSSGAPPPS (SEQ ID NO: 1095) or XGPPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid; or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSGAPPPK (SEQ ID NO: 1171) or XGPSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small aliphatic or apolar or slightly polar amino acid. In some embodiments, about 1 to about 21 amino acids may comprise a sequence that is identical to SEQ ID NO: 1095. 1096, 1170, 1171, or 1172, contain one or more conservative substitutions. In some embodiments, the acylated or alkylated amino acid is at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminal overhang analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminal overhang analog.
In any of the above exemplary embodiments, the amino acid at position 1 that confers GIP agonist activity may be an amino acid without an imidazole side chain. The amino acid in position 1 may be, for example, a large aromatic amino acid. In some embodiments, the large aromatic amino acid is any of the large aromatic amino acids described herein, including, for example, Tyr.
The analogs of the above exemplary embodiments can further comprise 1-6 additional amino acid modifications, e.g., any of the modifications described herein that increase or decrease activity at any of the GIP, GLP-1, and glucagon receptors, increase solubility, improve duration of action or half-life in circulation, delay onset, or increase stability.
In certain aspects, the glucagon analogs of the exemplary embodiments described above further comprise amino acid modifications at 1, 2, or all of positions 27, 28, and 29. The modifications at these positions may be any of those described herein with respect to these positions. For example, with respect to SEQ ID NO: 1227. 1228, 1229 or 1230, position 27 may be substituted with a large aliphatic amino acid (e.g. Leu, Ile or norleucine) or Met; position 28 may be substituted with another small aliphatic amino acid (e.g., Gly or Ala) or Asn; and/or position 29 may be substituted with another small aliphatic amino acid (e.g., Ala or Gly) or Thr. Alternatively, the analog may comprise such amino acid modifications at positions 27 and/or 28.
The analog may further comprise one or more of the following additional modifications:
(i) the amino acid at position 2 is any one of D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val or alpha-amino-N-butyric acid;
(ii) The amino acid at position 10 is Tyr, Trp, Lys, Orn, Glu, Phe or Val;
(iii) acyl is linked to Lys at position 10;
(iv) the amino acid at position 12 is Ile, Lys or Arg;
(v) the amino acid at position 16 is any one of Ser, Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly or AIB;
(vi) the amino acid at position 17 is Gln or Arg;
(vii) the amino acid at the 18 th position is any one of Ala, Arg, Ser, Thr or Gly;
(viii) the amino acid at position 20 is any one of Ala, Ser, Thr, Lys, citrulline, Arg, Orn or AIB or another alpha, alpha-disubstituted amino acid;
(ix) the amino acid at the 21 position is any one of Glu, Asp, homoglutamic acid and homocysteamine;
(x) The amino acid at position 23 is Val or Ile;
(xi) The amino acid at position 24 is any one of Gln, Asn, Ala, Ser, Thr or AIB; and
(xii) One or more conservative substitutions at any one of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 and 29.
In some embodiments, the analog comprises a combination of modifications (i) - (xii). Alternatively or additionally, the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gln with Glu), wherein the analog has less than 1% glucagon activity at the glucagon receptor. Alternatively or additionally, the analog can comprise an amino acid modification at position 7 (e.g., an amino acid substitution of Thr with an amino acid without a hydroxyl group (e.g., Abu or Ile)), wherein the analog has less than about 10% GLP-1 activity at the GLP-1 receptor.
For the exemplary embodiments, the analog can be covalently linked to a hydrophilic moiety. In some embodiments, the analog is covalently attached to a hydrophilic moiety at any of positions 16, 17, 20, 21, 24, 29, 40 or the C-terminal amino acid. In certain embodiments, the analog comprises a hydrophilic moiety covalently attached to the analog at position 24.
In some embodiments, the hydrophilic moiety is covalently attached to Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. Lys, Cys, Orn, homocysteine or acetyl-phenylalanine may be the amino acid sequence for SEQ ID NO: 1001. 1227, 1228, 1229 or 1230 are natural amino acids or may be substituted amino acids. In some embodiments, wherein the hydrophilic moiety is linked to Cys, the linkage may comprise the structure:
for analogs comprising a hydrophilic moiety, the hydrophilic moiety can be any of the hydrophilic moieties described herein. See, e.g., the teaching under the section "attachment of hydrophilic moieties". In some embodiments, the hydrophilic moiety is polyethylene glycol (PEG). In certain embodiments, the PEG has a molecular weight of about 1,000 daltons to about 40,000 daltons, e.g., about 20,000 daltons to about 40,000 daltons.
For the exemplary embodiments, the analogs may comprise modified amino acids within the C-terminal overhang in which the side chain is covalently attached to an acyl or alkyl group. The acylated or alkylated analogs may be identical to the acylated or alkylated peptides described in the "acylated and alkylated" section. In some embodiments, the acyl group is a C4-C30 fatty acyl group, e.g., a C10 fatty acyl group or alkyl group, a C12 fatty acyl group or alkyl group, a C14 fatty acyl group or alkyl group, a C16 fatty acyl group or alkyl group, a C18 fatty acyl group or alkyl group, a C20 acyl group or alkyl group, or a C22 acyl group or alkyl group. The acyl or alkyl group may be covalently attached to any amino acid of the analog, including but not limited to the amino acid at position 10 or 40 or the C-terminal amino acid. In some embodiments, the acyl group or alkyl group is covalently attached to a side chain of an amino acid of formula I, formula II, or formula III (e.g., a Lys residue). Acyl or alkyl and a peptide corresponding to SEQ ID NO: 1001. 1227, 1228, 1229 or 1230 are covalently linked to naturally occurring amino acids or may be linked to substituted amino acids. Acyl or alkyl is compared to the corresponding amino acid sequence of SEQ ID NO: 1095. 1096, 1171, or 1172 are covalently linked to the natural amino acid, or may be linked to a substituted amino acid.
In the above exemplary embodiments, where the analog comprises an acyl group or an alkyl group, the analog can be attached to the acyl group or alkyl group through a spacer as described herein. The spacer can be, for example, 3-10 atoms in length, and can be, for example, an amino acid (e.g., 6-aminocaproic acid, any of the amino acids described herein), a dipeptide (e.g., Ala-Ala, β Ala- β Ala, Leu-Leu, Pro-Pro, γ Glu- γ Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects, the total length of the spacer and acyl or alkyl group is from about 14 to about 28 atoms. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
In some very specific embodiments, the analogs of the invention comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 1099-.
In addition, specific examples of the analogs of the present invention include, but are not limited to, any of the analogs mentioned in tables 1-3.
In further exemplary embodiments, analogs of glucagon having GIP agonist activity comprise an acyl group or an alkyl group (e.g., an acyl group or an alkyl group that is non-natural to naturally occurring amino acids), wherein the acyl group or alkyl group is linked to a spacer, wherein (i) the spacer is linked to the amino acid side chain at position 10 of the analog; or (ii) the analog comprises an overhang of 1-21 amino acids C-terminal to amino acid 29, and the spacer is complementary to a spacer corresponding to the amino acid sequence of SEQ id no: an amino acid side chain attachment at one of the opposite positions 37-43 of 1001, wherein the analog has an EC50 for GIP receptor activation of about 10nM or less.
In such embodiments, the analog can comprise SEQ ID NO: 1001 having (i) an amino acid modification at position 1 that confers GIP agonist activity, (ii) an amino acid modification at 1, 2, or all of positions 27, 28, and 29, (iii) at least one of:
(A) The analogs comprise a lactam bridge between the amino acid side chains at positions i and i +4, where i is 12, 13, 16, 17, 20, or 24, or between the amino acid side chains at positions j and j +3, where j is 17;
(B) substitution of 1, 2, 3 or all amino acids at positions 16, 20, 21 and 24 of the analog with an α, α -disubstituted amino acid; or
(C) The analogs comprise (i) an amino acid substitution of Ser at position 16 with an amino acid of formula IV:
[ formula IV ],
wherein n is 1 to 7, wherein R1 and R2 are each independently selected from H, C1-C18Alkyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) NH2、(C1-C18Alkyl) SH, (C)0-C4Alkyl) (C3-C6) Cycloalkyl group, (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7And (C)1-C4Alkyl) (C3-C9Heteroaryl) in which R is7Is H or OH, the side chain of the amino acid of formula IV comprises a free amino group; and (ii) amino acid substitution of Gln at position 20 with an α, α -disubstituted amino acid.
And (iv) up to 6 additional amino acid modifications.
The α, α -disubstituted amino acid of the analogs of these embodiments may be any α, α -disubstituted amino acid, including but not limited to amino isobutyric Acid (AIB), amino acids disubstituted with the same or different groups selected from methyl, ethyl, propyl and n-butyl, or with cyclooctane or cycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). In certain embodiments, the α, α -disubstituted amino acid is AIB.
The amino acid of formula IV of the analogs of these embodiments can be any amino acid, for example, an amino acid of formula IV, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In certain embodiments, n is 2, 3, 4 or 5, in which case the amino acid is Dab, Orn, Lys or homolysine, respectively.
In any of the above exemplary embodiments, the amino acid modification at position 1 that confers GIP agonist activity may be a substitution of His with an amino acid without an imidazole side chain. The amino acid modification at position 1 may be, for example, a substitution of His with a large aromatic amino acid. In some embodiments, the large aromatic amino acid is any of the large aromatic amino acids described herein, including, for example, Tyr.
Further, with respect to the exemplary embodiments described above, the amino acid modifications at 1, 2, or all of positions 27, 28, and 29 can be any of those modifications at those positions described herein. For example, Met at position 27 can be substituted with a large aliphatic amino acid (optionally Leu), Asn at position 28 can be substituted with a small aliphatic amino acid (optionally Ala), and/or Thr at position 29 can be substituted with a small aliphatic amino acid (optionally Gly). Alternatively, the analog may comprise such amino acid modifications at positions 27 and/or 28.
The analogs of the above exemplary embodiments can further comprise 1-9 or 1-6 additional other amino acid modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional amino acid modifications, e.g., any of the modifications described herein that increase or decrease activity at any of GIP, GLP-1, and glucagon receptors, increase solubility, improve duration of action or half-life in circulation, delay onset, or increase stability. The analogue may further comprise, for example, an amino acid modification at position 12 (optionally substituted by Ile), and/or amino acid modifications at positions 17 and 18 (optionally substituted by Q at position 17 and substituted by A at position 18), and/or the addition of GPSSGAPPPS (SEQ ID NO: 1095) or XGPSGAPPPS (SEQ ID NO: 1096), or a derivative thereof with the amino acid sequence of SEQ ID NO: 1095 or 1096 is added to the C-terminus as compared to a sequence containing one or more conservative substitutions. The analog may comprise one or more of the following modifications:
(i) ser at position 2 substituted by D-Ser, Ala, D-Ala, Gly, N-methyl-Ser, AIB, Val or alpha-amino-N-butanoic acid;
(ii) tyr at position 10 is substituted with Trp, Lys, Orn, Glu, Phe, or Val;
(iii) acyl is linked to Lys at position 10;
(iv) lys at position 12 is substituted with Arg;
(v) ser at position 16 substituted with Glu, Gln, homoglutamic acid, homocysteic acid, Thr, Gly, Lys or AIB;
(vi) Arg at position 17 substituted with Gln;
(vii) arg at position 18 substituted with Ala, Ser, Thr, or Gly;
(viii) gln at position 20 is substituted with Ala, Ser, Thr, Lys, citrulline, Arg, Orn, or AIB;
(ix) asp at position 21 is substituted by Glu, homoglutamic acid, homocysteic acid;
(x) Val at position 23 is substituted with Ile;
(xi) Gln at position 24 is substituted with Asn, Ala, Ser, Thr or AIB; and
(xii) Conservative substitutions at any one of positions 2, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 28 and 29.
In some embodiments, the analog comprises a combination of modifications (i) - (xii). Alternatively or additionally, the analog can comprise an amino acid modification at position 3 (e.g., an amino acid substitution of Gln with Glu), wherein the analog has less than 1% glucagon activity at the glucagon receptor. Alternatively or additionally, the analog can comprise an amino acid modification at position 7 (e.g., Thr with an amino acid without a hydroxyl group (e.g., Abu or Ile)), a deletion of an amino acid from the C-terminus of the amino acid to position 27 or 28 (resulting in a 27 amino acid peptide or a 28 amino acid peptide), or a combination thereof, wherein the analog has less than about 10% GLP-1 activity at the GLP-1 receptor.
For the exemplary embodiments, the analog can be covalently linked to a hydrophilic moiety. In some embodiments, the analog is covalently attached to the hydrophilic moiety at any amino acid at position 16, 17, 20, 21, 24, 29, 40 or C-terminus. In certain embodiments, the analog comprises a C-terminal overhang (e.g., the amino acid sequence of SEQ ID NO: 1095) and an amino acid comprising a hydrophilic moiety is added such that the hydrophilic moiety is covalently attached to the analog at position 40.
In some embodiments, the hydrophilic moiety is covalently attached to Lys, Cys, Orn, homocysteine, or acetyl-phenylalanine of the analog. Lys, Cys, Orn, homocysteine or acetyl-phenylalanine may be the amino acid native to the glucagon sequence (SEQ ID NO: 1001) or may be a substitution of the amino acid sequence of SEQ ID NO: 1001, and a natural amino acid. In some embodiments, wherein the hydrophilic moiety is linked to Cys, the linkage to the hydrophilic moiety may comprise the following structure:
for analogs comprising a hydrophilic moiety, the hydrophilic moiety can be any of the hydrophilic moieties described herein. See, e.g., the teaching under the section "attachment of hydrophilic moieties". In some embodiments, the hydrophilic moiety is polyethylene glycol (PEG). In certain embodiments, the PEG has a molecular weight of about 1,000 daltons to about 40,000 daltons, e.g., about 20,000 daltons to about 40,000 daltons.
In exemplary embodiments where the analog comprises an acyl or alkyl group attached to the analog through a spacer, the spacer can be any of the spacers described herein. The spacer can be, for example, 3-10 atoms in length, and can be, for example, an amino acid (e.g., 6-aminocaproic acid, any of the amino acids described herein), a dipeptide (e.g., Ala-Ala, β Ala- β Ala, Leu-Leu, Pro-Pro, γ Glu- γ Glu), a tripeptide, or a hydrophilic or hydrophobic bifunctional spacer. In certain aspects, the total length of the spacer and acyl or alkyl group is from about 14 to about 28 atoms. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
An acyl or alkyl group is any acyl or alkyl group described herein, e.g., an acyl or alkyl group that is non-natural to a naturally occurring amino acid. In some embodiments, the acyl or alkyl is a C4-C30 fatty acyl group, e.g., a C10 fatty acyl group or alkyl group, a C12 fatty acyl group or alkyl group, a C14 fatty acyl group or alkyl group, a C16 fatty acyl group or alkyl group, a C18 fatty acyl group or alkyl group, a C20 acyl group or alkyl group, or a C22 acyl group or alkyl group, or a C4-C30 alkyl group. In particular embodiments, the acyl group is a C12-C18 fatty acyl group (e.g., a C14 or C16 fatty acyl group).
In some embodiments, the analog comprises an amino acid sequence from about 1 to about 21 amino acids C-terminal to amino acid 29 of the analog: GPSSGAPPPS (SEQ ID NO: 1095) or XGPPSSGAPPPS (SEQ ID NO: 1096), wherein X is any amino acid; or GPSSGAPPPK (SEQ ID NO: 1170) or XGPSGAPPPK (SEQ ID NO: 1171) or XGPSGAPPPSK (SEQ ID NO: 1172), wherein X is Gly or a small aliphatic or apolar or slightly polar amino acid. In some embodiments, about 1 to about 21 amino acids may comprise a sequence that is identical to SEQ ID NO: 1095. 1096, 1170, 1171, or 1172, contain one or more conservative substitutions. In some embodiments, the acylated or alkylated amino acid is at position 37, 38, 39, 40, 41, 42, or 43 of the C-terminal overhang analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the C-terminal overhang analog.
A GIP agonist can be a peptide comprising the amino acid sequence of any amino acid sequence (e.g., SEQ ID NO: 1005-1094), optionally with up to 1, 2, 3, 4, or 5 additional modifications that maintain GIP agonist activity. In certain embodiments, the GIP agonist comprises SEQ ID NO: 1099-1262.
Class 3 glucagon related peptides
In certain embodiments, the glucagon-related peptide is a class 3 glucagon-related peptide described herein and in international patent application No. PCT/US2009/47438 (filed 6.16.2009), international patent application publication No. WO 2008/101017 (published 8.21.2008), and U.S. provisional application No. 6I/090,412 and U.S. application No. 61/177,476, the contents of which are incorporated by reference in their entirety.
Some of the biological sequences (SEQ ID NO: 1-656) mentioned in the following sections relating to class 3 glucagon-like related peptides correspond to the sequence of SEQ ID NO: 1-656.
Activity of
The class 3 glucagon-related peptide may be a peptide that exhibits increased activity at the glucagon receptor, and in yet another embodiment has increased biophysical stability and/or water solubility. Additionally, in some embodiments, the class 3 glucagon-related peptide loses the selectivity of native glucagon for the glucagon receptor over the GLP-1 receptor, thus indicating a co-agonist of both receptors. Selected amino acid modifications within the class 3 glucagon-like peptide can control the relative activity of the peptide at the GLP-1 receptor versus the glucagon receptor. Thus, the class 3 glucagon-related peptide can be a glucagon/GLP-1 co-agonist having greater activity at the glucagon receptor than the GLP-1 receptor, a glucagon/GLP-1 co-agonist having about the same activity at both receptors, or a glucagon/GLP-1 co-agonist having greater activity at the GLP-1 receptor than the glucagon receptor. The latter class of co-agonists can be engineered to exhibit little or no activity at the glucagon receptor and retain the ability to activate the GLP-1 receptor at the same or better potency compared to native GLP-1. Any of these co-agonists may also include modifications that impart enhanced biophysical stability and/or water solubility.
The 3 rd glucagon-like peptide may be modified to produce a glucagon peptide having an activity at the GLP-1 receptor of at least about 1% (including at least about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%) to about 200% or more relative to native GLP-1 and an activity at the glucagon receptor of at least about 1% (including about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%) to about 500% or more relative to native glucagon. The amino acid sequence of native glucagon is SEQ ID NO: 1, the amino acid sequence of GLP-1(7-36) amide is SEQ ID NO: 52 and the amino acid sequence of GLP-1(7-37) acid is SEQ ID NO: 50. in exemplary embodiments, the class 3 glucagon-related peptide may have at least 10% of the activity of native glucagon at the glucagon receptor and at least 50% of the activity of native GLP-1 at the GLP-1 receptor, or at least 40% of the activity of native glucagon at the glucagon receptor and at least 40% of the activity of native GLP-1 at the GLP-1 receptor, or at least 60% of the activity of native glucagon at the glucagon receptor and at least 60% of the activity of native GLP-1 at the GLP-1 receptor.
The selectivity of a 3 rd class glucagon-related peptide for the glucagon receptor relative to the GLP-1 receptor can be expressed as the relative ratio of glucagon/GLP-1 activity (the activity of the peptide for the glucagon receptor relative to native glucagon divided by the activity of the peptide for the GLP-1 receptor relative to native GLP-1). For example, a glucagon-like peptide of class 3 having 60% of the activity of native glucagon at the glucagon receptor and 60% of the activity of native GLP-1 at the GLP-1 receptor has a glucagon/GLP-1 activity ratio of 1: 1. Exemplary ratios of glucagon/GLP-1 activity include about 1: 1, 1.5: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1, or about 1: 10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, or 1: 1.5. For example, a glucagon/GLP-1 activity ratio of 10: 1 indicates a 10-fold selectivity for the glucagon receptor over the GLP-1 receptor. Similarly, a GLP-1/glucagon activity ratio of 10: 1 indicates a 10-fold selectivity for the GLP-1 receptor over the glucagon receptor.
In some embodiments, the 3 rd glucagon-like peptide has less than about 10% of the activity of native glucagon at the glucagon receptor, e.g., about 1-10% or about 0.1-10% or greater than about 0.1% but less than about 10%, while having at least 20% of the activity of GLP-1 at the GLP-1 receptor. For example, exemplary class 3 glucagon-related peptides described herein have about 0.5%, about 1%, or about 7% of the activity of native glucagon while having at least 20% of the activity of GLP-1 at the GLP-1 receptor.
The class 3 glucagon-related peptide may be a glucagon peptide with increased or decreased activity at the glucagon receptor or the GLP-1 receptor or both. The class 3 glucagon-related peptide may be a glucagon peptide having an altered selectivity of the glucagon receptor relative to the GLP-1 receptor.
Thus, there is provided a high potency class 3 glucagon-related peptide disclosed herein with improved solubility and/or stability. Exemplary high potency glucagon-like-3-like related peptides have at least about 200% of the activity of native glucagon at the glucagon receptor, optionally between pH 6 and 8, or between 6 and 9, or between 7 and 9 (e.g., pH 7), are soluble at a concentration of at least 1mg/mL, optionally retain at least 95% of the starting peptide (e.g., 5% or less of the starting peptide is degraded or cleaved) after 24 hours at 25 ℃. By way of further example, exemplary class 3 glucagon-related peptides have an activity of greater than about 40% or greater than about 60% at both the glucagon and GLP-1 receptors (at a ratio of between about 1: 3 and 3: 1, or between about 1: 2 and 2: 1), optionally between pH 6 and 8, or between 6 and 9, or between 7 and 9 (e.g., pH 7), are soluble at a concentration of at least 1mg/mL, and optionally retain at least 95% of the starting peptide after 24 hours at 25 ℃. Another exemplary class 3 glucagon-like related peptide has greater than about 175% of the activity of native glucagon at the glucagon receptor and less than about 20% of the activity of native GLP-1 at the GLP-1 receptor, optionally between pH 6 and 8, or between 6 and 9, or between 7 and 9 (e.g., pH 7), is soluble at a concentration of at least 1mg/mL, and optionally retains at least 95% of the starting peptide after 24 hours at 25 ℃. Yet another exemplary class 3 glucagon-like related peptide has less than about 10% of the activity of native glucagon at the glucagon receptor and at least about 20% of the activity of native GLP-1 at the GLP-1 receptor, optionally between pH 6 and 8, or between 6 and 9, or between 7 and 9 (e.g., pH 7), is soluble at a concentration of at least 1mg/mL, and optionally retains at least 95% of the starting peptide after 24 hours at 25 ℃. Yet another exemplary glucagon-like peptide class 3 has less than about 10% but greater than 0.1%, 0.5%, or 1% of the activity of native glucagon at the glucagon receptor and at least about 50%, 60%, 70%, 80%, 90%, or 100% or more of the activity of native GLP-1 at the GLP-1 receptor, optionally between pH 6 and 8, or between 6 and 9, or between 7 and 9 (e.g., pH 7), is soluble at a concentration of at least 1mg/mL, and optionally retains at least 95% of the starting peptide after 24 hours at 25 ℃. In some embodiments, such class 3 glucagon-related peptides retain at least 22, 23, 24, 25, 26, 27, or 28 naturally occurring amino acids (e.g., 1-7, 1-5, or 1-3 modifications relative to naturally occurring glucagon) at the corresponding position in native glucagon.
Modifications affecting glucagon activity
Increased activity at the glucagon receptor is achieved by amino acid modification at position 16 of native glucagon (SEQ ID NO: 1). In some embodiments, the class 3 glucagon-related peptide is a glucagon agonist that has been modified relative to a wild-type peptide of His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1) to increase the potency of the peptide at the glucagon receptor. According to its ability to stimulate cAMP synthesis in a proven in vitro model assay (see example 5), the normally occurring serine at position 16 of native glucagon (SEQ ID NO: 1) can be substituted with a selected acidic amino acid to increase the potency of glucagon. More specifically, such substitutions increase the analog's potency at the glucagon receptor by at least 2-fold, 4-fold, 5-fold, and up to 10-fold or more. Such substitutions also increase the activity of the analog at the GLP-1 receptor to at least 5-fold, 10-fold, or 15-fold that of native glucagon, but maintain selectivity at the glucagon receptor over the GLP-1 receptor.
By way of non-limiting example, such increased potency may be obtained by substituting glutamic acid or another negatively charged amino acid having a side chain that is 4 atoms in length, or any of glutamine, homoglutamic acid, or homocysteine acid, or a charged amino acid having a side chain containing at least one heteroatom (e.g., N, O, S, P) and having a side chain length of about 4 (or 3-5) atoms for the naturally occurring serine at position 16. According to some embodiments, the serine residue at position 16 of native glucagon is substituted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid, homocysteine, threonine, or glycine. According to some embodiments, the serine residue at position 16 of native glucagon is substituted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid, and homocysteic acid, and in some embodiments, the serine residue is substituted with glutamic acid.
In some embodiments, the increased potency class 3 glucagon-like related peptide comprises SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7 or the peptide of SEQ ID NO: 5 glucagon agonist analogs. According to some embodiments, there is provided a class 3 glucagon-related peptide having an increased potency at the glucagon receptor as compared to wild-type glucagon, wherein the peptide comprises SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: 10, wherein the glucagon peptide retains its selectivity for the glucagon receptor over the GLP-1 receptor. In some embodiments, the glucagon-like receptor-like peptide class 3 with increased specificity for the glucagon receptor comprises SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10, or a glucagon agonist analog thereof, wherein the carboxy-terminal amino acid retains its native carboxylic acid group. According to some embodiments, the 3 rd glucagon-like peptide comprises NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 10), wherein the potency of the peptide on the glucagon receptor is increased to about 5-fold compared to native glucagon as determined by the in vitro cAMP assay of example 5.
Glucagon receptor activity can be decreased, maintained, or increased by amino acid modification at position 3, such as a substitution of the naturally occurring glutamine at position 3. In some embodiments, it has been demonstrated that substitution of the amino acid at position 3 with an acidic, basic, or hydrophobic amino acid (glutamic acid, ornithine, norleucine) substantially reduces or disrupts glucagon receptor activity. Analogs substituted with, for example, glutamic acid, ornithine or norleucine have about 10% or more, e.g., about 1-10% or about 0.1-10% or greater than about 0.1% but less than about 10%, of the activity of native glucagon at the glucagon receptor, while having at least 20% of the activity of GLP-1 at the GLP-1 receptor. For example, exemplary analogs described herein have about 0.5%, about 1%, or about 7% of the activity of native glucagon while having at least 20% of the activity of GLP-1 at the GLP-1 receptor. In particular, any of the class 3 glucagon related peptides described herein (including glucagon analogs, glucagon agonist analogs, glucagon co-agonists, and glucagon/GLP-1 co-agonist molecules) can be modified to contain a modification at position 3, e.g., substitution of Gln with Glu, to produce a peptide that is highly selective for the GLP-1 receptor as compared to the glucagon receptor, e.g., 10 times selective for the GLP-1 receptor as compared to the glucagon receptor.
In another embodiment, the glutamine at the naturally occurring 3-position of any 3 rd glucagon-like peptide can be substituted with a glutamine analog without substantial loss of activity at the glucagon receptor, and in some cases, the glucagon receptor activity is increased as described herein. In a specific embodiment, the amino acid at position 3 is substituted with dab (ac). For example, the glucagon agonist can comprise SEQ ID NO: 595. SEQ ID NO: 601. SEQ ID NO: 603. SEQ ID NO: 604. SEQ ID NO: 605 and SEQ ID NO: 606, or a pharmaceutically acceptable salt thereof.
Modifications at position 2 (e.g., AIB at position 2) and in some cases at position 1 have been observed to reduce glucagon activity. This decrease in glucagon activity can be restored as follows: by stabilizing the alpha helix of the C-terminal portion of glucagon, e.g., by the methods described herein, e.g., by covalent bonds between amino acid side chains (e.g., 12 and 16, 16 and 20, or 20 and 24) at positions "i" and "i + 4". In some embodiments, such covalent bond is a lactam bridge between the glutamic acid at position 16 and the lysine at position 20. In some embodiments, such covalent bond is an intramolecular bridge other than a lactam bridge. For example, suitable covalent bonding methods include any one or more of olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, use of α, ω -diaminoalkane linkers, formation of metal-atom bridges, and other methods of peptide cyclization.
Modifications affecting GLP-1 activity
By replacing the carboxylic acid of the C-terminal amino acid with an electrically neutral group (e.g. amide or ester), the activity towards the GLP-1 receptor is increased. In some embodiments, the glucagon-like peptide class 3 comprises SEQ ID NO: 20, wherein the carboxy-terminal amino acid has an amide group, replacing a carboxylic acid group present in the natural amino acid. These 3 rd glucagon-like peptides have potent activity at both the glucagon and GLP-1 receptors and thus act as co-agonists at both receptors. According to some embodiments, the glucagon-like-3-related peptide is a glucagon and GLP-1 receptor co-agonist, wherein the peptide comprises SEQ ID NO: 20, wherein the amino acid in position 28 is Asn or Lys and the amino acid in position 29 is Thr-amide.
Modification by stabilizing the alpha helix of the C-terminal portion of glucagon (e.g., near residues 12-29) results in increased activity at the GLP-1 receptor.
In some embodiments, such modifications allow the formation of intramolecular bridges between the side chains of two amino acids that are separated by 3 intervening amino acids (i.e., the amino acid at position "i" and the amino acid at position "i + 4", where i is any integer between 12 and 25), by 2 intervening amino acids (i.e., the amino acid at position "j" and the amino acid at position "j + 3", where j is any integer between 12 and 27), or by 6 intervening amino acids (i.e., the amino acid at position "k" and the amino acid at position "k + 7", where k is any integer between 12 and 22). In exemplary embodiments, the bridge or linker is about 8 (or about 7-9) atoms in length and is formed between the side chains of the amino acids at positions 12 and 16, or at positions 16 and 20, or at positions 20 and 24, or at positions 24 and 28. The 2 amino acid side chains may be linked to each other by non-covalent bonds (e.g. hydrogen bonding, ionic interactions, e.g. forming salt bridges) or by covalent bonds.
According to some embodiments, the 3 rd glucagon-like peptide has glucagon/GLP-1 receptor co-agonist activity and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11. 47, 48 and 49. In some embodiments, the side chains are covalently bound to each other, while in some embodiments, 2 amino acids are bound to each other to form a lactam ring.
According to some embodiments, the glucagon-like-peptide-3 comprises SEQ ID NO: 45, wherein at least one lactam ring is formed between the side chains of an amino acid pair selected from the group consisting of amino acid pairs 12 and 16, 16 and 20, 20 and 24 or 24 and 28. In some embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 20, wherein the peptide comprises an intramolecular lactam bridge formed between the amino acids at positions 12 and 16 or between the amino acids at positions 16 and 20. In some embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 20, wherein an intramolecular lactam bridge is formed between the amino acids at positions 12 and 16, between the amino acids at positions 16 and 20 or between the amino acids at positions 20 and 24, and the amino acid at position 29 is a glycine, wherein the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 20 is linked to the C-terminal amino acid. In yet another embodiment, the amino acid at position 28 is aspartic acid.
In some embodiments, stabilization of the alpha helical structure of the C-terminal portion of the class 3 glucagon-related peptide is achieved by forming an intramolecular bridge that is not a lactam bridge. For example, suitable covalent bonding methods include any one or more of the following: olefin metathesis, lanthionine-based cyclization, disulfide bridge or modified sulfur-containing bridge formation, use of alpha, omega-diaminoalkane linkers, formation of metal-atom bridges, and other methods for peptide cyclization that stabilize alpha helices.
In addition, one or more α, α -disubstituted amino acids can be purposefully introduced at positions that maintain the desired activity, resulting in enhanced activity at the GLP-1 receptor by stabilizing the α -helical structure in the C-terminal portion of the glucagon peptide (around amino acids 12-29). Such peptides may be considered herein as peptides without intramolecular bridges. In some aspects, stabilization of the alpha helix is achieved in such a manner that no intramolecular bridges (e.g., salt bridges) or covalent bonds need be introduced. In some embodiments, 1, 2, 3, 4 or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 of the glucagon peptide is substituted with an α, α -disubstituted amino acid. For example, substitution of aminoisobutyric Acid (AIB) at position 16 of the glucagon-like peptide class 3 in the absence of a salt bridge or lactam increases GLP-1 activity. In some embodiments, 1, 2, 3 or more of positions 16, 20, 21 or 24 are substituted with AIB.
Increased activity at the GLP-1 receptor can be achieved by amino acid modification at position 20. In some embodiments, the glutamine at position 20 is replaced with another hydrophilic amino acid (e.g., lysine, citrulline, arginine, or ornithine) having a side chain that is charged or has hydrogen bonding capability and is at least about 5 (or about 4-6) atoms in length.
In a nucleic acid comprising SEQ ID NO: 26 has been shown to have increased activity at the GLP-1 receptor in a glucagon-like peptide class 3 of the C-terminal overhang. Further improvements in the amino acid sequences comprising seq id NO: 26, GLP-1 activity of such glucagon-like peptide class 3.
The GLP-1 potency can be further suitably increased by modifying the amino acid at position 10 to Trp.
Combinations of modifications that increase GLP-1 receptor activity can provide higher GLP-1 activity than if only any of such modifications were used. For example, the glucagon-like-3-related peptide may comprise modifications at positions 16, 20, and the C-terminal carboxylic acid group, optionally with covalent bonds between amino acids at positions 16 and 20; may contain modifications at the 16-position and the C-terminal carboxylic acid group; may comprise modifications at positions 16 and 20, optionally with covalent bonds between the amino acids at positions 16 and 20; or may comprise modifications at the 20-position and the C-terminal carboxylic acid group; with the optional proviso that the amino acid at position 12 is not Arg; or with the optional proviso that the amino acid at position 9 is not Glu.
Modifications affecting solubility
Addition of hydrophilic moieties
The class 3 glucagon-related peptides can be further modified to improve the solubility and stability of the peptides in aqueous solution at physiological pH while maintaining high biological activity relative to native glucagon. The hydrophilic moieties described herein can be linked to a glucagon-like 3-peptide as discussed further herein.
According to some embodiments, it is contemplated that the polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: the introduction of hydrophilic groups at positions 17, 21 and 24 of the glucagon-like peptide class 3 of 10 improves the solubility and stability of high potency glucagon analogues in solutions having physiological pH. The introduction of such groups also extends the duration of action, for example as measured by an extended half-life in circulation.
In some embodiments, the glucagon-like-3-like peptide comprises a sequence selected from the group consisting of seq id no: SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18 and SEQ ID NO: 19, wherein the side chain of the amino acid residue at one of positions 16, 17, 21 or 24 of said glucagon-like peptide type 3 further comprises a polyethylene glycol chain having a molecular weight selected from the group consisting of about 500 to about 40,000 daltons. In some embodiments, the polyethylene glycol chain has a molecular weight selected from about 500 to about 5,000 daltons. In another embodiment. The polyethylene glycol chains have a molecular weight of about 10,000 to about 20,000 daltons. In still other exemplary embodiments, the polyethylene glycol chains have a molecular weight of from about 20,000 to about 40,000 daltons.
Suitable hydrophilic moieties include any water soluble polymer known in the art, including the hydrophilic moieties described herein, homopolymers or copolymers of PEG, and monomethyl-substituted polymers of PEG (mPEG). According to some embodiments, the hydrophilic group comprises a Polyethylene (PEG) chain. More specifically, in some embodiments, the glucagon-like peptide class 3 comprises seq id NO: 6 or SEQ ID NO: 7, wherein the PEG chain is covalently linked to the side chains of the amino acids present at positions 21 and 24 of the glucagon-like-3-peptide and the carboxy-terminal amino acid of the glucagon-like-3-peptide has a carboxylic acid group. According to some embodiments, the polyethylene glycol chains have an average molecular weight selected from the range of about 500 to about 10,000 daltons.
According to some embodiments, the pegylated type 3 glucagon-related peptide comprises two or more polyethylene glycol chains covalently bound to the type 3 glucagon-related peptide, wherein the glucagon chains have an overall molecular weight of from about 1,000 to about 5,000 daltons. In some embodiments, the pegylated glucagon agonist comprises a sequence consisting of SEQ ID NO: 5 or SEQ ID NO: 5, wherein the PEG chains are covalently attached to amino acid residues at positions 21 and 24, and wherein the 2 PEG chains have a total molecular weight of about 1,000 to about 5,000 daltons.
Charged C terminal
Can be prepared, for example, by mapping SEQ ID NO: 20 (preferably at a position C-terminal to position 27) incorporates 1, 2, 3 or more charged amino acids, further improving the glucagon peptide comprising SEQ ID NO: 20, solubility of glucagon related peptide class 3. Such charged amino acids can be introduced by, for example, substituting the natural amino acid with a charged amino acid at position 28 or 29, or by, for example, adding a charged amino acid after position 27, 28, or 29. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. Other modifications, such as conservative substitutions, can be made to the class 3 glucagon related peptide that still maintain glucagon activity. In some embodiments, provided is SEQ ID NO: 20, wherein the analog differs from the glucagon related peptide of SEQ ID NO: 20, and in some embodiments, the analog differs from the amino acid substitution at position 20 of SEQ id no: 20.
Acylation/alkylation
According to some embodiments, the glucagon peptide is modified to comprise an acyl group or an alkyl group, for example a C4-C30 acyl group or alkyl group. In some aspects, the acyl group or alkyl group is not naturally present on the amino acid. In particular aspects, the acyl group or alkyl group is non-natural to any naturally occurring amino acid. Acylation or alkylation may extend the half-life in circulation and/or delay onset and/or extend the duration of action and/or improve resistance to proteases, such as DPP-IV. The activity of the glucagon-like peptide class 3 at the glucagon receptor and the GLP-1 receptor is maintained, if not significantly improved, after acylation. Furthermore, the potency of the acylated analogs is comparable, if not significantly improved, to the non-acylated form of the class 3 glucagon-related peptide.
In some embodiments, the present invention provides glucagon related peptide class 3 modified to comprise an acyl or alkyl group covalently attached to the amino acid at position 10 of the glucagon peptide. The glucagon peptide may further comprise a spacer between the amino acid at position 10 of the glucagon-like related peptide type 3 and the acyl or alkyl group. Any of the foregoing glucagon-like peptide class 3 may comprise 2 acyl groups or 2 alkyl groups, or a combination thereof.
In a particular aspect of the invention, the acylated glucagon-like 3-peptide comprises SEQ ID NO: 534-544 and 546-549.
Truncation of the C-terminus
In some embodiments, the class 3 glucagon-related peptides described herein are further modified by truncation or deletion of 1 or 2 amino acids (i.e., positions 29 and/or 28) of the C-terminus of the glucagon peptide without affecting activity and/or potency at the glucagon and GLP-1 receptors. In this aspect, the 3 rd glucagon-like related peptide may comprise amino acids 1-27 or 1-28 of the native glucagon peptide (SEQ ID NO: 1), optionally with one or more modifications as described herein.
In some embodiments, the truncated glucagon-like peptide of class 3 comprises seq id NO: 550 or SEQ ID NO: 551. in another embodiment, the truncated glucagon agonist peptide comprises SEQ ID NO: 552 or SEQ ID NO: 553.
C-terminal protrusion end
According to some embodiments, the class 3 glucagon-related peptide disclosed herein is modified by adding a second peptide (e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28) to the carboxy terminus of the glucagon peptide. In some embodiments, the polypeptide has an amino acid sequence selected from SEQ ID NOs: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68 and SEQ ID NO: 69, covalently bound by a peptide bond to a second peptide, wherein the second peptide comprises a sequence selected from the group consisting of SEQ ID NO: 26. SEQ ID NO: 27 and SEQ ID NO: 28, in sequence. In yet another embodiment, in the glucagon related peptide type 3 comprising a C-terminal overhang, the threonine at position 29 of the native glucagon peptide is replaced with glycine. Has a glycine substitution for threonine at position 29 and comprises SEQ id no: 26, the potency at the GLP-1 receptor of the carboxy-terminal overhang modified to comprise the amino acid sequence of SEQ ID NO: 26 is 4 times greater than native glucagon at the carboxy-terminal overhang. The potency at the GLP-1 receptor can be further enhanced by substituting alanine for the native arginine at position 18.
Thus, the glucagon-like peptide class 3 can have the amino acid sequence of SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28 carboxyl-terminal overhang. According to some embodiments, the polypeptide comprising SEQ ID NO: 33 or SEQ ID NO: 20, further comprising the glucagon peptide of SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28. More specifically, the glucagon-like peptide class 3 comprises a sequence selected from the group consisting of SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 15 further comprising the sequence of SEQ ID NO: 27(KRNRNNIA) or seq id NO: 28. More specifically, the glucagon peptide comprises a sequence selected from the group consisting of seq id NO: 10. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 55 and SEQ ID NO: 56 further comprising the sequence of SEQ ID NO: 26(GPSSGAPPPS) or SEQ ID NO: 29. In some embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 64.
Other modifications
Any of the modifications described herein with respect to class 3 glucagon related peptides that increase or decrease glucagon receptor activity and increase GLP-1 receptor activity can be used alone or in combination. Combinations of modifications that increase the activity of the GLP-1 receptor generally provide higher GLP-1 activity than if only any one of such modifications were used. Any of the above modifications may also be combined with other modifications described herein with respect to the class 3 glucagon-related peptide that confer other desirable properties, such as increased solubility and/or stability and/or extended duration of action. Alternatively, any of the above modifications may be combined with other modifications described herein with respect to the class 3 glucagon related peptide that do not substantially affect solubility or stability or activity. Exemplary modifications include, but are not limited to:
(A) solubility is improved, for example, by introducing 1, 2, 3, or more charged amino acids into the C-terminal portion of native glucagon, preferably at a position C-terminal to position 27. Such charged amino acids can be introduced, for example, by substituting the natural amino acid with a charged amino acid at position 28 or 29, or by adding a charged amino acid, for example, after position 27, 28 or 29. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. In other embodiments, 1, 2, 3, or all of the charged amino acids have a positive charge. Such modifications increase solubility, e.g., provide at least 2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 30-fold or greater solubility relative to native glucagon when measured after 24 hours at 25 ℃ at a given pH (e.g., pH 7) of between about 5.5 and 8.
(B) By adding a hydrophilic moiety as described herein (e.g., a polyethylene glycol chain), for example, at position 16, 17, 20, 21, 24, or 29 of the peptide or at the C-terminal amino acid, solubility is improved and the duration of action or half-life in circulation is extended.
(C) Stability is improved by modification of the aspartic acid at position 15, for example by deletion or substitution with glutamic acid, homoglutamic acid, cysteic acid or homocysteic acid. Such modifications may reduce degradation or lysis at a pH in the range of 5.5-8, particularly in acidic or basic buffers, e.g. after 24 hours at 25 ℃, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the starting peptide.
(D) Stability is improved by modification of the methionine at position 27, for example by substitution with leucine or norleucine. Such modifications can reduce oxidative degradation. Stability may also be improved by Gln modifications at positions 20 or 24, for example by substitution with Ser, Thr, Ala or AIB. Such modifications can reduce degradation by deamidation of Gln. Stability can be improved by modification of the Asp at position 21, for example by substitution with Glu. Such modifications may reduce degradation that occurs through dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid.
(E) Resistance to dipeptidyl peptidase IV (DPP IV) cleavage is increased by modifying the amino acid at position 1 or 2 with a DPP-IV resistant amino acid as described herein and including modifying the amino acid at position 2 with N-methyl-alanine.
(F) Conservative or non-conservative substitutions, additions or deletions that do not affect activity, e.g., conservative substitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; a deletion at one or more of positions 27, 28 or 29; or the deletion of amino acid 29, optionally in combination with a C-terminal amide or ester replacing the C-terminal carboxylic acid group;
(G) adding a C-terminal overhang as described herein;
(H) extending the half-life in circulation and/or extending the duration of action and/or delaying onset of action as described herein, e.g., by acylation or alkylation of the glucagon peptide;
(I) homo-or heterodimerization as described herein.
Other modifications include substitution of the His in position 1 with a large aromatic amino acid (e.g., Tyr, Phe, Trp, or amino-Phe); ser at position 2 substituted with Ala; tyr at position 10 is substituted with Val or Phe; lys at position 12 is substituted with Arg; asp at position 15 substituted with Glu; ser at position 16 is substituted with Thr or AIB.
A glucagon-like-3-peptide having GLP-1 activity comprising a non-conservative substitution of His at position 1 with a large aromatic amino acid (e.g., Tyr) can retain GLP-1 activity, provided that the alpha helix is stabilized by an intramolecular bridge (e.g., any of the intramolecular bridges described herein).
Conjugates and fusions
The class 3 glucagon-related peptide may be linked to the conjugate moiety, optionally by covalent bonding and optionally by a linker.
The 3 rd glucagon-like peptide may also be a component of a fusion peptide or fusion protein in which the second peptide or polypeptide is fused to a terminus (e.g., the carboxy terminus of the 3 rd glucagon-like peptide).
More specifically, the fused glucagon-like-3-peptide can comprise SEQ id no: 55. SEQ ID NO: 9 or SEQ ID NO: 10 further comprising the glucagon peptide of SEQ ID NO: 26(GPSSGAPPPS), SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28 (KRNR). In some embodiments, the nucleic acid sequence of SEQ ID NO: 26(GPSSGAPPPS), SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28(KRNR) is bonded via a peptide bond to amino acid 29 of the class 3 glucagon-like peptide. Applicants found that in a glucagon-like related peptide fusion peptide type 3 comprising a C-terminal overhang peptide of exendin-4 (e.g. SEQ ID NO: 26 or SEQ ID NO: 29), the substitution of the natural threonine residue in position 29 with glycine significantly improves GLP-1 receptor activity. Such amino acid substitutions can be used in combination with other modifications disclosed herein with respect to class 3 glucagon-related peptides to increase the affinity of the glucagon analog for the GLP-1 receptor. For example, the T29G substitution may be combined with S16E and N20K amino acid substitutions, optionally with a lactam bridge between amino acids 16 and 20, and optionally with the addition of a PEG chain as described herein. In some embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 64. In some embodiments, the glucagon-like fusion peptide type 3 peptide portion of the glucagon fusion peptide is selected from the group consisting of SEQ ID NO: 55. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5, wherein the PEG chain is selected from the range of 500-. More specifically, in some embodiments, the glucagon-like-peptide segment No. 3 is selected from the group consisting of SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: 63, wherein the PEG chain is selected from the range of 500-5,000. In some embodiments, the glucagon-like-3-like peptide is a peptide comprising SEQ ID NO: 55 and SEQ ID NO: 65, wherein seq id NO: 65 to SEQ ID NO: 55 at the carboxy terminus.
According to some embodiments, the nucleic acid sequence of SEQ ID NO: 10, to increase the GLP-1 receptor potency to a point where the relative activities at the glucagon and GLP-1 receptors are nearly equivalent. Thus, in some embodiments, the glucagon-like 3-peptide comprises a terminal amino acid comprising an amide group substituted for a carboxylic acid group present on a natural amino acid. The relative activity of the class 3 glucagon-related peptides on the corresponding glucagon and GLP-1 receptors can be adjusted as follows: by further modifying the class 3 glucagon-related peptide to produce analogs having about 40% to about 500% or more of the activity of native glucagon at the glucagon receptor and about 20% to about 200% or more of the activity of native GLP-1 at the GLP-1 receptor, e.g., to a 50-fold, 100-fold or greater increase in the normal activity of glucagon at the GLP-1 receptor. In some embodiments, the glucagon peptides described herein have up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native glucagon at the glucagon receptor. In some embodiments, the glucagon peptides described herein have up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of native GLP-1 at the GLP-1 receptor.
Exemplary embodiments
According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 55, wherein the analog differs from the sequence of SEQ ID NO: 55, wherein the glucagon peptide has at least 20% of the activity of native GLP-1 at the GLP-1 receptor.
According to some embodiments, there is provided a glucagon/GLP-1 receptor co-agonist comprising the sequence:
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 33), wherein Xaa in position 15 is selected from the following amino acids: asp, Glu, cysteamine acid, homoglutamic acid andhomocysteine, Xaa at position 16, selected from the following amino acids: ser, Glu, Gln, homoglutamic acid and homocysteic acid, Xaa at position 20 is Gln or Lys, Xaa at position 24 is Gln or Glu, Xaa at position 28 is Asn, Lys or an acidic amino acid, Xaa at position 29 is Thr, Gly or an acidic amino acid, and R is COOH or CONH2Provided that if serine is at position 16, Lys is at position 20, or if serine is at position 16, Glu is at position 24 and Lys is at either position 20 or 28. In some embodiments, the glucagon/GLP-1 receptor co-agonist comprises SEQ ID NO: 33, wherein the amino acid at position 28 is aspartic acid and the amino acid at position 29 is glutamic acid. In another embodiment, the amino acid at position 28 is a native asparagine, the amino acid at position 29 is a glycine and the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 65 and the amino acid sequence of SEQ ID NO: 33 are covalently linked at the carboxy terminus.
In some embodiments, there is provided a nucleic acid comprising SEQ ID NO: 33, wherein an additional acidic amino acid is added to the carboxy terminus of the peptide. In yet another embodiment, the carboxy-terminal amino acid of the glucagon analog has an amide replacing the carboxylic acid group of the natural amino acid. In some embodiments, the glucagon analog comprises a sequence selected from the group consisting of: SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43 and SEQ ID NO: 44.
according to some embodiments, there is provided SEQ ID NO: 33, wherein the analog differs from the glucagon peptide analog of SEQ ID NO: 33, with the proviso that if the amino acid in position 16 is serine, then either the 20-position is lysine or a lactam bridge is formed between the amino acid in position 24 and the amino acid in position 20 or 28. According to some embodiments, the analog differs from SEQ ID NO: 33. in some embodiments, the nucleic acid sequence of SEQ id no: 33 differ from the sequence by 1-2 amino acids selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, or in some embodiments by a single amino acid selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, with the proviso that if the amino acid at position 16 is serine, then either position 20 is lysine or a lactam bridge is formed between the amino acid at position 24 and the amino acid at position 20 or 28.
According to another embodiment, there is provided a GLP-1 receptor agonist with relative selectivity comprising the sequence NH 2-His-Ser-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 53), wherein Xaa in position 3 is selected from the following amino acids: glu, Orn or Nle, Xaa at position 15 is selected from the following amino acids: asp, Glu, cysteic acid, homoglutamic acid, and homocysteic acid, Xaa at position 16 being selected from the following amino acids: ser, Glu, Gln, homoglutamic acid and homocysteic acid, Xaa at position 20 is Gln or Lys, Xaa at position 24 is Gln or Glu, Xaa at position 28 is Asn, Lys or acidic amino acid, Xaa at position 29 is Thr, Gly or acidic amino acid, R is COOH and CONH2SEQ ID NO: 26 or SEQ ID NO: 29, with the proviso that if serine is at position 16, Lys is at position 20, or if serine is at position 16, Glu is at position 24 and Lys is at either position 20 or 28. In some embodiments, the amino acid at position 3 is glutamic acid. In some embodiments, the acidic amino acid substituted at position 28 and/or 29 is aspartic acid or glutamic acid. In some embodiments, the glucagon peptides (including co-agonist peptides) comprise the amino acid sequence of SEQ ID NO: 33, further comprising an additional acidic amino acid added to the carboxy-terminus of the peptide. In yet another embodiment, the carboxy-terminal amino acid of the glucagon analog has an amide replacing the carboxylic acid group of the natural amino acid.
According to some embodiments, there is provided a glucagon/GLP-1 receptor co-agonist comprising a modified glucagon peptide selected from the group consisting of: NH (NH)2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 34), wherein position 15Xaa above is selected from the following amino acids: asp, Glu, cysteic acid, homoglutamic acid, and homocysteic acid, Xaa at position 16 being selected from the following amino acids: ser, Glu, Gln, homoglutamic acid and homocysteic acid, Xaa at position 20 is Gln or Lys, Xaa at position 24 is Gln or Glu, Xaa at position 28 is Asn, Asp or Lys, R is COOH or CONH2Xaa at position 29 is Thr or Gly, R is COOH, CONH2SEQ ID NO: 26 or SEQ id no: 29, with the proviso that if serine is at position 16, Lys is at position 20, or if serine is at position 16, Glu is at position 24 and Lys is at either position 20 or 28. In some embodiments, R is CONH2Xaa at position 15 is Asp and Xaa at position 16 is selected from the group consisting of the following amino acids: glu, Gln, homoglutamic acid and homocysteic acid, Xaa at positions 20 and 24 are each Gln, Xaa at position 28 is Asn or Asp, and Xaa at position 29 is Thr. In some embodiments, Xaa at positions 15 and 16 are each Glu, Xaa at positions 20 and 24 are each Gln, Xaa at position 28 is Asn or Asp, Xaa at position 29 is Thr, and R is CONH2。
It is reported that certain positions of the native glucagon peptide can be modified while maintaining at least some of the activity of the parent peptide. Thus, applicants contemplate that the peptide located in SEQ ID NO: 11, one or more of the amino acids at positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 may be substituted with an amino acid other than that present in the native glucagon peptide, while still retaining activity at the glucagon receptor. In some embodiments, the methionine residue present at position 27 of the native peptide is replaced with leucine or norleucine to prevent oxidative degradation of the peptide. In another embodiment, the amino acid at position 20 is substituted with Lys, Arg, Orn, or citrulline (Citrullene) and/or position 21 is substituted with Glu, homoglutamic acid, or homocysteamine.
In some embodiments, provided is SEQ ID NO: 20, wherein 1-6 amino acids selected from position 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 27, 28 or 29 of the analog differ from the amino acid sequence of SEQ ID NO: 1 with the proviso that if the amino acid at position 16 is serine then Lys is present at position 20 or if serine is present at position 16 then Glu is present at position 24 and Lys is present at either position 20 or 28. According to another embodiment, there is provided SEQ ID NO: 20, wherein 1-3 amino acids selected from position 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27, 28, or 29 of the analog differ from the amino acid sequence of SEQ id no: 1 corresponding amino acid. In another embodiment, provided is SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: 11, wherein 1-2 amino acids selected from position 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20 or 21 of the analog differ from the amino acid sequence of SEQ ID NO: 1, and in yet another embodiment, 1-2 different amino acids, as compared to the amino acid present in the native glucagon sequence (SEQ ID NO: 1), represent conservative amino acid substitutions. In some embodiments, provided is SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 15, wherein the glucagon peptide further comprises 1, 2, or 3 amino acid substitutions at a position selected from the group consisting of positions 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27, or 29. In some embodiments, the substitution at position 2, 5, 7, 10, 11, 13, 14, 16, 17, 18, 19, 20, 21, 27, or 29 is a conservative amino acid substitution.
According to some embodiments, there is provided a glucagon/GLP-1 receptor co-agonist comprising a variant of the sequence of SEQ ID NO 33, wherein 1 to 10 amino acids selected from positions 16, 17, 18, 20, 21, 23, 24, 27, 28 and 29, respectively, of the variant are different from the amino acid sequence of SEQ ID NO: 1 corresponding amino acid. According to some embodiments, there is provided a variant of the sequence of SEQ ID NO 33, wherein the variant differs from SEQ ID NO: 33. according to some embodiments, there is provided a glucagon/GLP-1 receptor co-agonist comprising a variant of the sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected from positions 17 to 26 of the variant differ from the amino acid sequence of SEQ ID NO: 1 corresponding amino acid. According to some embodiments, there is provided a variant of the sequence of SEQ ID NO 33, wherein the variant differs from SEQ ID NO: 33. according to some embodiments, there is provided a variant of the sequence of SEQ ID NO 33, wherein the variant differs from the sequence of SEQ ID NO: 33, wherein the substituted amino acid is selected from Ala, Ser, Thr and Gly. According to some embodiments, there is provided a variant of the sequence of SEQ ID NO 33, wherein the variant differs from SEQ ID NO: 33. SEQ ID NO: 55 includes such variations. In another embodiment, glucagon/GLP-1 receptor co-agonists are provided comprising variants of the sequence of SEQ ID NO 33, wherein 1-2 amino acids selected from positions 17-22 of the variant differ from the amino acid sequence of SEQ ID NO: 1, and in yet another embodiment, variants of SEQ ID NO 33 are provided, wherein the variants differ from the amino acid sequence of SEQ ID NO: 33. according to some embodiments, there is provided a glucagon/GLP-1 receptor co-agonist comprising the sequence:
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Xaa-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 51), wherein Xaa at position 15 is Asp, Glu, cysteic acid, homoglutamic acid or homocysteine, Xaa at position 16 is Ser, Glu, Gln, homoglutamic acid or homocysteic acid, Xaa at position 20 is Gln, Lys, Arg, Orn or citrulline, Xaa at position 21 is Asp, Glu, homoglutamic acid or homocysteic acid, Xaa at position 24 is Gln or Glu, Xaa at position 28 is Asn, Lys or an acidic amino acid, Xaa at position 29 is Thr or an acidic amino acid, r is COOH or CONH2. In some embodiments, R is CONH2. According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 47. SEQ ID NO: 48 or SEQ ID NO: 49, wherein the variant differs from said sequence by an amino acid substitution at position 20. In some embodiments, the amino acid substitution is selected from the group consisting of a substitution at position 20 with Lys, Arg, Orn, or citrulline.
In some embodiments, there is provided a nucleic acid comprising SEQ ID NO: 34, wherein the analog differs from the glucagon peptide of SEQ ID NO: 34. in some embodiments, the serine residue is substituted with aminoisobutyric acid, D-alanine, and in some embodiments, the serine residue is substituted with aminoisobutyric acid. Such modifications inhibit cleavage by dipeptidyl peptidase IV while maintaining the original potency of the parent compound (e.g., at least 75%, 80%, 85%, 90%, 95% or more of the potency of the parent compound). In some embodiments, the solubility of the analog is increased, for example, by introducing 1, 2, 3, or more charged amino acids into the C-terminal portion of native glucagon, preferably at a position C-terminal to position 27. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. In another embodiment, the analog further comprises an acidic amino acid substituted for the natural amino acid at position 28 or 29 or added to the amino acid sequence of SEQ ID NO: 34 the carboxyl terminal acidic amino acid of the peptide.
In some embodiments, the glucagon analogs disclosed herein are further modified at position 1 or 2 to reduce sensitivity to cleavage by dipeptidyl peptidase IV. In some embodiments, provided is SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 15, wherein the analog differs from the parent molecule by a substitution at position 2 and has reduced sensitivity (i.e., resistance) to cleavage by dipeptidyl peptidase IV. More specifically, in some embodiments, position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of: d-serine, D-alanine, valine, amino-N-butyric acid, glycine, N-methylserine and aminoisobutyric acid. In some embodiments, position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of: d-serine, D-alanine, glycine, N-methylserine and aminoisobutyric acid. In another embodiment, position 2 of the analog peptide is substituted with an amino acid selected from the group consisting of: d-serine, glycine, N-methylserine and aminoisobutyric acid. In some embodiments, the amino acid at position 2 is not D-serine. In some embodiments, the glucagon peptide comprises SEQ ID NO: 21 or SEQ ID NO: 22.
In some embodiments, provided is SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 15, wherein the analog differs from the parent molecule by a substitution at position 1 and has reduced sensitivity (i.e., resistance) to cleavage by dipeptidyl peptidase IV. More specifically, position 1 of the analog peptide is substituted with an amino acid selected from the group consisting of: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. In another embodiment, there is provided a polypeptide comprising SEQ ID NO: 34, wherein the analog differs from the glucagon peptide of SEQ ID NO: 34. in some embodiments, the solubility of the analog is increased, for example, by introducing 1, 2, 3, or more charged amino acids into the C-terminal portion of native glucagon, preferably at a position C-terminal to position 27. In exemplary embodiments, 1, 2, 3, or all of the charged amino acids carry a negative charge. In another embodiment, the analog further comprises an acidic amino acid substituted for the natural amino acid at position 28 or 29 or added to the amino acid sequence of SEQ ID NO: 34 the carboxyl terminal acidic amino acid of the peptide. In some embodiments, the acidic amino acid is aspartic acid or glutamic acid.
In some embodiments, the glucagon/GLP-1 receptor co-agonist comprises the sequence of seq id NO: 20, further comprising an additional carboxy-terminal extension of 1 amino acid or a sequence selected from SEQ ID NO: 26. SEQ ID NO: 27 and SEQ ID NO: 28. In which a single amino acid is added to SEQ ID NO: in the carboxy-terminal embodiment of 20, the amino acid is typically selected from one of the 20 common amino acids, while in some embodiments, the additional carboxy-terminal amino acid has an amide group that replaces the carboxylic acid of the natural amino acid. In some embodiments, the additional amino acid is selected from the group consisting of glutamic acid, aspartic acid, and glycine.
In an alternative embodiment, glucagon/GLP-1 receptor co-agonists are provided wherein the peptide comprises at least one lactam ring formed between the side chains of a glutamic acid residue and a lysine residue, wherein the glutamic acid residue and the lysine residue are separated by 3 amino acids. In some embodiments, the carboxy-terminal amino acid of the lactam-bearing glucagon peptide has an amide group of a carboxylic acid that replaces the natural amino acid. More specifically, in some embodiments, glucagon and GLP-1 co-agonists are provided comprising a modified glucagon peptide selected from the group consisting of:
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO：66)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO：67)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO：68)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Xaa-R(SEQ ID NO：69)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Asn-Thr-R(SEQ ID NO：16)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R(SEQ ID NO：17)
NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R(SEQ ID NO：18)
Wherein Xaa at position 28 is Asp or Asn, Xaa at position 29 is Thr or Gly, and R is selected from COOH and CONH2Glutamic acid, aspartic acid, glycine, SEQ ID NO: 26. SEQ ID NO: 27 and SEQ ID NO: 28 and the lactam bridge is formed in SEQ ID NO: 66 between Lys at position 12 and Glu at position 16 in SEQ ID NO: 67 between Glu at position 16 and Lys at position 20 of SEQ ID NO: 68 between Lys at position 20 and Glu at position 24 in SEQ ID NO: 69 between Glu at position 24 and Lys at position 28 in SEQ ID NO: between Lys at position 12 and Glu at position 16 and between Lys at position 20 and Glu at position 24 of SEQ ID NO: 17 between Lys at position 12 and Glu at position 16 and between Glu at position 24 and Lys at position 28, and in SEQ ID NO: between Glu at position 16 and Lys at position 20 of 18 and between Glu at position 24 and Lys at position 28. In some embodiments, R is selected from COOH, CONH2Glutamic acid, aspartic acid and glycine, wherein the amino acid at the position 28 is Asn, and the amino acid at the position 29 is threonine. In some embodiments, R is CONH2The amino acid at position 28 is Asn and the amino acid at position 29 is threonine. In another embodiment, R is selected from SEQ ID NO: 26. SEQ ID NO: 29 and SEQ ID NO: the amino acids at positions 65 and 29 are glycine.
In yet another embodiment, the glucagon/GLP-1 receptor co-agonist is selected from the group consisting of SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18, wherein the peptide further comprises an additional carboxy-terminal extension of 1 amino acid or a sequence selected from SEQ id no: 26. SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the terminal overhang comprises SEQ ID NO: 26. SEQ ID NO: 29 or SEQ ID NO: 65 and the glucagon peptide comprises the sequence of SEQ ID NO: 55, or a sequence of seq id no. In some embodiments, the glucagon/GLP-1 receptor co-agonist comprises SEQ ID NO: 33, wherein the amino acid at position 16 is glutamic acid, the amino acid at position 20 is lysine, the amino acid at position 28 is asparagine, and SEQ ID No: 26 or SEQ ID NO: 29 and SEQ ID NO: 33 are linked at the carboxy terminus.
In which a single amino acid is added to SEQ ID NO: in the carboxy-terminal embodiment of 20, the amino acid is typically selected from one of the 20 common amino acids, and in some embodiments, the amino acid has an amide group of a carboxylic acid that replaces the natural amino acid. In some embodiments, the additional amino acid is selected from glutamic acid and aspartic acid and glycine. In embodiments where the glucagon agonist analog further comprises a carboxy-terminal extension, the carboxy-terminal amino acid of the extension terminates in an amide group or ester group rather than a carboxylic acid in some embodiments.
In another embodiment, the glucagon/GLP-1 receptor co-agonist comprises the sequence: NH (NH)2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Xaa-CONH2(SEQ ID NO: 19) wherein Xaa at position 30 represents any amino acid. In some embodiments, Xaa is selected from one of the 20 common amino acids, and in some embodiments, the amino acid is glutamic acid, aspartic acid, or glycine. Can be prepared by ligating the PEG chain to seq id NO: the amino acid side chains at positions 17, 21, 24 or 30 of 19 are covalently linked to further enhance the solubility of the peptide. In yet another embodiment, the peptide comprises a sequence selected from SEQ ID NOs: 26. SEQ ID NO: 27 and SEQ ID NO: 28, and an additional carboxy-terminal overhang. According to some embodiments, the glucagon/GLP-1 receptor co-agonist comprises SEQ ID NO: 30. SEQ ID NO: 31 and SEQ ID NO: 32, or a sequence of the sequence of 32.
The sequence of SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 64 are further site-specifically modified within the glucagon sequence to provide a glucagon agonist group with varying degrees of GLP-1 agonism (aginism). Thus, peptides with nearly identical in vitro potency for various receptors were prepared and characterized. Likewise, peptides with a 10-fold selectivity increase in potency for each of the two receptors have been identified and characterized. As described above, substitution of the serine residue at position 16 with glutamic acid increases the potency of native glucagon at both the glucagon and GLP-1 receptors, but maintains approximately 10-fold selectivity at the glucagon receptor. A glucagon analog having about 10-fold selectivity for the GLP-1 receptor was additionally obtained by substituting glutamic acid for the native glutamine at position 3 (SEQ ID NO: 22).
The solubility of the glucagon/GLP-1 co-agonist peptide in aqueous solution at physiological pH can be further improved while maintaining high biological activity relative to native glucagon by introducing hydrophilic groups at positions 16, 17, 21 and 24 of the peptide, or by adding a single modified amino acid (i.e., an amino acid modified to contain a hydrophilic group) at the carboxy terminus of the glucagon/GLP-1 co-agonist peptide. According to some embodiments, the hydrophilic group comprises a Polyethylene (PEG) chain. More specifically, in some embodiments, the glucagon peptide comprises SEQ id no: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ id no: 18, wherein the PEG chain is covalently linked to the side chain of amino acid 16, 17, 21, 24, 29 or the C-terminal amino acid of the glucagon peptide, with the proviso that if the peptide comprises seq id NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 or SEQ ID NO: 13, the polyethylene glycol chain is covalently bound to an amino acid residue at position 17, 21 or 24 if the peptide comprises SEQ ID NO: 14 or SEQ ID NO: 15, the polyethylene glycol chain is covalently bound to an amino acid residue at position 16, 17 or 21, and if the peptide comprises SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18, the polyethylene glycol chain is covalently bound to the amino acid residue at position 17 or 21.
In some embodiments, the glucagon peptide comprises SEQ ID NO: 11. SEQ ID NO: 12 or SEQ ID NO: 13, wherein the PEG chain is covalently linked to the side chain of amino acid 17, 21, 24 or the C-terminal amino acid of the glucagon peptide, and the carboxy-terminal end of the peptide has an amide group replacing the carboxylic acid group of the natural amino acid. In some embodiments, the glucagon/GLP-1 receptor co-agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18 and SEQ ID NO: 19, wherein the PEG chain is identical to the sequence of SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 17, 21 or 24 of 19; or SEQ ID NO: 14 and SEQ ID NO: bit 16, 17 or 21 of 15; or SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18 at position 17 or 21. In another embodiment, the glucagon/GLP-1 receptor co-agonist peptide comprises SEQ ID NO: 11 or SEQ ID NO: 19, wherein the PEG chain is covalently linked to the side chain of amino acid 17, 21 or 24 or the C-terminal amino acid of the glucagon peptide.
According to some embodiments, and following the conditional restrictions described in the preceding paragraphs, the glucagon co-agonist peptide is modified to contain one or more amino acid substitutions at the 16, 17, 21, 24 or 29 or C-terminal amino acid, wherein the natural amino acid is substituted with an amino acid having a side chain suitable for crosslinking with a hydrophilic moiety, including, for example, PEG. The natural peptide may be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non-naturally occurring amino acids refer to amino acids that do not naturally occur in vivo, but which can be incorporated into the peptide structures described herein. Alternatively, an amino acid having a side chain suitable for crosslinking with a hydrophilic moiety (including, e.g., PEG) can be added to the carboxy terminus of any of the glucagon analogs disclosed herein. According to some embodiments, in the glucagon/GLP-1 receptor co-agonist peptide, an amino acid substitution is made at a position selected from 16, 17, 21, 24, or 29, replacing the natural amino acid with an amino acid selected from lysine, cysteine, ornithine, homocysteine, and acetylphenylalanine, wherein the substituted amino acid further comprises a PEG chain covalently attached to a side chain of the amino acid. In some embodiments, the polypeptide is selected from SEQ ID NOs: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18 and SEQ ID NO: 19 to comprise a PEG chain covalently attached to an amino acid side chain at position 17 or 21 of the glucagon peptide. In some embodiments, the pegylated glucagon/GLP-1 receptor co-agonist further comprises SEQ ID NO: 26. SEQ ID NO: 27 or SEQ ID NO: 29.
In another embodiment, the glucagon peptide comprises SEQ ID NO: 55 or SEQ ID NO: 56, further comprising a sequence identical to SEQ ID NO: 55 or SEQ ID NO: 56 to the C-terminal amino acid of SEQ ID NO: 26. SEQ ID NO: 29 or SEQ ID NO: 65 and optionally further comprising a PEG chain covalently attached to the side chain of amino acid 17, 18, 21, 24 or 29 or the C-terminal amino acid of the peptide. In another embodiment, the glucagon peptide comprises SEQ ID NO: 55 or SEQ ID NO: 56, wherein the PEG chain is covalently linked to an amino acid side chain at position 21 or 24 of the glucagon peptide, and the peptide further comprises SEQ ID NO: 26 or SEQ ID NO: 29C-terminal of which protrudes.
In another embodiment, the glucagon peptide comprises SEQ ID NO: 55. SEQ ID NO: 33 or SEQ ID NO: 34, wherein an additional amino acid is added to seq id NO: 33 or SEQ ID NO: 34 and the PEG chain is covalently linked to the side chain of the added amino acid. In yet another embodiment, the pegylated glucagon analog further comprises a sequence that is identical to SEQ ID NO: 33 or SEQ ID NO: 34, C-terminal amino acid of SEQ ID NO: 26 or SEQ ID NO: 29C-terminal of which protrudes. In another embodiment, the glucagon peptide comprises SEQ ID NO: 19, wherein the PEG chain is covalently linked to the side chain of the amino acid at position 30 of the glucagon peptide, and the peptide further comprises a sequence that is complementary to the sequence of SEQ id no: 19 to the C-terminal amino acid of SEQ ID NO: 26 or SEQ ID NO: 29C-terminal of which protrudes.
The polyethylene glycol chain may be in the form of a straight chain, or may be branched. According to some embodiments, the polyethylene glycol chains have an average molecular weight selected from the range of about 500 to about 10,000 daltons. In some embodiments, the polyethylene glycol chains have an average molecular weight range selected from the range of about 1,000 to about 5,000 daltons. In an alternative embodiment, the polyethylene glycol chains have an average molecular weight selected from the range of about 10,000 to about 20,000 daltons. According to some embodiments, the pegylated glucagon peptide comprises two or more polyethylene glycol chains covalently bound to the glucagon peptide, wherein the total molecular weight of the glucagon chains is from about 1,000 to about 5,000 daltons. In some embodiments, the pegylated glucagon agonist comprises a sequence consisting of SEQ ID NO: 5 or SEQ ID NO: 5, wherein the PEG chains are covalently attached to amino acid residues at positions 21 and 24, and wherein the 2 PEG chains have a total molecular weight of about 1,000 to about 5,000 daltons.
In certain exemplary embodiments, the glucagon peptide comprises a glucagon peptide comprising SEQ ID NO: 1, and comprises an acylated or alkylated amino acid at position 10. In some embodiments, the amino acid at position 10 is acylated or alkylated with a C4-C30 fatty acid. In certain aspects, the amino acid at position 10 comprises an acyl or alkyl group that is non-natural to naturally occurring amino acids.
In certain embodiments, the glucagon peptide comprising an acylated or alkylated amino acid at position 10 comprises a stabilized alpha helix. Thus, in certain aspects, the glucagon peptides comprise an acyl or alkyl group and an intramolecular bridge described herein, e.g., a covalent intramolecular bridge (e.g., a lactam bridge) between the side chains of the amino acid at position i and the amino acid at position i +4, wherein i is 12, 16, 20, or 24. Alternatively or additionally, the glucagon peptide comprises an acyl or alkyl group as described herein, and 1, 2, 3 or more of positions 16, 20, 21 and/or 24 of the glucagon peptide is substituted with an α, α -disubstituted amino acid (e.g., AIB). In some cases, the non-native glucagon peptide comprises Glu at position 16 and Lys at position 20, wherein optionally the lactam bridge connects Glu and Lys, and optionally the glucagon peptide further comprises one or more modifications selected from: gln at position 17, Ala at position 18, Glu at position 21, Ile at position 23, and Ala at position 24.
Furthermore, in any embodiment wherein the glucagon peptide comprises an acylated or alkylated amino acid at position 10, the glucagon peptide may further comprise a C-terminal amide in place of the C-terminal alpha carboxylic acid.
In some embodiments, the glucagon peptides comprising an acyl group or an alkyl group described herein further comprise amino acid substitutions at position 1, 2, or 1 and 2, wherein the amino acid substitutions confer DPP-IV protease resistance. For example, the His at position 1 may be substituted with an amino acid selected from: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. Alternatively or additionally, the Ser at position 2 may be substituted with an amino acid selected from: d-serine, alanine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine and aminoisobutyric acid. In some embodiments, the amino acid at position 2 is not D-serine.
A glucagon peptide comprising an amino acid at position 10 acylated or alkylated as described herein may comprise an amino acid sequence substantially identical to SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. For example, the glucagon peptide comprises SEQ ID NO: 1. in certain embodiments, the amino acid sequence of the acylated or alkylated glucagon peptide is identical to SEQ ID NO: 1 (e.g., greater than 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or almost 100% identity to SEQ ID NO: 1). In certain specific embodiments, the glucagon peptide is a peptide comprising the amino acid sequence of SEQ ID NO: 55. The glucagon peptide may be SEQ ID NO: 55(55 has 1 or 2 amino acid modifications), 2-4, 9-18, 20, 23-25, 33, 40-44, 53, 56, 61, 62, 64, 66-514, and 534.
The acyl or alkyl groups of these embodiments may be any of the acyl or alkyl groups described herein. For example, the acyl group can be a C4-C30 (e.g., C8-C24) fatty acyl group, and the alkyl group can be a C4-C30 (e.g., C8-C24) alkyl group.
The amino acid to which the acyl group or alkyl group is attached may be any of the amino acids described herein, for example an amino acid of any of formula I (e.g. Lys), formula II and formula III.
In some embodiments, the acyl group or alkyl group is directly attached to the amino acid at position 10. In some embodiments, the acyl group or alkyl group is linked to the amino acid at position 10 via a spacer, for example a spacer of 3-10 atoms in length, such as an amino acid or dipeptide. Suitable spacers for attachment of acyl or alkyl groups are described herein.
According to some embodiments, the glucagon-like 3-peptide may be an analog of any of the foregoing glucagon-like 3-peptides described herein, which analog has agonist activity at the GIP receptor. The level of activity of the analogs at the glucagon receptor, GLP-1 receptor, and GIP receptor, the potency at each of these receptors, and the selectivity at each of these receptors may be in accordance with the teachings of class 2 glucagon related peptides described herein. See section 2 for teachings of glucagon-related peptide part titled "activity".
In some embodiments of the invention, analogs of glucagon peptides are provided that have agonist activity at the GIP receptor. In certain embodiments, the analog comprises the amino acid sequence of SEQ id no: 1 and the C-terminal 1-21 amino acid extension of amino acid 29.
In certain aspects, the analogs comprise at least one amino acid modification and up to 15 amino acid modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid modifications, up to 10 amino acid modifications). In certain embodiments, the analogs comprise at least one amino acid modification up to 10 amino acid modifications and other conservative amino acid modifications. Conservative amino acid modifications are described herein.
In some aspects, at least one amino acid modification provides a stable alpha helical structure to the C-terminal portion of the analog. Modifications to obtain stable alpha helical structures are described herein. See, e.g., the teaching under the section entitled Stabilization/Intramolecular bridges of alpha helices. In some aspects, the analog comprises an intramolecular bridge (e.g., covalent intramolecular bridge, non-covalent intramolecular bridge) between the 2 amino acid side chains of the analog. In certain aspects, the intramolecular bridge connects the amino acid side chains at positions i and i +4, where i is 12, 13, 16, 17, 20, or 24. In other aspects, the intramolecular bridge connects the amino acid side chains at positions j and j +3, where j is 17, or connects the amino acid side chains at positions k and k +7, where k is any integer between 12 and 22. In certain embodiments, the intramolecular bridge is a covalent intramolecular bridge, such as a lactam bridge. In a particular aspect, the lactam bridge connects the amino acid side chains at positions 16 and 20. In particular aspects, one amino acid at positions 16 and 20 is a positively charged amino acid and the other is a negatively charged amino acid. For example, the analog may comprise a lactam bridge connecting the side chains of Glu at position 16 and Lys at position 20. In other aspects, the negatively charged amino acid and the positively charged amino acid form a salt bridge. In this case, the intramolecular bridge is a non-covalent intramolecular bridge.
In particular aspects, the amino acid modification that provides a stable alpha helix is SEQ ID NO: 1 with an α, α -disubstituted amino acid. Suitable α, α -disubstituted amino acids, including, for example, AIB, for stabilizing an α helix are described herein. In some aspects, seq id NO: 1, 2, 3 or more amino acids at positions 16, 20, 21 and 24 of 1 are substituted with α, α -disubstituted amino acids (e.g., AIB). In a specific embodiment, the amino acid at position 16 is AIB.
Analogs having agonist activity to GIP receptors can comprise other modifications, such as any of the modifications described herein. For example, amino acid modifications can increase or decrease activity at one or both of the GLP-1 receptor and the glucagon receptor. Amino acid modifications may improve the stability of the peptide, e.g. improve resistance to degradation by DPP-IV proteases, stabilize the bond between amino acids 15 and 16. Amino acid modifications can increase the solubility of the peptide and/or alter the time of action of the analog on any of the GIP, glucagon, and GLP-1 receptors. Combinations of any of these modification types may be present in analogs having agonist activity to the GIP receptor.
Thus, in some aspects, the analog comprises SEQ ID NO: 1, the amino acid sequence of which is: gln at position 17, Ala at position 18, Glu at position 21, Ile at position 23, and Ala or Cys at position 24. In some aspects, the analog comprises a C-terminal amide replacing the C-terminal alpha carboxy group. In certain embodiments, the analogs comprise amino acid substitutions at position 1, position 2, or positions 1 and 2 that confer DPP-IV protease resistance. Suitable amino acid substitutions are described herein. For example DMIA in position 1 and/or d-Ser or AIB in position 2. In some embodiments, the amino acid at position 2 is not D-serine.
Additionally or alternatively, the analog may comprise one or a combination of the following modifications: (a) ser at position 2 substituted with Ala; (b) gln at position 3 is substituted with Glu or glutamine analogue; (c) thr at position 7 is substituted by Ile; (d) tyr at position 10 is substituted with Trp or an amino acid comprising an acyl or alkyl group that is non-natural to the naturally occurring amino acid; (e) lys at position 12 is substituted with Ile; (f) asp at position 15 substituted with Glu; (g) ser at position 16 substituted with Glu; (h) gln at position 20 is substituted by Ser, Thr, Ala, AIB; (i) gln at position 24 substituted by Ser, Thr, Ala, AIB; (j) met at position 27 is substituted by Leu or Nle; (k) asn at position 29 is substituted with a charged amino acid (optionally Asp or Glu); and (l) substitution of Thr at position 29 with Gly or a charged amino acid (optionally Asp or Glu).
In certain aspects, the analog does not comprise an amino acid modification at position 1 that provides GIP agonist activity. In some aspects, the amino acid at position 1 is not a large aromatic amino acid, such as Tyr. In some embodiments, the amino acid at position 1 is an amino acid comprising an imidazole ring, e.g., His, an analog of His. In certain embodiments, the analog is not any of the compounds disclosed in U.S. patent application No. 61/151,349. In certain aspects, the analog comprises SEQ ID NO: 657 and 669.
As for analogs having agonist activity to the GIP receptor, the analogs contain an overhang of 1-21 amino acids (e.g., 5-19, 7-15, 9-12 amino acids). The overhang of the analog may comprise any amino acid sequence, provided that the overhang is 1-21 amino acids. In some aspects, the overhang is 7-15 amino acids, while in other aspects, the overhang is 9-12 amino acids. In some embodiments, the overhang comprises (i) SEQ ID NO: 26 or 674, (ii) an amino acid sequence substantially identical to SEQ ID NO: 26 or 674 (ii) having high sequence identity (e.g., at least 80%, 85%, 90%, 95%, 98%, 99%), or (iii) having one or more conservative amino acid modifications.
In some embodiments, at least one amino acid of the overhang is acylated or alkylated. The amino acid comprising an acyl or alkyl group can be located at any position of the analog overhang. In certain embodiments, the acylated or alkylated amino acid of the overhang is at one of positions 37, 38, 39, 40, 41 or 42 (numbering according to SEQ ID NO: 1) of the analog. In certain embodiments, the acylated or alkylated amino acid is located at position 40 of the analog.
In exemplary embodiments, the acyl group or alkyl group is an acyl group or alkyl group that is not native to a naturally occurring amino acid. For example, the acyl or alkyl group can be a C4-C30 (e.g., C12-C18) fatty acyl group or a C4-C30 (e.g., C12-C18) alkyl group. The acyl or alkyl group can be any of the acyl or alkyl groups discussed herein.
In some embodiments, the acyl group or alkyl group is directly attached to the amino acid, e.g., through the side chain of the amino acid. In other embodiments, the acyl group or alkyl group is attached to the amino acid through a spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic bifunctional spacer). In certain aspects, the spacer is 3 to 10 atoms in length. In some embodiments, the amino acid spacer is not γ -Glu. In some embodiments, the dipeptide spacer is not gamma-Glu-gamma-Glu.
Further, in exemplary embodiments, the amino acid to which the acyl group or alkyl group is attached can be any of the amino acids described herein, including, for example, an amino acid of formula I, formula II, or formula III. The acylated or alkylated amino acid may be, for example, Lys. Suitable acyl or alkyl containing amino acids are described herein, as well as suitable acyl and alkyl groups. See, e.g., the teachings under the headings acylation and alkylation.
In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 amino acids) of an overhang are positively charged amino acids, such as amino acids of formula IV, e.g., Lys. The term "positively charged amino acid" as used herein refers to any amino acid, naturally occurring or non-naturally occurring, that comprises a positive charge on its side chain atom at physiological pH. In certain aspects, the positively charged amino acids are located at any of positions 37, 38, 39, 40, 41, 42, and 43. In a specific embodiment, the positively charged amino acid is located at position 40.
In other cases, the overhang is acylated or alkylated as described herein and comprises 1-6 positively charged amino acids as described herein.
In still other embodiments, the analog having agonist activity to a GIP receptor comprises (i) a sequence of SEQ ID NO: 1, (ii) an overhang of 1-21 amino acids (e.g., 5-18, 7-15, 9-12 amino acids) at the C-terminus of analog 29, and (iii) an amino acid comprising an acyl or alkyl group that is non-natural to naturally occurring amino acids, which is located outside the C-terminus overhang (e.g., at any of positions 1-29). In some embodiments, the analog comprises an acylated or alkylated amino acid at position 10. In particular aspects, the acyl or alkyl is a C4-C30 fatty acyl or C4-C30 alkyl. In some embodiments, the acyl group or alkyl group is attached through a spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobic bifunctional spacer). In certain aspects, the analogs comprise amino acid modifications that stabilize the alpha helix, such as a salt bridge between Glu at position 16 and Lys at position 20, or an alpha, alpha-disubstituted amino acid at any 1, 2, 3 or more of positions 16, 20, 21 and 24. In particular aspects, the analogs further comprise amino acid modifications that confer resistance to DPP-IV proteases, e.g., DMIA at position 1, AIB at position 2. Analogs comprising other amino acid modifications are contemplated herein.
In certain embodiments, the analog having GIP receptor activity has at least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%) of the activity of native GIP for the GIP receptor. In some embodiments, the analog has greater than 20% (e.g., greater than 50%, greater than 75%, greater than 100%, greater than 200%, greater than 300%, greater than 500%) of the activity of native GIP for the GIP receptor. In some embodiments, the analogs have appreciable agonist activity at one or both of the GLP-1 and glucagon receptors. In some aspects, the selectivity for these receptors (the GIP receptor and the GLP-1 receptor and/or the glucagon receptor) is within a factor of 1000. For example, an analog having GIP receptor activity can be less than 500-fold, 100-fold, within 50-fold, within 25-fold, within 15-fold, within 10-fold selective for the GLP-1 receptor than for the GIP receptor and/or the glucagon receptor.
In particular aspects, the analog comprises SEQ ID NO: 657 and 669.
According to some embodiments, the glucagon-like peptide type 3 comprises the amino acid sequence of native glucagon (SEQ ID NO: 1) comprising the following modifications: AIB at position 2, Glu at position 3, Lys at position 10, Glu at position 16, Gln at position 17, Ala at position 18, Lys at position 20, Glu at position 21, Ile at position 23, Ala at position 24; wherein Lys at position 10 is acylated with a C14 or C16 fatty acid, and wherein the C terminal carboxylic acid is replaced with an amide. In a specific embodiment, this glucagon-like peptide class 3 is linked via its N-terminal amino acid to the dipeptide D-Lys-sarcosine.
According to some embodiments, the glucagon-like-peptide-3 comprises SEQ ID NO: 70-514, 517-534 or 554 consisting essentially of or consisting of the amino acid sequence of any one of SEQ ID NOs: 70-514, 517-534 or 554, optionally with up to 1, 2, 3, 4 or 5 additional modifications that retain GLP-1 agonist and/or glucagon agonist activity. In certain embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 562 684 and 1701 1776. In some embodiments, the glucagon-like-3-like peptide comprises SEQ ID NO: 1801-1908.
Class 4 glucagon related peptides
In certain embodiments, the glucagon related peptide is a glucagon related peptide class 4 (see, e.g., international (PCT) patent application No. PCT/US2008/080973, incorporated herein by reference in its entirety).
All biological sequences mentioned in the following sections (SEQ ID NO: 1301-: 1-71.
Activity of
According to some embodiments, a class 4 glucagon-related peptide (hereinafter "class 4 peptide") is provided. In certain aspects, class 4 peptides having glucagon antagonist activity are provided. Glucagon antagonists can be used in any situation where inhibition of glucagon agonism is desired. The most direct and obvious use is in the treatment of diabetes to lower blood glucose, where glucagon antagonism has been demonstrated in preclinical models of hyperglycemia. The glucagon antagonists may be further modified to improve the biophysical stability and/or water solubility of the compounds, while maintaining antagonist activity of the parent compound. In certain aspects, the class 4 peptide is defined as a pure glucagon antagonist.
The term "glucagon antagonist" refers to a compound that counteracts glucagon activity or prevents glucagon function. For example, glucagon antagonists inhibit the maximal response obtained by glucagon at the glucagon receptor by at least 60% (e.g., at least 70% inhibition) and preferably at least 80%. In some embodiments, the glucagon antagonist inhibits the maximal response obtained by glucagon at the glucagon receptor by at least 90%. In a specific embodiment, the glucagon antagonist inhibits the maximal response obtained by glucagon at the glucagon receptor by 100%. In addition, glucagon antagonists at a concentration of about 1 μ M exhibit less than about 20% of the maximum agonist activity obtained by glucagon at the glucagon receptor. In some embodiments, the glucagon antagonist has less than about 10% of the maximum agonist activity obtained by glucagon at the glucagon receptor. In a specific embodiment, the glucagon antagonist has less than about 5% of the maximum agonist activity obtained by glucagon at the glucagon receptor. In yet another specific embodiment, the glucagon antagonist has 0% of the maximum agonist activity obtained by glucagon at the glucagon receptor.
A "pure glucagon antagonist" is a glucagon antagonist that does not produce any detectable activation of glucagon or GLP-1 receptor activity as measured by cAMP production using a validated in vitro model assay (see, e.g., PCT/US 2008/080973). For example, a pure glucagon antagonist has less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0%) of the maximal agonist activity obtained by glucagon at the glucagon receptor, and has less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1%, and about 0%) of the maximal agonist activity obtained by GLP-1 at the GLP-1 receptor.
Thus, in some aspects, a class 4 peptide having pure glucagon antagonist activity is provided. According to some embodiments, the glucagon antagonist has activity to reduce glucagon receptor glucagon induced cAMP production by at least 50% of maximum when the glucagon receptor is contacted with 0.8nM glucagon simultaneously with the glucagon antagonist, as determined by cAMP production in an in vitro assay. In some embodiments, the glucagon antagonist reduces glucagon receptor glucagon-induced cAMP production by at least 80% of a maximum amount.
The class 4 peptide is believed to be suitable for any of the uses described previously in relation to glucagon antagonists. Thus, class 4 peptides described herein are useful for treating hyperglycemia or other metabolic diseases caused by elevated glucagon or high blood glucose levels. According to some embodiments, the patient to be treated with a class 4 peptide disclosed herein is a domesticated animal, while in another embodiment, the patient to be treated is a human. Studies have shown that the lack of glucagon inhibition in diabetic patients causes postprandial hyperglycemia in part by accelerating glycogenolysis. Analysis of blood glucose during the Oral Glucose Tolerance Test (OGTT) in the presence or absence of somatostatin-induced glucagon inhibition indicated a significant increase in glucose in subjects with higher glucagon levels. Thus, the class 4 peptides of the present invention are useful for treating hyperglycemia, and are expected to be useful for treating various types of diabetes, including insulin-dependent or non-insulin-dependent type I diabetes, type II diabetes, or gestational diabetes, and alleviating complications of diabetes, including nephropathy, retinopathy, and vasculopathy.
In some embodiments, the terminal 10 amino acids of Exendin-4 (i.e., the sequence of SEQ ID NO: 1319 (GPSSGAPPPS)) are linked to the carboxy-terminus of the class 4 peptide. These fusion proteins are expected to have pharmacological activity for suppressing appetite and inducing weight loss/maintenance. According to some embodiments, the class 4 peptides disclosed herein may be further modified to comprise a peptide sequence identical to SEQ ID NO: 1342, class 4 peptide of SEQ ID NO: 1319 (GPSSGAPPPS) and administered to an individual to induce weight loss or to help maintain weight. More specifically, class 4 peptides comprise a sequence selected from the group consisting of: SEQ ID NO: 1302. SEQ ID NO: 1303. SEQ ID NO: 1304. SEQ ID NO: 1305. SEQ ID NO: 1306. SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1336. SEQ ID NO: 1339. SEQ ID NO: 1340. SEQ ID NO: 1341. SEQ ID NO: 1342. SEQ ID NO: 1343 and SEQ ID NO: 1344 and further comprising SEQ ID NO: 1319 (GPSSGAPPPS) for suppressing appetite and inducing weight loss/maintaining weight. In some embodiments, the administered class 4 peptide comprises SEQ ID NO: 1346 or SEQ ID NO: 1347.
Such methods of reducing appetite or promoting weight loss are expected to be useful for reducing weight, preventing weight gain, or treating various causes of obesity (including drug induced obesity) and reducing complications associated with obesity including vascular disease (coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, type II diabetes onset, hyperlipidemia, and musculoskeletal disease.
The class 4 peptides of the invention may be administered alone or in combination with other antidiabetic or antiobesity agents. Antidiabetic agents known in the art or under investigation include insulin; sulfonylureas, such as tolbutamide (Orinase), acetohexamide (demelor), tolazamide (Tolinase), chlorpropamide (trypticase), glipizide (glicotrol), glyburide (dabinetol), dabrymide (dabeta), glyburide (Micronase), dalianon (Glynase), glimepiride (alamide), or gliclazide (damacron)); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones, such as rosiglitazone (vindia), pioglitazone (Actos) or troglitazone (e.g. forest (Rezulin)) or other PPAR γ inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (acarbose/xylobay); exenatide (Byetta) or pramlintide; dipeptidyl peptidase-4 (DPP-4) inhibitors, such as vildagliptin or sitagliptin; SGLT (sodium dependent glucose transporter 1) inhibitors; or an FBPase (fructose 1, 6-bisphosphatase) inhibitor.
Antiobesity agents known in the art or under investigation include appetite suppressants including phenethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), amfepramoneBenzometrizineBenzphetamineSibutramineRimonabantOther cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (profac); qnexa (topiramate and phentermine); excalia (bupropion and zonisamide) or contextual (bupropion and naltrexone); or a lipase inhibitor similar to cenicrobile (orlistat) or cetilistat (also known as ATL-962) or GT 389-255.
Class 4 peptides of the invention may also be administered to patients with catabolic wasting (catabolic wasting). It is estimated that more than half of cancer patients experience catabolic wasting characterized by involuntary progressive weight loss, weakness, and low body fat and muscle levels. This syndrome is also common in AIDS patients and can also be present in bacterial and parasitic diseases, rheumatoid arthritis and chronic diseases of the intestine, liver, lung and heart. Catabolic wasting is often associated with anorexia and can be manifested as a result of an aging condition or physical trauma. Catabolic wasting is a symptom that reduces quality of life, worsens the underlying condition, and is the leading cause of death. Applicants contemplate that class 4 peptides disclosed herein can be administered to patients to treat catabolic wasting.
Pharmaceutical compositions comprising a class 4 peptide disclosed herein can be formulated and administered to a patient using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also includes pharmaceutical compositions comprising one or more of the class 4 peptides disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise a class 4 peptide as the sole pharmaceutically active ingredient, or the class 4 peptide may be combined with one or more other active agents. According to some embodiments, there is provided a composition comprising a class 4 peptide of the invention and a compound that activates a GLP-1 receptor (e.g., GLP-1 analog, exendin-4 analog, or derivatives thereof). According to some embodiments there is provided a composition comprising a class 4 peptide of the invention and insulin or an insulin analogue. Alternatively, a nucleic acid molecule comprising SEQ id no: 1342, which further comprises the amino acid sequence of SEQ ID NO 1319 (gps gappps) linked to amino acid 24 of SEQ ID NO 1342, and anti-obesity peptides, are provided for use in inducing weight loss or preventing weight gain. Suitable anti-obesity peptides include anti-obesity peptides disclosed in U.S. Pat. Nos. 5,691,309, 6,436,435 or U.S. patent application 20050176643, including but not limited to GLP-1, GIP (gastric inhibitory peptide), MP1, PYY, MC-4, Leptin (Leptin).
Class 4 peptide structures
In some embodiments, a class 4 glucagon-related peptide is provided in which the aspartic acid normally present at position 9 (position 9 of glucagon, SEQ ID NO: 1301) is substituted with a glutamic acid or a cysteic acid-type derivative. More specifically, in some aspects, the first amino acid deletion (des-His) and the aspartic acid at position 9 is substituted with glutamic acid to produce a class 4 peptide. Glucagon-like-4-peptide with a sulfonic acid substituent substituting for the amino acid at position 9 of glucagon behaves like a carboxylic acid type amino acid, but has little critical difference in physical properties (e.g., solubility). Homocysteine (hCysosO) when substituting isostearic acid in position 9 in the conventional des-His, G1u9, class 4 peptide3) Retaining partial antagonist and weak agonist.
In some embodiments, class 4 peptides are provided in which the first 2-5 amino acids are removed and position 9 (numbering according to SEQ ID NO: 1301) is hCys (SO)3) Homoglutamic acid, beta-homoglutamic acid or alkylcarboxylic acid derivatives of cysteine having the following structure to give compounds which act as hormone antagonists with high specificity, efficacy and non-contaminating agonist properties:
Wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4Alkynyl.
According to some embodiments, the polypeptide comprising a sequence identical to SEQ ID NO: 1301 the wild type sequence compared to a class 4 peptide of a glucagon peptide modified by: deletion of 2-5 amino acid residues from the N-terminus and substitution of the aspartic acid residue at position 9 of the native protein with glutamic acid, homoglutamic acid, β -homoglutamic acid, a sulfonic acid derivative of cysteine, or an alkyl carboxylic acid derivative of cysteine having the structure:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4Alkynyl.
In a specific embodiment, the class 4 peptide comprising a deletion of 2-5 amino acid residues from the N-terminus and substitution of Asp 9 of native glucagon is further modified by up to 3 amino acid modifications. Alternatively or additionally, class 4 peptides may comprise 1, 2 or 3 conservative amino acid modifications. Alternatively, class 4 peptides may comprise one or more amino acid modifications selected from:
A. one or two amino acids or the N-terminal or C-terminal amino acid at positions 10, 20 and 24 (amino acid numbering according to SEQ ID NO: 1301) of class 4 peptides are substituted with an amino acid covalently linked to an acyl or alkyl group through an ester, ether, thioether, amide or alkylamine linkage;
B. one or two amino acids or the N-or C-terminal amino acid at positions 16, 17, 20, 21 and 24 (amino acid numbering according to SEQ ID NO: 1301) of class 4 peptides are substituted with an amino acid selected from the group consisting of: cys, Lys, ornithine, homocysteine and acetyl-phenylalanine (Ac-Phe), wherein the amino acid is covalently bound to a hydrophilic moiety;
C. Adding an amino acid covalently bound to a hydrophilic moiety to the N-terminus or C-terminus of the class 4 peptide;
asp at position D.15 (numbering according to SEQ ID NO: 1301) is substituted with cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid;
ser at position E.16 (numbering according to SEQ ID NO: 1301) is replaced by cysteic acid, glutamic acid, homoglutamic acid and homocysteine;
F. according to SEQ ID NO: 1301, amino acid number, one or more AIBs in positions 16, 20, 21 and 24 are substituted;
G. according to SEQ ID NO: number 1301, amino acid at position 29 or deletion of amino acids at positions 28 and 29;
(ii) each or both of Asn at position 28 and Thr at position 29 (amino acid numbering according to SEQ ID NO: 1301) is substituted with a charged amino acid; and/or in SEQ id no: 1301, 1-2 charged amino acids are added at the C terminal;
i.27 (numbering according to SEQ ID NO: 1301) Met is replaced by Leu or norleucine;
J. converting a polypeptide having the sequence of SEQ ID NO: 19-21 and 53 to the amino acid sequence of any one of SEQ ID NOs: end C of 1301; wherein Thr at position 29 (numbering according to SEQ ID NO: 1301) is Thr or Gly; and
K.C the terminal carboxylic acid is replaced by an amide or an ester.
In a specific embodiment, the class 4 peptide comprises amino acid modifications of A, B or C described above, or a combination thereof. In yet another specific embodiment, the class 4 peptide comprises amino acid modifications of any of the above D-K, or combinations thereof, in addition to amino acid modifications of A, B, and/or C.
In some embodiments, the class 4 peptide comprises a glucagon peptide, wherein the first 5 amino acids have been removed from the N-terminus and the remaining N-terminal amino group is replaced with a hydroxyl group ("PLA 6 analog"), resulting in a glucagon peptide of SEQ ID NO: 1339. Applicants have found that substitution of phenyl-lactic acid for phenylalanine in class 4 peptide analogs in which the first 5 amino acids are deleted and the glutamic acid at position 9 (relative to native glucagon) is substituted further increases the potency of those class 4 peptide analogs.
In some embodiments, the amino acid sequence of SEQ ID NO: 1339, class 4 peptide:
wherein X6Is C1-C3Alkyl radical, C2-C3Alkenyl (alkene) or C2-C3Alkynyl, in some embodiments, X6Is C1-C3Alkyl, and in another embodiment, X6Is C2An alkyl group. In some embodiments, the class 4 peptide comprises a glucagon peptide in which the first 5 amino acids have been removed from the N-terminus and the aspartic acid residue at position 4 (position 9 of native glucagon) has been substituted with cysteamine or homocysteine. In some embodiments, the class 4 peptide comprises a glucagon peptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1339. SEQ ID NO: 1307 and SEQ ID NO: 1308 . In some embodiments, the class 4 peptide comprises a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1308, wherein the amino acid in position 4 is homocysteine.
In another embodiment, the amino acid sequence of SEQ ID NO is further modified by substituting the aspartic acid residue at position 4 (position 9 of native glucagon) with glutamic acid, homoglutamic acid, β -homoglutamic acid, or an alkyl carboxylic acid derivative of cysteine having the structure: 1339, class 4 peptide:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4Alkynyl. In a specific embodiment, X5Is C1Or C2An alkyl group.
However, applicants have found that in the case of PLA substitution of the N-terminal phenylalanine in a glucagon analogue lacking 1-5 (i.e. a glucagon analogue lacking the first 5 amino acids), no further substitution of the native aspartic acid residue at position 4 (position 9 of native glucagon) is required to produce an analogue with pure antagonism. This result was unexpected in light of the teachings of the prior art that it was necessary to replace the native aspartic acid residue at position 4 to produce a high affinity, potent antagonist of glucagon (2-29) analogs. Use of PLA substitutions to improve the relative potency of Asp9 analogues to Glu9 and hCys (SO)3H)9 the potency of the analogue is comparable.
Substitution of phenylalanine residues with other phenylalanine analogs, including 3, 4-2F-phenylalanine (3, 4-2F-Phe), 2-naphthylalanine (2-Nal), N-acyl-phenylalanine (Ac-Phe), α -methylhydrocinnamic acid (MCA), and benzylmalonic acid (BMA), is not as effective as substitution of PLA.
PLA substituted at sites other than position 6 (numbered according to the amino acids of native glucagon), including positions 4 and 5, showed that PLA6 analogs were considerably more potent antagonists than glucagon analogs with a slightly extended N-terminus. The invention also includes analogs in which the N-terminal amino group is substituted with an acylated and alkylated "O-terminal" peptide.
In addition, PLA6 substitution not only increases the potency of the antagonist, but also plays a decisive role in pegylation. PLA6 analogs can be selectively pegylated without restoring glucagon agonism. In the absence of PLA substitution, pegylation of the analogs unexpectedly induced glucagon agonism. This glucagon agonism was absent in the pegylated PLA6 analog. Several sites for pegylation were investigated, including positions 3, 6 and 19 (positions 8, 11 and 19 of native glucagon) and the N-terminal amino acid residue. In some embodiments, pegylation is at position 19 (position 24 of native glucagon) because this site shows the most potent and selective glucagon antagonism.
In some embodiments, class 4 peptides comprise the general structure of a-B-C, wherein a is selected from:
(i) phenyl Lactic Acid (PLA);
(ii) oxy derivatives of PLA;
(iii) a 2-6 amino acid peptide, wherein 2 consecutive amino acids of the peptide are linked by an ester or ether bond;
b represents SEQ ID NO: 1301, wherein i is 3, 4, 5, 6 or 7, optionally comprising one or more amino acid modifications selected from:
(iv) asp at position 9 (amino acid numbering according to SEQ ID NO: 1301) is substituted with Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β -homoglutamic acid or an alkyl carboxylic acid derivative of cysteine having the structure:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4Alkynyl.
(v) One or two of the amino acids at positions 10, 20 and 24 (amino acid numbering according to SEQ ID NO: 1301) is substituted with an amino acid covalently linked to an acyl or alkyl group through an ester, ether, thioether, amide or alkylamine linkage;
(vi) one or two amino acids at positions 16, 17, 20, 21 and 24 (according to the amino acid numbering of SEQ ID NO: 1301) are substituted with an amino acid selected from the group consisting of: cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid is covalently linked to a hydrophilic moiety;
(vii) Asp at position 15 (numbering according to SEQ ID NO: 1301) is substituted with cysteamine acid, glutamic acid, homoglutamic acid and homocysteamine acid;
(viii) ser at position 16 (numbering according to SEQ ID NO: 1301) is replaced by cysteic acid, glutamic acid, homoglutamic acid and homocysteine;
(ix) according to SEQ ID NO: 1301, amino acid number, one or more of positions 16, 20, 21 and 24 is substituted with AIB;
and C is selected from:
(x)X；
(xi)X-Y；
(xii) X-Y-Z; and
(xiii)X-Y-Z-R10，
wherein X is Met, Leu or Nle; y is Asn or a charged amino acid; z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetylphenylalanine (Ac-Phe) or a charged amino acid; wherein R10 is selected from SEQ ID NO: 1319-1321 and 1353; and
(xiv) Any one of (x) - (xiii), wherein the C-terminal carboxylic acid is replaced with an amide.
In a particular aspect, the class 4 peptide comprises an oxy derivative of PLA. As used herein, "oxy derivative of PLA" refers to a compound comprising a PLA modified structure in which the hydroxyl group is replaced with an O-R11Replacement of wherein R11Is a chemical moiety. In this regard, the oxy derivative of PLA may be, for example, an ester of PLA or an ether of PLA.
Methods for preparing oxy derivatives of PLA are known in the art. For example, if the oxy derivative is an ester of PLA, the ester can be formed by reacting the hydroxyl group of PLA with a carbonyl group bearing a nucleophile. The nucleophile may be any suitable nucleophile, including but not limited to an amine or a hydroxyl group. Thus, the ester of PLA may comprise the structure of formula IV below:
Formula IV
Wherein R7 is an ester formed when the hydroxyl group of PLA reacts with a nucleophile-bearing carbonyl.
The carbonyl group carrying the nucleophile, which reacts with the hydroxyl group of the PLA to form an ester, can be, for example, a carboxylic acid derivative, or an active ester of a carboxylic acid. The carboxylic acid derivative may be, but is not limited to, an acid chloride, an acid anhydride, an amide, an ester, or a nitrile. The active ester of a carboxylic acid may be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), carbodiimide or hexafluorophosphate. In some embodiments, the carbodiimide is 1, 3-Dicyclohexylcarbodiimide (DCC), 1' -Carbonyldiimidazole (CDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or 1, 3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate salt is selected from benzotriazol-1-yl-oxy-tris (dimethylamino) hexafluorophosphate(BOP), benzotriazol-1-yl-oxytripyrazole hexafluorophosphatePyrrolidinyl radical(PyBOP) 2- (1H-7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethylurea hexafluorophosphate(HATU) and o-benzotriazole-N, N, N ', N' -tetramethyl-urea hexafluorophosphate(HBTU)。
Methods for preparing ethers from reaction with hydroxyl groups (e.g., the hydroxyl groups of PLA) are also known in the art. For example, the hydroxyl group of PLA can react with a haloalkyl or tosylated alkyl alcohol to form an ether linkage.
In general, R11Is a moiety that does not reduce the activity of class 4 peptides. In some embodiments, the chemical moiety increases the activity, stability and/or solubility of the class 4 peptide.
In a particular embodiment, the chemical moiety bound to the PLA through an oxygen-containing linkage (e.g., through an ester or ether linkage) is a polymer (e.g., a polyalkylene glycol), a carbohydrate, an amino acid, a peptide, or a lipid, such as a fatty acid or a steroid.
In a particular embodiment, the chemical moiety is an amino acid, which is optionally a constituent of a peptide, such that formula IV is an depsipeptide. In this aspect, the PLA may be located at a position other than the N-terminal amino acid residue of the class 4 peptide, such that the class 4 peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) amino acids N-terminal to the PLA residue. For example, a class 4 peptide may comprise a PLA at position n, where n is position 2, 3, 4, 5, or 6 of the class 4 peptide.
The amino acid at the N-terminus of the PLA residue may be synthetic or naturally occurring. In a particular embodiment, the amino acid that is the N-terminus of the PLA is a naturally occurring amino acid. In some embodiments, the amino acid that is the N-terminus of PLA is the N-terminal amino acid of native glucagon. For example, class 4 peptides may comprise SEQ ID NO: 1354-1358, wherein PLA is linked to threonine via an ester bond:
SEQ ID NO：1354 His-Ser-Gln-Gly-Thr-PLA
SEQ ID NO：1355 Ser-Gln-Gly-Thr-PLA
SEQ ID NO：1356 Gln-Gly-Thr-PLA
SEQ ID NO：1357 Gly-Thr-PLA
SEQ ID NO：1358 Thr-PLA
In an alternative embodiment, one or more of the N-terminal amino acids may be substituted with an amino acid other than that of native glucagon. For example, if the class 4 peptide comprises PLA as the amino acid at position 5 or 6, the amino acid at position 1 and/or 2 can be an amino acid that reduces susceptibility to cleavage by dipeptidyl peptidase IV. More specifically, in some embodiments, the 1-position of the class 4 peptide is an amino acid selected from the group consisting of: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. More specifically, in some embodiments, position 2 of the antagonist peptide is an amino acid selected from the group consisting of: d-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB). In addition, for example, if the class 4 peptide comprises PLA as the amino acid at position 4, 5 or 6, the amino acid at position 3 of the class 4 peptide may be glutamic acid compared to the native glutamine residue of native glucagon. In an exemplary embodiment of the invention, the class 4 peptide comprises at the N-terminus the amino acid sequence of SEQ ID NO: 1359-1361.
For class 4 peptides comprising compounds of formula IV, the polymer can be any polymer provided that it is reactive with the hydroxyl groups of PLA. The polymer may be a polymer that naturally or normally contains a nucleophile-bearing carbonyl group. Alternatively, the polymer may be a polymer derivatized to contain carbonyl groups bearing carbonyl groups. The polymer may be a derivatized polymer of any one of: a polyamide; a polycarbonate; polyalkylene and derivatives thereof including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates; polymers of acrylates and methacrylates including poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate); polyvinyl polymers including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly (vinyl acetate), and polyvinyl pyrrolidone; polyglycolide; a polysiloxane; polyurethanes and their copolymers; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitro cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and cellulose sulfate; polypropylene; polyethylene, including poly (ethylene glycol), poly (ethylene oxide), and poly (ethyl terephthalate), and polystyrene.
The polymer may be a biodegradable polymer including synthetic biodegradable polymers (e.g., polymers of lactic and glycolic acids, polyanhydrides, polyorthoesters, polyurethanes, poly (butyric acid), poly (valeric acid), and (lactide-caprolactone) copolymers) and natural biodegradable polymers (e.g., alginates and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, etc.; hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), and any copolymers or mixtures thereof. In general, these materials degrade by enzymatic hydrolysis or in vivo exposure to water, by surface or bulk erosion.
The polymer may be a bioadhesive polymer, such as a bioerodible hydrogel (see h.s.sawhney, c.p.pathak and j.a.hubbell, by Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein), poly (hyaluronic acid), casein, gelatin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).
In some embodiments, the polymer is a water soluble polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl amylcellulose, methylcellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly-hydroxyalkyl methacrylates, hydroxymethyl methacrylates, methacrylic acid copolymers, polymethacrylic acid, polymethyl methacrylate, maleic anhydride/methyl vinyl ether copolymers, polyvinyl alcohol, sodium polyacrylate and calcium polyacrylate, Polyacrylic acid, acidic carboxyl polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymers, methyl vinyl ether-maleic anhydride copolymers, carboxymethylamide, potassium methacrylate divinylbenzene copolymers, polyoxyethylene glycols, polyethylene oxide and derivatives, salts and combinations thereof.
In a particular embodiment, the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).
The saccharide can be any saccharide, provided that it comprises or is made to comprise a carbonyl group having an alpha leaving group. For example, the saccharide can be a saccharide derivatized to include a carbonyl group having an alpha leaving group. In this regard, the saccharide can be a derivatized form of a monosaccharide (e.g., glucose, galactose, fructose), disaccharide (e.g., sucrose, lactose, maltose), oligosaccharide (e.g., raffinose, stachyose), polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.
For class 4 peptides comprising a compound of formula IV, the lipid can be any lipid comprising a carbonyl group with an alpha leaving group. For example, the lipid can be a lipid derivatized to include a carbonyl group. In this aspect, the lipid can be a derivative of a fatty acid (e.g., a C4-C30 fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine), a glycerolipid (e.g., mono-, di-, tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., steroid, cholesterol), an isopentenol lipid, a sugar lipid (saccharalid), or a polyketide, an oil, a wax, cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.
In some embodiments, R7 has a molecular weight of about 100kDa or less, e.g., about 90kDa or less, about 80kDa or less, about 70kDa or less, about 60kDa or less, about 50kDa or less, about 40kDa or less. Thus, the molecular weight of R7 may be about 35kDa or less, about 30kDa or less, about 25kDa or less, about 20kDa or less, about 15kDa or less, about 10kDa or less, about 5kDa or less, or about 1 kDa.
In an alternative embodiment, the class 4 peptide comprises as a peptide of 2 to 6 amino acids, wherein 2 consecutive amino acids of the peptide are connected by an ester or ether bond. Ester or ether linkages may be between, for example, amino acids 2 and 3, 3 and 4, 4 and 5, or 5 and 6. Optionally, the peptide may be further modified by covalent attachment to another chemical moiety, including attachment to a polymer (e.g., a hydrophilic polymer), alkylation, or acylation.
For class 4 peptides comprising the general structure a-B-C, B represents an amino acid of native glucagon, e.g., SEQ ID NO: 1301 wherein i is 3, 4, 5, 6 or 7, optionally comprising one or more amino acid modifications. In a specific embodiment, B represents an optionally further modified SEQ ID NO: 1301 amino acids 7 to 26 of.
In some embodiments, B is modified by up to 3 amino acid modifications. For example, the sequences shown in SEQ ID NO: 1301, B of the natural amino acid sequence is modified by one or more conservative amino acid modifications.
In another embodiment, B comprises one or more amino acid modifications selected from (iv) - (ix) described herein. In a specific embodiment, B comprises one or both of amino acid modifications (v) and (vi). In another specific embodiment, B comprises, in addition to (v) and (vi), one or a combination of amino acid modifications selected from (iv), (vii), (viii), and (ix).
In another specific embodiment, the class 4 peptide comprises one or more charged amino acids at the C-terminus. For example, Y and/or Z may be charged amino acids, such as Lys, Arg, His, Asp, and Glu. In yet another embodiment, the class 4 peptide comprises 1-2 charged amino acids (e.g., Lys, Arg, His, Asp, and Glu) at the C-terminus of Z. In a particular aspect, Z followed by 1-2 charged amino acids does not contain R10.
In some embodiments, the class 4 peptide comprises a hydrophilic moiety covalently bound to an amino acid residue of the class 4 peptide, as described herein. For example, according to SEQ ID NO: 1301, class 4 peptides may comprise a hydrophilic moiety covalently attached to the amino acid at position 1, 16, 20, 21 or 24. In another embodiment, the hydrophilic moiety is attached to the C-terminal amino acid of class 4 peptide (which in some cases is 1 or 11 amino acids C-terminal to Z). In yet another embodiment, if A is PLA, PLA-Phe, or PLA-Thr-Phe, the hydrophilic moiety is attached to the PLA, wherein the PLA is modified to include the hydrophilic moiety. In another embodiment, an amino acid comprising a hydrophilic moiety is added to the N-terminus or C-terminus of the class 4 peptide. In another embodiment, class 4 peptides comprise an acyl group or an alkyl group as described herein. For example, according to SEQ ID NO: number 1301, acylation or alkylation may occur outside the amino acid side chain at position 10, 20 or 24. In an alternative embodiment, the acylation or alkylation occurs outside the side chain of the C-terminal amino acid of the class 4 peptide (which in some cases is 1 or 11 amino acids C-terminal to Z). In yet another embodiment, if A is PLA, PLA-Phe or PLA-Thr-Phe, the PLA is modified to contain an acyl group or an alkyl group.
Exemplary embodiments
Class 4 peptides may comprise any synthetic or naturally occurring amino acid, provided that at least 2 consecutive amino acids of the peptide are linked by an ester or ether bond. In a specific embodiment, the peptide comprises an amino acid of native glucagon. For example, the peptide may comprise j-6 of native glucagon (SEQ ID NO: 1301), wherein j is 1, 2, 3, 4 or 5. Alternatively, the peptide may comprise the amino acid sequence set forth in SEQ ID NO: 1301 is based on an amino acid sequence with one or more amino acid modifications at the N-terminus. The amino acids in position 1 and/or 2 are amino acids that reduce the susceptibility to cleavage by dipeptidyl peptidase IV. For example, the peptide may comprise an amino acid at position 1 of the class 4 peptide selected from the group consisting of: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. More specifically, in some embodiments, position 2 of the antagonist peptide is an amino acid selected from the group consisting of: d-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB). Furthermore, for example, the amino acid at position 3 of the class 4 peptide may be glutamic acid, as opposed to the native glutamine residue of native glucagon. Thus, class 4 peptides may comprise the amino acid sequence:
Xaa1-Xaa2-Xaa3-Thr-Gly-Phe(SEQ ID NO：1368)；
Xaa2-Xaa3-Thr-Gly-Phe (SEQ ID NO: 1369); or
Xaa3-Thr-Gly-Phe(SEQ ID NO：1370)；
Wherein Xaa1Selected from: his, D-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine; xaa2Selected from: ser, D-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB); and Xaa3Is Gln or Glu.
The invention also includes embodiments wherein the C-terminal amino acid of the class 4 peptide has an amide group substituted for a carboxylic acid group present on the natural amino acid.
In some embodiments in which class 4 peptide is pegylated, class 4 peptide comprises a truncated glucagon peptide, specifically a 6-29 peptide in which the "N-terminal" amino acid is PLA (phenyl-lactic acid). Such glucagon derivatives have unique advantages. They are more potent peptides than peptides with native N-terminal phenylalanine and inhibit any glucagon agonism caused by pegylation, which is not observed with native phenylalanine. Finally, although the existing literature identifies that substitution of the native aspartic acid at position 9 is required for antagonist activity, applicants have discovered that substitution at PLA is desirable6The unexpected result of not requiring such substitution in the (6-29) glucagon analogues.
In some embodiments, the amino acid of class 4 peptides is substituted with at least one cysteine residue, wherein the side chain of the cysteine residue is further modified with a thiol-reactive reagent, including, for example, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl, and haloacyl. These thiol-reactive reagents may contain carboxyl, keto, hydroxyl and ether groups as well as other hydrophilic moieties such as polyethylene glycol units. In an alternative embodiment, the amino acid of class 4 peptides is substituted with lysine, and the side chain of the substituted lysine residue is further modified using an amine reactive reagent, such as an aldehyde of an active ester of a carboxylic acid (succinimidyl, anhydride, etc.) or a hydrophilic moiety (e.g., polyethylene glycol). According to some embodiments, the lysine residue corresponding to position 12 of the native peptide is substituted with arginine and a single lysine substitution is inserted at one of the amino acids corresponding to position 1, 16, 17, 20, 21, 24 or 29 of the native peptide, or a lysine is added to the N-terminus or C-terminus of the class 4 peptide.
In another embodiment, the methionine residue corresponding to position 27 of the native peptide is changed to leucine or norleucine to prevent oxidative degradation of the peptide.
In some embodiments, the class 4 peptides described herein are further modified by truncation or deletion of one or two amino acids from the C-terminus of the glucagon peptide (i.e., truncation of the amino acids at position 29 or 28 and 29 of native glucagon) without affecting activity and/or potency at the glucagon receptor. In this aspect, a class 4 peptide as described herein can, for example, consist essentially of, or consist of, amino acids 1-27, 1-28, 2-27, 2-28, 3-27, 3-28, 4-27, 4-28, 5-27, 5-28, 6-27, or 6-28 of a native glucagon peptide (SEQ ID NO: 1301) having one or more modifications that result in the activity of a class 4 peptide as described herein.
The class 4 peptides disclosed herein also include amino acid substitutions at positions known to be non-critical to the function of the glucagon peptide. In some embodiments, the substitution is selected from SEQ ID NO: 1339 conservative amino acid substitutions at 1, 2 or 3 of positions 2, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 19, 22, 23 or 24. In some embodiments, the class 4 peptide comprises SEQ ID NO: 1342, wherein the glucagon peptide further comprises an amino acid sequence that is complementary to the sequence of SEQ ID NO: 1342 other amino acid substitutions at 1-3 amino acid positions selected from positions 2, 5, 6, 8, 9, 12, 13 and 14. In some embodiments, in SEQ ID NO: the substitutions at positions 2, 5, 6, 8, 9, 12, 13 and 14 of 1342 are conservative amino acid substitutions. In some embodiments, the amino acid corresponding to position 16, 17, 20, 21, 24, or 29 of the native peptide, more specifically the amino acid at position 21 and/or 24, is substituted with a cysteine or lysine, wherein the PEG chain is covalently attached to the substituted cysteine or lysine residue.
According to some embodiments, the modified class 4 peptide comprises two or more polyethylene glycol chains covalently attached to the peptide, wherein the total molecular weight of the glucagon chain is from about 1,000 to about 5,000 daltons. In some embodiments, the pegylated class 4 peptide comprises a sequence selected from SEQ ID NOs: 1312 and SEQ ID NO: 1322, wherein said peptide comprises a polyethylene glycol chain linked to amino acids at positions 11 and 19, and 2 PEG chains have a total molecular weight of about 1,000 to about 5,000 daltons.
According to some embodiments, there is provided a class 4 peptide comprising a modified glucagon peptide selected from the group consisting of:
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO：1309)，
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Gln-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO：1310)，
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO: 1311) and
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO: 1312), wherein Xaa ═ aspartic acid, glutamic acid, cysteic acid or homocysteine at position 4, Xaa ═ Lys or Arg at position 7, Xaa at position 10 is aspartic acid, cysteic acid, glutamic acidHomoglutamic acid and homocysteic acid; xaa at position 11 is Ser, Lys, Cys, Orn, homocysteine or acetylphenylalanine, Xaa at position 16 is Asp, Lys, Cys, Orn, homocysteine or acetylphenylalanine, Xaa at position 19 is Gln, Lys, Cys, Orn, homocysteine and acetylphenylalanine, Xaa at position 22 is Met, Leu or Nle, R1Is OH or NH2，R2Is COOH or CONH2Wherein the peptide is as set forth in SEQ ID NO: 1309 at position 11 of SEQ ID NO: 1310 at position 16 of SEQ ID NO: 1311 and at position 19 of SEQ ID NO: 1312 with the proviso that if Xaa ═ aspartic acid at position 4, then R is pegylated at positions 16 and 19 of 13121Is OH. According to some embodiments, the peptide comprises SEQ ID NO: 1309. SEQ ID NO: 1310 or SEQ ID NO: 1311, wherein R is1Is OH, R2Is CONH2. In some embodiments, the peptide comprises SEQ ID NO: 1309. SEQ ID NO: 1310 or SEQ ID NO: 1311, wherein R is1Is OH, R2Is CONH2The amino acid at position 4 is aspartic acid, and in yet another embodiment such peptides comprise a peptide comprising SEQ ID NO: carboxy-terminal overhang of 1319 sequence.
According to some embodiments, the peptide comprises a sequence selected from: SEQ ID NO: 1309. SEQ ID NO: 1310. SEQ ID NO: 1313. SEQ ID NO: 1314 and SEQ ID NO: 1316, wherein the peptide is represented in SEQ ID NO: 1309 and SEQ ID NO: 1313, pegylated at position 11, and substituted amino acid sequence of SEQ ID NO: 1310, and pegylation at position 16 of SEQ ID NO: 1310 and SEQ ID NO: 1314 is pegylated at position 19. In some embodiments, the glucagon agonist comprises SEQ ID NO: 1313 or SEQ ID NO: 1314 of the peptide of (b). In some embodiments, the C-terminal amino acid of a class 4 peptide disclosed herein has an amide group replacing a carboxylic acid group present on a natural amino acid. According to some embodiments, the class 4 peptide comprises SEQ ID NO: 1318, or a pharmaceutically acceptable salt thereof.
According to some embodiments, there is provided a class 4 peptide, wherein the plasma protein is covalently linked to an amino acid side chain of the peptide to improve the solubility, stability and/or pharmacokinetics of the glucagon peptide. For example, serum albumin can be covalently bound to a class 4 peptide provided herein. In some embodiments, the plasma protein is covalently bound to an amino acid corresponding to position 16, 17, 20, 21, 24 or 29 of the native glucagon peptide. More specifically, in some embodiments, the plasma protein binds to an amino acid corresponding to position 16 or 24 of the native glucagon peptide, wherein the class 4 peptide comprises the sequence: SEQ ID NO: 1303. SEQ ID NO: 1304. SEQ ID NO: 1305. SEQ ID NO: 1306. SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1309. SEQ ID NO: 1311. SEQ ID NO: 1312. SEQ ID NO: 1322. SEQ ID NO: 1323. SEQ ID NO: 1324. SEQ ID NO: 1325. SEQ ID NO: 1326. SEQ ID NO: 1327. SEQ ID NO: 1328. SEQ ID NO: 1336 and SEQ ID NO: 1339. in some embodiments, the class 4 peptide comprises a peptide selected from the group consisting of: SEQ ID NO: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311 and SEQ ID NO: 1312.
According to some embodiments, there is provided a class 4 peptide, wherein a linear amino acid sequence representing the Fc portion of an immunoglobulin molecule has been covalently linked to an amino acid side chain of a class 4 peptide disclosed herein to improve the solubility, stability and/or pharmacokinetics of the glucagon peptide. For example, an amino acid sequence representing the Fc portion of an immunoglobulin molecule may be covalently bound to the amino acid sequence of SEQ ID NO: 1307. SEQ ID NO: 1339 position 11, 12, 15, 16, 19, 21 or 24 of the glucagon peptide or glucagon analog thereof. In some embodiments, the Fc peptide is covalently bound to SEQ ID NO: 1306. SEQ ID NO: 1307. SEQ ID NO: 1308 or SEQ ID NO: 1336 position 11 or 19 of the class 4 peptide. The Fc portion is usually isolated from IgG, but Fc peptide fragments from any immunoglobulin should function equally. In some embodiments, the glucagon peptide is selected from the group consisting of SEQ ID NO: 1303. SEQ ID NO: 1304. SEQ ID NO: 1305. SEQ ID NO: 1307. SEQ ID NO: 1308 and SEQ ID NO: 1339, wherein the Fc portion is attached to a position corresponding to position 16, 17, 20, 21, 24 or 29 of the native glucagon peptide. In some embodiments, the class 4 peptide comprises a sequence selected from SEQ ID NOs: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311 and SEQ ID NO: 1312, wherein the Fc peptide hybridizes to the sequence of SEQ id no: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311 and SEQ ID NO: 1312 at the two 11 and 19 positions.
In certain embodiments of the invention, the class 4 peptide comprises SEQ ID NO: 1362. amino acid sequence of any one of 1364-1367 and 1371.
Modifications to improve solubility
Class 4 peptides may be further modified to improve the solubility of the peptide in aqueous solution at physiological pH while maintaining glucagon antagonist activity in some aspects. Introduction of hydrophilic groups at the corresponding positions of the native peptide at positions 1, 16, 17, 20, 21, 24 and 29 or at the C-terminus can improve the solubility of the resulting class 4 peptide in solutions having physiological pH while maintaining the parent compound antagonist activity. Thus, in some embodiments, the class 4 peptides disclosed herein are further modified to comprise one or more hydrophilic groups covalently attached to the side chain of an amino acid corresponding to amino acids 1, 16, 17, 20, 21, 24 and 29 of the native glucagon peptide, or the N-or C-terminal amino acid. In yet another embodiment, the amino acid side chains corresponding to amino acids 16 and 24 of the native glucagon peptide are covalently bound to a hydrophilic group, and in some embodiments, the hydrophilic group is polyethylene glycol (PEG).
Applicants have also found that native glucagon can be modified by introducing a charge at its carboxy terminus to increase the solubility of the peptide while maintaining the agonist properties of the peptide. The increased solubility allows the preparation and storage of glucagon solutions at around neutral pH. Formulating glucagon solutions at a relatively neutral pH (e.g., a pH of about 6.0 to about 8.0) improves the long-term stability of class 4 peptides.
Moreover, applicants contemplate that similar modifications can be made to the class 4 peptides disclosed herein to increase their solubility in aqueous solution at relatively neutral pH (e.g., pH of about 6.0 to about 8.0) while maintaining antagonist properties of the parent protein. Accordingly, some embodiments of the invention relate to SEQ ID NO: 1339 which has been further modified relative to the natural amino acids present at positions 6-29 of wild-type glucagon (SEQ ID NO: 1301) by substituting a charged amino acid for the natural uncharged amino acid or adding a charged amino acid to the carboxy terminus, thereby adding charge to the peptide. According to some embodiments, the nucleic acid sequence of SEQ ID NO: 1339, 1-3 uncharged natural amino acids of class 4 peptides are replaced with charged amino acids. In some embodiments, the charged amino acid is selected from the group consisting of lysine, arginine, histidine, aspartic acid, and glutamic acid. More specifically, applicants have discovered that substitution of the corresponding amino acid normally present at position 28 and/or 29 of native glucagon with a charged amino acid, and/or the addition of 1-2 charged amino acids to the carboxy terminus of a class 4 peptide, improves the solubility and stability of the class 4 peptide in aqueous solution at physiologically relevant pH (i.e., pH of about 6.5 to about 7.5). Thus, such modifications of class 4 peptides disclosed herein are expected to have a similar effect on solubility in aqueous solutions, particularly at a pH in the range of about 5.5 to about 8.0, while maintaining the biological activity of the parent peptide.
According to some embodiments, the SEQ ID NO: 1339, class 4 peptide: by substituting the natural amino acid at the position corresponding to the corresponding position 28 and/or 29 of natural glucagon with a negatively charged amino acid (e.g., aspartic acid or glutamic acid) and optionally adding a negatively charged amino acid (e.g., aspartic acid or glutamic acid) to the carboxy terminus of the peptide. In an alternative embodiment, the SEQ ID NO: 1339, class 4 peptide: by substituting the natural amino acid corresponding to position 29 of the natural glucagon with a positively charged amino acid (e.g., lysine, arginine or histidine) and optionally adding one or two positively charged amino acids (e.g., lysine, arginine or histidine) to the carboxy terminus of the peptide. According to some embodiments, there is provided a class 4 peptide having improved solubility and stability, wherein the peptide comprises SEQ ID NO: 1341, with the proviso that SEQ ID NO: 1341, at least one amino acid at position 23 or 24 is substituted with an acidic amino acid, and/or an additional acidic amino acid is added to SEQ ID NO: 1341 carboxyl terminus. In some embodiments, the acidic amino acids are independently selected from Asp, Glu, cysteic acid, and homocysteine.
According to some embodiments, there is provided a class 4 peptide with improved solubility and stability, wherein the antagonist comprises SEQ ID NO: 1341. SEQ ID NO: 1342. SEQ ID NO: 1343 or SEQ ID NO: 1344, wherein at least one amino acid at position 23 or 24 is substituted with a non-natural amino acid residue (i.e., at least one amino acid present at position 23 or 24 of the analog is an acidic amino acid that is different from the amino acid present at the corresponding position of SEQ ID No. 1307). According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: a glucagon agonist of sequence 1341 or 1342, with the proviso that if the amino acid at position 23 is asparagine and the amino acid at position 24 is threonine, the peptide further comprises 1-2 amino acids independently selected from Lys, Arg, His, Asp, or Glu, which are added to the carboxy terminus of the class 4 peptide.
In another embodiment, the amino acid sequence of SEQ ID NO: 1342, and in some embodiments the hydrophilic moiety is linked to the amino acid at position 11, 16 or 19, and in yet another embodiment the hydrophilic moiety is linked to amino acid 19. In some embodiments, the hydrophilic moiety is the Fc portion of a plasma protein or immunoglobulin, while in an alternative embodiment, the hydrophilic moiety is a hydrophilic hydrocarbon chain. In some embodiments, the hydrophilic moiety is polyethylene glycol having a molecular weight selected from the range of about 1,000 to about 5,000 daltons. In another embodiment, the hydrophilic moiety is polyethylene glycol and has a molecular weight of at least about 20,000 daltons. In some embodiments, the polyethylene-modified class 4 peptide comprises SEQ ID NO: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311. SEQ ID NO: 1312. SEQ ID NO: 1343. SEQ ID NO: 1344 or SEQ ID NO: 1345.
Modifications to improve stability
The Asp-Ser sequence at positions 15-16 of native glucagon is identified as the only unstable dipeptide, which leads to early chemical cleavage of the native hormone in aqueous buffer. For example, more than 50% of native glucagon can cleave into fragments when kept in 0.01N HCl at 37 ℃ for 2 weeks. 2 strips released cleaved peptides 1-15 and 16-29 had no glucagon-like biological activity, thus representing a limitation on aqueous pre-formulations of glucagon and related analogs. Selective chemical substitution of Asp with Glu at position 15 of the native glucagon peptide was observed to almost eliminate chemical cleavage of the 15-16 peptide bond.
Thus, it is expected that similar modifications can be made to the class 4 peptides of the invention to reduce their susceptibility to premature chemical cleavage in aqueous buffer. According to some embodiments, the class 4 peptide described herein may be further modified to improve its stability in aqueous solution by replacing the native aspartic acid at position 15 of the native glucagon peptide with an amino acid selected from the group consisting of cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid. According to some embodiments, the nucleic acid sequence of SEQ ID NO: 1339 the aspartic acid residue at position 10 of the class 4 peptide may be substituted with an amino acid selected from the group consisting of cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid, and in some embodiments, the amino acid sequence of SEQ ID NO: 1339 by glutamic acid. According to some embodiments, there is provided a class 4 peptide having improved stability in aqueous solution, wherein the antagonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1336. SEQ ID NO: 1340 and SEQ ID NO: 1342. In yet another embodiment, the class 4 peptide is amidated.
According to some embodiments, increasing stability by reducing degradation of a class 4 peptide described herein may also be achieved by substituting glutamic acid, cysteic acid, homoglutamic acid, or homocysteic acid for serine at position 16 (according to numbering of native glucagon). In a specific embodiment, the serine at position 16 (numbered according to the native glucagon sequence) is replaced with glutamic acid. In a more specific aspect, class 4 peptides comprising such modifications comprise a C-terminal carboxylic acid and are not amidated.
According to some embodiments, there is provided a class 4 peptide comprising a glucagon peptide selected from the group consisting of: SEQ ID NO: 1307. SEQ ID NO: 1336. SEQ ID NO: 1339. SEQ ID NO: 1340. SEQ ID NO: 1341. SEQ ID NO: 1342. SEQ ID NO: 1343 and SEQ ID NO: 1344 which is further modified by one or more further amino acid substitutions at positions corresponding to positions 11, 12, 15, 16, 19 and/or 24 of the native glucagon peptide, wherein said amino acid substitutions comprise substitutions with amino acids having side chains suitable for cross-linking with hydrophilic moieties, including, for example, PEG. The peptide may be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non-naturally occurring amino acids refer to amino acids that are not naturally occurring in vivo, but which can nevertheless be incorporated into the peptide structures described herein. In some embodiments, a class 4 peptide is provided, wherein the peptide comprises SEQ ID NO: 1307. SEQ ID NO: 1336. SEQ ID NO: 1339. SEQ ID NO: 1340. SEQ ID NO: 1341. SEQ ID NO: 1342. SEQ ID NO: 1343 and SEQ ID NO: 1344, and further comprising a polyethylene glycol chain bound to the corresponding 21 or 24 position of the native glucagon peptide. In yet another embodiment, the C-terminus of the class 4 peptide is modified to replace the carboxylic acid group with an amide group.
Fusion peptides and conjugates
The present disclosure also includes a class 4 peptide fusion peptide, wherein the second peptide is fused to the C-terminus of the class 4 peptide. More specifically, the fusion peptide may comprise SEQ ID NO: 1344, further comprising the amino acid sequence of SEQ ID NO: 1319(GPSSGAPPPS), SEQ ID NO: 1320(Lys Arg Asn Arg Asn Asn Ile Ala) or SEQ ID NO: 1321(Lys Arg Asn Arg). In some embodiments, the nucleic acid sequence of SEQ ID NO: 1319(GPSSGAPPPS) is linked by a peptide bond to SEQ ID NO: 1342, amino acid 24 of the class 4 peptide. In another embodiment, the fusion peptide comprises SEQ ID NO: 1307. SEQ ID NO: 1336. SEQ ID NO: 1339. SEQ ID NO: 1340. SEQ ID NO: 1341 or SEQ ID NO: 1343, further comprising the peptide of SEQ ID NO: 1319 (GPSSGAPPPS). In another embodiment, the fusion peptide comprises SEQ ID NO: 1307. SEQ ID NO: 1336. SEQ ID NO: 1337. SEQ ID NO: 1338. SEQ ID NO: 1339. SEQ ID NO: 1341 or SEQ ID NO: 1343, further comprising the peptide of SEQ ID NO: 1320. SEQ ID NO: 1321 or SEQ ID NO: 1353, or a pharmaceutically acceptable salt thereof. In some embodiments, the class 4 peptide fusion peptide comprises a sequence selected from SEQ ID NOs: 1346 and SEQ id no 1347. In yet another embodiment, the C-terminus of the fusion peptide is modified to replace the carboxylic acid group with an amide group.
In some embodiments, a class 4 peptide fusion peptide is provided, wherein the class 4 peptide portion of the fusion peptide is selected from the group consisting of SEQ ID NO: 1303. SEQ ID NO: 1304. SEQ ID NO: 1305. SEQ ID NO: 1306. SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1309. SEQ ID NO: 1311. SEQ ID NO: 1312. SEQ ID NO: 1313. SEQ ID NO: 1314. SEQ ID NO: 1315. SEQ ID NO: 1310. SEQ ID NO: 1316. SEQ ID NO: 1317. SEQ ID NO: 1318 and SEQ ID NO: 1339, SEQ ID NO: 1319 to the carboxy-terminus of the class 4 peptide moiety, and wherein the PEG chain, if present, is selected from the range of 500-40,000 daltons. More specifically, in some embodiments, the class 4 peptide segment is selected from the group consisting of SEQ id nos: 1313. SEQ ID NO: 1314. SEQ ID NO: 1315. SEQ ID NO: 1316. SEQ ID NO: 1346 and SEQ ID NO: 1347, wherein the PEG chain is selected from about 500 to about 5,000 daltons, more particularly, in some embodiments, about 1,000 daltons. In yet another embodiment, the C-terminus is modified to replace the carboxylic acid group with an amide group.
Class 4 peptides may additionally comprise 1-2 charged amino acids added to the carboxy terminus. Wherein 1-2 charged amino acids are added to the amino acid sequence of SEQ ID NO: 1344, the amino acid is a negatively charged amino acid, including, for example, glutamic acid and aspartic acid. In some embodiments, the class 4 peptide comprises SEQ ID NO: 1342, wherein at least one of positions 27 and 28, corresponding to the native glucagon peptide, comprises an amino acid selected from the group consisting of aspartic acid and glutamic acid, and wherein SEQ ID NO: 1342 is optionally modified to include additional 1-2 negatively charged amino acids added to the carboxy terminus. In some embodiments, the negatively charged amino acid is glutamic acid or aspartic acid.
Class 4 peptides disclosed herein can be combined with other active agents, including, for example, insulin, to treat diseases or conditions characterized by excessive glucagon activity. In some embodiments, a class 4 peptide that has been modified to covalently bind to a PEG chain having a molecular weight greater than 10,000 daltons may be administered in combination with insulin to help maintain stable blood glucose levels in a diabetic patient. The class 4 peptide of the present disclosure may be co-administered with insulin as a single composition, administered simultaneously as separate solutions, or the insulin and class 4 peptide may be administered at different times from each other. In some embodiments, the composition comprising insulin and the composition comprising a class 4 peptide are administered within 12 hours of each other. The exact ratio of class 4 peptide to insulin administered will depend in part on the glucagon level of the patient being measured and can be determined by routine experimentation.
Dimeric peptides
The present disclosure also includes multimers of the modified class 4 peptides disclosed herein. Two or more modified class 4 peptides can be linked together using standard linkers and methods known to those skilled in the art. For example, a dimer is formed between two modified class 4 peptides by using a bifunctional thiol crosslinker and a bifunctional amine crosslinker, particularly for class 4 peptides (e.g., SEQ ID NO: 1309, SEQ ID NO: 1310, SEQ ID NO: 1311, and SEQ ID NO: 1312) substituted (e.g., at positions 11, 16, or 19) with cysteine, lysine, ornithine, homocysteine, or acetylphenylalanine residues. The dimer may be a homodimer, or may be a heterodimer. In some embodiments, the dimer is present in a peptide independently selected from SEQ ID NO: 1308. SEQ ID NO: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311. SEQ ID NO: 1312. SEQ ID NO: 1345. SEQ ID NO: 1346 or SEQ ID NO: 1347, wherein the 2 peptides are linked to each other by a linker linked to position 11 of each peptide, position 16 of each peptide, or position 19 of each peptide, or any combination thereof. In some embodiments, the bond is a disulfide bond between Cys11 and Cys11 or Cys19 and Cys19 or Cys11 and Cys19 residues of the respective class 4 peptide.
Similarly, the peptide may be represented in a sequence independently selected from SEQ ID NOs: 1303. SEQ ID NO: 1304. SEQ ID NO: 1305. SEQ ID NO: 1306. SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1309. SEQ ID NO: 1310. SEQ ID NO: 1311. SEQ ID NO: 1312. SEQ ID NO: 1336. SEQ ID NO: 1337. SEQ ID NO: 1338. SEQ ID NO: 1339 and SEQ ID NO: a dimer is formed between 2 of the class 4 peptides of 1342, wherein a bond is formed between amino acid positions independently selected from positions 16, 21 and 24 relative to the native glucagon peptide.
According to some embodiments, there is provided a class 4 peptide dimer comprising 2 class 4 peptides (each comprising the sequence of SEQ id no: 1346), wherein the 2 antagonists are linked to each other by a disulfide bond through the amino acid at position 25. In another embodiment, a class 4 peptide dimer is provided comprising 2 class 4 peptides (each comprising the sequence of SEQ ID NO: 1347) wherein the 2 antagonists are linked to each other by disulfide bonds through the amino acid at position 35. In some embodiments, the polypeptide encoded by SEQ ID NO: 1346 and SEQ ID NO: class 4 peptides of 1347 form dimers in which the amino acid at position 10 is glutamic acid.
In some embodiments, the dimer comprises a homodimer of a class 4 peptide fusion peptide selected from: SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1336. SEQ ID NO: 1337. SEQ ID NO: 1340. SEQ ID NO: 1339. NO: 1340. SEQ ID NO: 1341. SEQ ID NO: 1342 and said class 4 peptide. According to some embodiments, there is provided a dimer comprising a first class 4 peptide bound to a second class 4 peptide by a linker, wherein the first and second peptides of the dimer are independently selected from the group consisting of SEQ ID NO: 1307. SEQ ID NO: 1308. SEQ ID NO: 1336. SEQ ID NO: 1337. SEQ ID NO: 1339. SEQ ID NO: 1340. SEQ ID NO: 1341 and SEQ ID NO: 1342 and pharmaceutically acceptable salts of said glucagon polypeptides. In another embodiment, the first and second class 4 peptides of the dimer are independently selected from the group consisting of SEQ ID NOs: 1307. SEQ ID NO: 1308. SEQ ID NO: 1336 and SEQ ID NO: 1339.
In another embodiment, the dimer comprises a homodimer of a class 4 peptide selected from: SEQ ID NO: 1323. SEQ ID NO: 1324. SEQ ID NO: 1325. SEQ ID NO: 1326. SEQ ID NO: 1327. SEQ ID NO: 1328. SEQ ID NO: 1329. SEQ ID NO: 1330. SEQ ID NO: 1331. in another embodiment, a class 4 peptide dimer is provided, wherein the first and second peptides of the dimer comprise amino acid sequences independently selected from SEQ ID NOs: 1323. SEQ ID NO: 1324. SEQ ID NO: 1325. SEQ ID NO: 1326. SEQ ID NO: 1327 and SEQ ID NO: 1328. In another embodiment, the dimer comprises a sequence selected from SEQ ID NOs: 1309. SEQ ID NO: 1311 and SEQ ID NO: 1312, wherein the peptide further comprises a polyethylene glycol chain covalently bound to the glucagon peptide at position 11 or 19.
The glucagon-like-4-like peptide can comprise SEQ ID NO: the amino acid sequence of any one of 1301-1371, optionally with up to 1, 2, 3, 4 or 5 additional modifications that maintain glucagon antagonist activity.
Class 5 glucagon related peptides
In certain embodiments, the glucagon related peptide is a glucagon related peptide class 5 (see, e.g., international (PCT) patent application No. PCT/US2008/081333, incorporated herein by reference in its entirety).
All biological sequences mentioned in the following sections (SEQ ID NO: 1401-1518) correspond to the sequences of SEQ ID NO: 1-118.
Activity of
In certain aspects, the class 5 glucagon-related peptide (hereinafter "class 5 peptide") may be a glucagon antagonist/GLP-1 agonist. Glucagon antagonists/GLP-1 agonists are used in any setting where inhibition of glucagon agonism is desired while also stimulating GLP-1 activity is desired. For example, glucagon antagonist activity in combination with GLP-1 stimulation may be useful in the treatment of diabetes, where glucagon antagonism has been demonstrated to cause blood glucose lowering and GLP-1 activity is associated with insulin production in a preclinical model of hyperglycemia. Compounds exhibiting GLP-1 activity are also known to be useful for treating obesity and preventing weight gain.
In certain aspects, it is believed that the class 5 peptide is suitable for any of the uses previously described for the other glucagon antagonists/GLP-1 agonists. These two activities have been separately shown to be highly desirable properties for the treatment of metabolic syndrome, in particular diabetes and obesity. Glucagon antagonist activity can be used in any environment where inhibition of glucagon agonism is desired. The presence of GLP-1 agonism also inhibits the endogenous secretion of glucagon from the pancreas, while stimulating insulin synthesis and secretion. The two pharmacological effects act in a synergistic manner to normalize abnormal metabolism. Thus, class 5 peptides are useful for treating hyperglycemia, or other metabolic disorders caused by elevated or high levels of glucagon. According to some embodiments, the patient to be treated with a glucagon antagonist/GLP-1 agonist, such as a class 5 peptide disclosed herein, is a domesticated animal, while in another embodiment, the patient to be treated is a human. Studies have shown that the lack of glucagon inhibition in diabetic patients causes postprandial hyperglycemia in part by accelerating glycogenolysis. Analysis of blood glucose during the Oral Glucose Tolerance Test (OGTT) in the presence or absence of somatostatin-induced glucagon inhibition indicated a significant increase in glucose in subjects with higher glucagon levels. Thus, the glucagon antagonists/GLP-1 agonists or class 5 peptides described herein are useful for treating hyperglycemia, and are expected to be useful for treating various types of diabetes, including insulin-dependent or non-insulin-dependent type I diabetes, type II diabetes, or gestational diabetes, and alleviating the complications of diabetes, including nephropathy, retinopathy, and vasculopathy.
Such methods of reducing appetite or promoting weight loss are expected to be useful for reducing weight, preventing weight gain, or treating various causes of obesity (including drug induced obesity) and reducing complications associated with obesity including vascular disease (coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, type II diabetes onset, hyperlipidemia, and musculoskeletal disease.
Pharmaceutical compositions comprising class 5 peptides can be formulated and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also includes pharmaceutical compositions comprising one or more of the class 5 peptides disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise a class 5 peptide as the sole pharmaceutically active ingredient, or the class 5 peptide may be combined with one or more other active agents. According to some embodiments, there is provided a composition comprising a class 5 peptide and insulin or an insulin analog. Alternatively, a polypeptide comprising SEQ ID NO: 1415 or SEQ ID NO: 1451 and an anti-obesity peptide, for use in inducing weight loss or preventing weight gain, said sequences further comprising a sequence identical to SEQ ID NO: 1415 or SEQ ID NO: 1451 amino acid 24 linked SEQ ID NO: 1421(GPSSGAPPPS) or SEQ ID NO: 1450 amino acid sequence. Suitable anti-obesity peptides include anti-obesity peptides disclosed in U.S. patent 5,691,309, 6,436,435 or U.S. patent application 20050176643.
Class 5 peptide structures
According to some embodiments, there is provided a class 5 peptide comprising a glucagon peptide, said glucagon peptide having been modified by: the alpha helical structure of the C-terminal part of the compound (around amino acids 12-29 according to the amino acid numbering of wild-type glucagon SEQ ID NO: 1401) is stabilized by deletion of the first 1-5 amino acid residues (e.g.amino acid 1, amino acid 2, amino acid 3, amino acid 4, amino acid 5) of the N-terminus and for example linked to each other via hydrogen bonding or ionic interaction (e.g.formation of a salt bridge) or via a covalent bond, by side chains of amino acid pairs selected from the group consisting of amino acid pairs of 12 and 16, 16 and 20, 20 and 24 and 28 (corresponding to the native glucagon peptide sequence). Alternatively, the alpha helix near stabilizing residues 12-29 is achieved by introducing one or more alpha, alpha-disubstituted amino acids at positions that retain the desired activity. In some embodiments, 1, 2, 3, 4 or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 (numbered according to the amino acids of wild-type glucagon) of the class 5 peptide or analog thereof is substituted with an α, α -disubstituted amino acid. For example, substitution of 16 position (according to amino acid numbering of wild-type glucagon) of class 5 peptides or analogs thereof with aminoisobutyric Acid (AIB) in the absence of a salt bridge or lactam provides a stable alpha helix. In some embodiments, 1, 2, 3 or more of positions 16, 20, 21 or 24 (according to the amino acid numbering of wild-type glucagon) are substituted with AIB.
According to some embodiments, a class 5 peptide is provided, wherein the peptide has at least 80% of the maximal agonism obtained by native GLP-1 at the GLP-1 receptor and has glucagon antagonist activity that reduces the maximal glucagon-induced cAMP production at the glucagon receptor by at least about 50% as measured by cAMP production in an in vitro assay. In some embodiments, the class 5 peptide has at least 90% of the activity of native GLP-1 at the GLP-1 receptor and has glucagon antagonist activity that reduces maximal glucagon-induced cAMP production at the glucagon receptor by at least about 80%.
According to some embodiments, the class 5 peptide comprises SEQ ID NO: 1402, wherein the peptide has a sequence that is complementary to a sequence of SEQ ID NO: 1402 further comprises amino acid substitutions at 1-3 amino acid positions selected from the group consisting of positions 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 22, and 24, having at least 90% of the activity of native GLP-1 at the GLP-1 receptor, and having glucagon antagonist activity that reduces maximal glucagon-induced cAMP production at the glucagon receptor by at least about 80%.
In some embodiments, the alpha-helical structure of the C-terminal portion of the class 5 peptide (around amino acids 12-29 according to the amino acid numbering of wild-type glucagon) is stabilized, for example, by forming a covalent or non-covalent intramolecular bridge, or by substituting and/or inserting amino acids near positions 12-29 with alpha helix stabilizing amino acids (e.g., alpha-disubstituted amino acids). In some embodiments, 1, 2, 3, 4 or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 (numbered according to the amino acids of wild-type glucagon) of the class 5 peptide or analog thereof is substituted with an α, α -disubstituted amino acid, such as aminoisobutyric Acid (AIB). For example, substitution of class 5 peptides or analogs thereof at position 16 (according to amino acid numbering of wild-type glucagon) with aminoisobutyric Acid (AIB) in the absence of a salt bridge or lactam provides a stable alpha helix.
In some embodiments, the class 5 peptide comprises SEQ ID NO: 1415 or SEQ ID NO: 1451, more specifically comprising a sequence selected from: SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407. SEQ ID NO: 1408. SEQ ID NO: 1409. SEQ ID NO: 1416. SEQ ID NO: 1417. SEQ ID NO: 1418. SEQ ID NO: 1419. SEQ ID NO: 1422. SEQ ID NO: 1423. SEQ ID NO: 1424 and SEQ ID NO: 1425. in yet another embodiment, the class 5 peptide comprises SEQ ID NO: 1415 or SEQ ID NO: 1451, wherein the peptide has a sequence relative to SEQ ID NO: 1415 or SEQ ID NO: 1451 further comprises amino acid substitutions at 1-3 amino acid positions selected from positions 1, 2, 5, 6, 8, 9, 12, 13 and 14. In some embodiments, the substitutions at positions 1, 2, 5, 6, 8, 9, 12, 13, and 14 are conservative amino acid substitutions. In some embodiments, the nucleic acid sequence of SEQ ID NO: 1405 or SEQ ID NO: 1406 threonine at position 24 is substituted with glycine.
According to some embodiments, class 5 peptides represent further modifications of the peptide, wherein in addition to the N-terminal deletion, the phenylalanine at position 6 of the native glucagon peptide is modified, e.g. to comprise a hydroxyl group in place of the N-terminal amino group. In yet another embodiment, the natural carboxylic acid of the C-terminal amino acid is replaced with a charge neutral group (e.g., amide or ester).
According to some embodiments, a class 5 peptide is prepared in which the first 3-5 amino acids of native glucagon are deleted; the amino acid at position 9 relative to the native glucagon peptide is substituted with an amino acid selected from the group consisting of glutamic acid, homoglutamic acid, β -homoglutamic acid, a sulfonic acid derivative of cysteine, or an alkyl carboxylic acid derivative of cysteine having the structure:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4An alkynyl group; and the alpha-helical structure of the C-terminal portion of glucagon (in the vicinity of amino acids 12-29 according to the amino acid numbering of wild-type glucagon) is stabilized, for example by forming a lactam bridge between the side chains of amino acids 12 and 16 or between amino acids 16 and 20, relative to the native glucagon peptide. Examples of pairs of amino acids capable of covalent bonding to form 7-atom bridges are detailed throughout this disclosure. In some embodiments, the sulfonic acid derivative of cysteine is cysteic acid or homocysteine.
In some embodiments, there is provided a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1405 between the side chains of amino acids 7 and 11 of SEQ ID NO: 1406 between 11 and 15 of SEQ ID NO: between positions 15 and 19 of 1407 and between SEQ ID NO: 1408 and a lactam ring formed between positions 19 and 24, each of said sequences being further modified to comprise a hydrophilic moiety covalently attached to the peptide. More specifically, in some embodiments, each lactam-bearing class 5 peptide is modified by covalent attachment of a polyethylene glycol chain. For example, for a polypeptide comprising SEQ ID NO: 1405, a peptide of class 5, which is pegylated at a position selected from the group consisting of 12, 15, 16, 19 and 24; for a polypeptide comprising SEQ ID NO: 1406 of a class 5 peptide pegylated at a position selected from the group consisting of 12, 16, 19 and 24; for a polypeptide comprising SEQ ID NO: 1407 a peptide of class 5 pegylated at a position selected from the group consisting of 11, 12, 16 and 24; for a polypeptide comprising SEQ ID NO: 1408 a class 5 peptide pegylated at a position selected from 11, 12, 15 and 16. According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 1447 or SEQ ID NO: 1448, wherein the peptide has a sequence relative to SEQ ID NO: 1447 or SEQ ID NO: the 1448 sequence is pegylated at a position selected from the group consisting of 12, 16, 19, and 24. In yet another embodiment, the polypeptide is produced by contacting SEQ ID NO: 1421 to the carboxy terminus of the peptide to compare SEQ ID NO: 1447 or SEQ ID NO: 1448 is further modified.
As detailed above, in certain aspects, a class 5 peptide is provided in which the first 5 amino acids of native glucagon are deleted, the amino group of the N-terminal amino acid (phenylalanine) is replaced with a hydroxyl group (i.e., the first amino acid is phenyl-lactic acid), and the side chains of one or more amino acid pairs selected from positions 12 and 16, 16 and 20, 20 and 24, and 24 and 28 are linked to each other, thereby stabilizing the class 5 peptide alpha helix.
According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 1402, which sequence is encoded by a peptide of SEQ ID NO: the serine residue at position 11 of 1402 (position 16 according to the amino acid numbering of native glucagon) is modified by substitution with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid, homocysteine, threonine or glycine. According to some embodiments, the nucleic acid sequence of SEQ ID NO: the serine residue at position 11 of 1402 is substituted with an amino acid selected from the group consisting of glutamic acid, glutamine, homoglutamic acid, and homocysteic acid, and in some embodiments, the serine residue is substituted with glutamic acid. According to some embodiments, the class 5 peptide comprises SEQ ID NO: 1438.
In some embodiments, a class 5 peptide is provided, wherein an intramolecular bridge is formed between the 2 amino acid side chains to stabilize the amino acid sequence of SEQ ID NO: 1402 peptide three-dimensional structure of the carboxy terminus. More specifically, selected from SEQ ID NOs: 1402 to each other, and side chains of one or more amino acids of the amino acid pairs 7 and 11, 11 and 15, 15 and 19, or 19 and 23, thereby stabilizing the C-terminal part of the alpha helix. The 2 side chains may be linked to each other by hydrogen bonding, ionic interactions (e.g. formation of salt bridges) or by covalent bonds. According to some embodiments, the size of the linker is 7-9 atoms, and in some embodiments, the size of the linker is 8 atoms. In some embodiments, the class 5 peptide is selected from SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 and SEQ ID NO: 1408. in some embodiments, the C-terminal amino acid of the class 5 peptide has an amide group substituted for a carboxylic acid group present on the natural amino acid.
According to some embodiments, there is provided a class 5 peptide, wherein the analog comprises SEQ id no: 1409. In some embodiments, the peptide of SEQ ID NO: 1409 three-dimensional structure of the carboxy terminus of the peptide. In some embodiments, the 2 amino acid side chains are joined to each other to form a lactam ring. The size of the lactam ring may vary with the length of the amino acid side chain, and in some embodiments, the lactam is formed by the linkage of the side chain of a lysine amino acid to a glutamic acid side chain. In some embodiments, the C-terminal amino acid of the class 5 peptide has an amide group substituted for a carboxylic acid group present on the natural amino acid.
The order of the amide bond in the lactam ring may be reversed (e.g., the lactam ring may be formed between the Lys12 and Glu16 side chains or between Glu 12 and Lys 16). According to some embodiments, there is provided SEQ ID NO: 1409 wherein at least one lactam ring is substituted at a position selected from the group consisting of SEQ ID NO: amino acid pair side chains of amino acid pairs 7 and 11, 11 and 15, 15 and 19, or 19 and 23 of 1409. In some embodiments, a class 5 peptide is provided, wherein the peptide comprises SEQ ID NO: 1410, further comprising the sequence set forth in SEQ ID NO: 1410 between the amino acids at positions 7 and 11, or between the amino acids at positions 11 and 15, or between the amino acids at positions 15 and 19. In some embodiments, a class 5 peptide is provided, wherein the peptide comprises SEQ ID NO: 1411, said sequence further comprising the sequence set forth in SEQ ID NO: an intramolecular lactam bridge formed between the amino acids at positions 7 and 11, or between the amino acids at positions 11 and 15 of 1411. In some embodiments, the class 5 peptide comprises SEQ ID NO: 1417.
Providing a polypeptide comprising SEQ ID NO: 1405, wherein the peptide of SEQ ID NO: 1405 aspartic acid at position 10 (position 15 of native glucagon) is substituted with glutamic acid, an amino acid of the general structure:
wherein X6Is C1-C3Alkyl radical, C2-C3Alkylene or C2-C3Alkynyl, in some embodiments, X6Is C1-C3Alkyl, and in another embodiment, X6Is C2An alkyl group. In some embodiments, provided is SEQ ID NO: 1409, wherein the peptide of SEQ ID NO: position 10 of 1409 (position 15 of native glucagon) is substituted with an amino acid selected from the group consisting of glutamic acid, cysteic acid, homocysteine and homoglutamic acid. In yet another embodiment, the nucleic acid sequence of SEQ ID NO: position 10 of 1409 is substituted with an amino acid selected from the group consisting of cysteamine acid and homocysteine acid. In some embodiments, provided is SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408, a class 5 peptide derivative of SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408 is substituted at position 10 with an amino acid selected from glutamic acid, cysteic acid, homocysteine and homoglutamic acid. In some embodiments, the C-terminal amino acid of the class 5 peptide has an amide group substituted for a carboxylic acid group present on the natural amino acid.
In some embodiments, the amino acid of the class 5 peptide is substituted with at least one cysteine residue, wherein the side chain of the cysteine residue is further modified with a thiol-reactive reagent, including, for example, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl, and haloacyl. These thiol-reactive reagents may contain carboxyl, keto, hydroxyl and ether groups as well as other hydrophilic moieties such as polyethylene glycol units. In an alternative embodiment, the amino acid of class 5 peptides is substituted with lysine, and the side chain of the substituted lysine residue is further modified using an amine reactive reagent, such as an aldehyde of an active ester of a carboxylic acid (succinimidyl, anhydride, etc.) or a hydrophilic moiety (e.g., polyethylene glycol). According to some embodiments, the polypeptide corresponding to SEQ ID NO: 1405 the lysine residue at position 7 of the peptide is substituted with arginine and a single lysine substitution is inserted corresponding to SEQ ID NO: 1405 in one of the amino acids at positions 12, 15, 16, 19 and 24.
In another embodiment, the methionine residue corresponding to position 22 of a class 5 peptide disclosed herein is converted to leucine or norleucine to prevent oxidative degradation of the peptide.
In some aspects, the class 5 peptide further comprises amino acid substitutions at positions known to be non-critical to the function of the glucagon analog. In some embodiments, the substitution is a conservative amino acid substitution at 1, 2, or 3 positions selected from 2, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 19, 22, 23, or 24. In some embodiments, the amino acid at position 21 and/or 24 corresponding to native glucagon peptide 16, 17, 20, 21, 24 or 29, more particularly relative to native glucagon, is substituted with a cysteine or lysine, wherein the PEG chain is covalently linked to the substituted cysteine or lysine residue.
According to some embodiments, there is provided a polypeptide comprising the sequence set forth by SEQ ID NO: 1409 by substitution at a position corresponding to position 11, 12, 15, 16, 19 and/or 24 of the peptide with one or more other amino acids (including, for example, substitution with cysteine) so as to be further modified, wherein the amino acid substitution comprises an amino acid having a side chain suitable for crosslinking with a hydrophilic moiety (including, for example, PEG). The native glucagon may be substituted with a naturally occurring amino acid or a synthetic (non-naturally occurring) amino acid. Synthetic or non-naturally occurring amino acids refer to amino acids that are not naturally occurring in vivo, but which can nevertheless be incorporated into the peptide structures described herein. In some embodiments, a class 5 peptide is provided, wherein the peptide comprises SEQ ID NO: 1409 and further comprising a polyethylene glycol chain bound to position 16 or 19 of the peptide. In yet another embodiment, the C-terminus of the glucagon analog is modified to replace the carboxylic acid group with an amide group.
According to some embodiments, there is provided a class 5 peptide comprising a glucagon analog selected from the group consisting of:
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Xaa-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO：1439)
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Gln-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO：1413)，
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO: 1414) and
R1-Phe-Thr-Ser-Xaa-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R2(SEQ ID NO: 1412) wherein Xaa at position 4 is aspartic acid, glutamic acid, cysteic acid or homocysteine, Xaa at position 10 is Asp, Glu, cysteic acid, homoglutamic acid and homocysteine, Xaa at position 16 is Asp, Cys, Orn, homocysteine or acetylphenylalanine, Xaa at position 19 is Gln, Cys, Orn, homocysteine and acetylphenylalanine, Xaa at position 22 is Met, Leu or Nle, R1Is OH or NH2And R is2Is Gly Pro SerSer Gly Ala Pro Pro Pro Ser (SEQ ID NO: 1421), Gly Pro Ser Ser GlyAla Pro Pro Pro Ser Xaa (SEQ ID NO: 1450; wherein Xaa is Cys, Orn, homocysteine or acetylphenylalanine), COOH or CONH2Wherein the peptide is optionally as set forth in SEQ ID NO: at position 16 of 1413SEQ ID NO: 1414 and SEQ ID NO: 1412 at positions 16 and 19. In some embodiments, the nucleic acid sequence of SEQ id no: the Thr at position 24 of 1412-1414 and 1439 was substituted with Gly. According to some embodiments, the peptide comprises SEQ ID NO: 13 or SEQ ID NO: 1414 in which R is1Is OH. According to some embodiments, the peptide comprises SEQ ID NO: 1413 or SEQ ID NO: 1414 in which R is1Is OH, R2Is CONH2. According to some embodiments, the peptide comprises SEQ ID NO: 1413 or SEQ ID NO: 1414 in which R is1Is OH, R2Is CONH2And threonine at position 24 is substituted with glycine.
In some embodiments, the class 5 peptide is further modified to comprise one or more amino acids of native GLP-1 by substitution of the native glucagon residue at the corresponding amino acid position. For example, class 5 peptides may comprise one or more amino acid substitutions at any one of positions 2, 3, 17, 18, 21, 23 and 24 (according to the amino acid numbering of native glucagon). In a specific embodiment, the class 5 peptide is modified by one or more of the following amino acid substitutions: ser2, Gln3, Arg17, Arg at position 18, Asp at position 21, Val at position 23, Ile, and Gln at position 24, are replaced by Ala (amino acid positions correspond to the native glucagon sequence). In a specific embodiment, the class 5 peptide is modified by replacing Ser2 with Ala and Gln3 (according to amino acid numbering of native glucagon) with Glu. In another specific embodiment, the class 5 peptide is modified with all of the following amino acid substitutions: arg17 was replaced by Gln, Arg at position 18 was replaced by Ala, Asp at position 21 was replaced by Glu, Val at position 23 was replaced by Ile and Gln at position 24 was replaced by Ala (according to the amino acid numbering of native glucagon). In yet another specific embodiment, the class 5 peptide is modified to comprise a Glu at position exactly 21 (numbering according to SEQ ID NO: 1401). Thus, class 5 peptides may comprise SEQ ID NO: 1460-1470, 1473-1478, 1480-1488, 1490-1496, 1503, 1504, 1506 and 1514-1518.
Also provided herein is a class 5 peptide or conjugate thereof comprising: (1) an alpha helix stabilized by the methods described herein (e.g., by intramolecular bridges, or incorporation of one or more alpha, alpha-disubstituted amino acids, or acidic amino acids at position 16 (numbering according to SEQ ID NO: 1401), or combinations thereof); (2) a C-terminal amide or ester replacing the C-terminal carboxylic acid, and (3) the general structure of A-B-C,
wherein A is selected from
(i) Phenyl Lactic Acid (PLA);
(ii) oxy derivatives of PLA; and
(iii) a peptide of 2 to 6 amino acids, wherein 2 consecutive amino acids of the peptide are linked by an ester or ether bond;
wherein B represents SEQ ID NO: 1401, wherein p is 3, 4, 5, 6 or 7, optionally comprising one or more amino acid modifications described herein, including, for example, any of the modifications described for class 5 peptides. For example, the one or more modifications may be selected from:
(iv) asp at position 9 (amino acid numbering according to SEQ ID NO: 1401) is substituted with Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β -homoglutamic acid or an alkyl carboxylic acid derivative of cysteine having the structure:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4An alkynyl group;
(v) one or two of the amino acids at positions 10, 20 and 24 (amino acid numbering according to SEQ ID NO: 1401) are substituted with an amino acid covalently linked to an acyl or alkyl group through an ester, ether, thioether, amide or alkylamine linkage;
(vi) One or two of the amino acids at positions 16, 17, 20, 21 and 24 (amino acid numbering according to SEQ ID NO: 1401) is substituted with an amino acid selected from Cys, Lys, ornithine, homocysteine and acetyl-phenylalanine (Ac-Phe), wherein the amino acid is covalently linked to a hydrophilic moiety;
(vii) asp at position 15 (numbering according to SEQ ID NO: 1401) is substituted with cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid;
(viii) ser at position 16 (numbering according to SEQ ID NO: 1401) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteine;
(ix) arg at position 17 by Gln, Arg at position 18 by Ala, Asp at position 21 by Glu, Val at position 23 by Ile, and Gln at position 24 by Ala (amino acid numbering according to SEQ ID NO: 1401);
(x) Replacement of Ser at position 16 with Glu, Gln at position 20 with Glu, or Gln at position 24 with Glu (according to amino acid numbering of SEQ ID NO: 1401);
wherein C (of the general structure A-B-C) is selected from:
(vii)X；
(viii)X-Y；
(ix)X-Y-Z；
(x)X-Y-Z-R10；
wherein X is Met, Leu or Nle; y is Asn or a charged amino acid; z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetylphenylalanine (Ac-Phe) or a charged amino acid; wherein R10 is selected from SEQ ID NO: 1421. 1426, 1427, and 1450.
In a particular aspect, the peptide comprises an oxy derivative of PLA. As used herein, "oxy derivative of PLA" refers to a compound comprising a PLA modified structure in which the hydroxyl group is replaced with an O-R11Replacement of wherein R11Is a chemical moiety. In this regard, the oxy derivative of PLA may be, for exampleAn ester of PLA or an ether of PLA.
Methods for preparing oxy derivatives of PLA are known in the art. For example, if the oxy derivative is an ester of PLA, the ester can be formed by reacting the hydroxyl group of PLA with a carbonyl group bearing a nucleophile. The nucleophile may be any suitable nucleophile, including but not limited to an amine or a hydroxyl group. Thus, the ester of PLA may comprise the structure of formula IV below:
formula IV
Wherein R7 is an ester formed when the hydroxyl group of PLA reacts with a nucleophile-bearing carbonyl.
The nucleophile-bearing carbonyl (which reacts with the hydroxyl group of the PLA to form an ester) can be, for example, a carboxylic acid derivative, or an active ester of a carboxylic acid. The carboxylic acid derivative may be, but is not limited to, an acid chloride, an acid anhydride, an amide, an ester, or a nitrile. The active ester of a carboxylic acid may be, for example, N-hydroxysuccinimide (NHS), tosylate (Tos), carbodiimide or hexafluorophosphate. In some embodiments, the carbodiimide is 1, 3-Dicyclohexylcarbodiimide (DCC), 1' -Carbonyldiimidazole (CDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or 1, 3-diisopropylcarbodiimide (DICD). In some embodiments, the hexafluorophosphate salt is selected from benzotriazol-1-yl-oxy-tris (dimethylamino) hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate(PyBOP) 2- (1H-7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethylurea hexafluorophosphate(HATU) and hexafluoro-phospho-o-benzotriazole-N, N, N ', N' -tetramethyl-urea(HBTU)。
Methods for preparing ethers by reaction with hydroxyl groups (e.g., of PLA) are also known in the art. For example, the hydroxyl group of PLA can react with a haloalkyl or tosylated alkyl alcohol to form an ether linkage.
In a particular embodiment, the chemical moiety bound to the PLA through an oxygen-containing linkage (e.g., through an ester or ether linkage) is a polymer (e.g., a polyalkylene glycol), a sugar, an amino acid, a peptide, or a lipid, such as a fatty acid or a steroid.
In a particular embodiment, the chemical moiety is an amino acid, which is optionally a constituent of a peptide, such that formula IV is an depsipeptide. In this aspect, the PLA may be located at a position other than the N-terminal amino acid residue of the peptide, such that the peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) amino acids N-terminal to the PLA residue. For example, the peptide may comprise PLA at position n, where n is position 2, 3, 4, 5, or 6 of the peptide.
The amino acid at the N-terminus of the PLA residue may be synthetic or naturally occurring. In a particular embodiment, the amino acid that is the N-terminus of the PLA is a naturally occurring amino acid. In some embodiments, the amino acid that is the N-terminus of PLA is the N-terminal amino acid of native glucagon. For example, the peptide may comprise SEQ ID NO: 1452-1456, wherein the PLA is linked to the threonine by an ester bond:
SEQ ID NO：1452 His-Ser-Gln-Gly-Thr-PLA
SEQ ID NO：1453 Ser-Gln-Gly-Thr-PLA
SEQ ID NO：1454 Gln-Gly-Thr-PLA
SEQ ID NO：1455 Gly-Thr-PLA
SEQ ID NO：1456 Thr-PLA
In an alternative embodiment, one or more of the N-terminal amino acids may be substituted with an amino acid other than that of native glucagon. For example, if the peptide comprises PLA as the amino acid at position 5 or 6, the amino acid at position 1 and/or 2 may be an amino acid that reduces susceptibility to cleavage by dipeptidyl peptidase IV. More specifically, in some embodiments, position 1 of the peptide is an amino acid selected from the group consisting of: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. More specifically, in some embodiments, the 2-position of the antagonist/agonist peptide is an amino acid selected from the group consisting of: d-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB). Further, for example, if the peptide comprises PLA as the amino acid at position 4, 5 or 6, the amino acid at position 3 of the peptide may be glutamic acid, as opposed to the native glutamine residue of native glucagon. In an exemplary embodiment of the invention, the peptide comprises at the N-terminus the sequence of SEQ ID NO: 1457-1459.
For peptides comprising compounds of formula IV, the polymer can be any polymer provided that it can react with the hydroxyl groups of PLA. The polymer may be a polymer that naturally or normally contains a nucleophile-bearing carbonyl group. Alternatively, the polymer may be a polymer derivatized to contain carbonyl groups bearing carbonyl groups. The polymer may be a derivatized polymer of any one of: a polyamide; a polycarbonate; polyalkylene and derivatives thereof including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates; polymers of acrylates and methacrylates including poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate); polyvinyl polymers including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly (vinyl acetate), and polyvinyl pyrrolidone; polyglycolide; a polysiloxane; polyurethanes and their copolymers; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitro cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and cellulose sulfate; polypropylene; polyethylene, including poly (ethylene glycol), poly (ethylene oxide), and poly (ethyl terephthalate), and polystyrene.
The polymer may be a biodegradable polymer, including synthetic biodegradable polymers (e.g., polymers of lactic and glycolic acids, polyanhydrides, polyorthoesters, polyurethanes, poly (butyric acid), poly (valeric acid), and (lactide-caprolactone) copolymers) and natural biodegradable polymers (e.g., alginates and other polysaccharides, including dextran and cellulose, collagen, chemical derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, etc.; hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamines and hydrophobic proteins)), as well as any copolymers or mixtures thereof. In general, these materials degrade by enzymatic hydrolysis or in vivo exposure to water, by surface or bulk erosion.
The polymer may be a bioadhesive polymer, such as a bioerodible hydrogel (see h.s.sawhney, c.p.pathak and j.a.hubbell, by Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein), poly (hyaluronic acid), casein, gelatin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).
In some embodiments, the polymer is a water soluble polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl amylcellulose, methylcellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, poly-hydroxyalkyl methacrylates, hydroxymethyl methacrylates, methacrylic acid copolymers, polymethacrylic acid, polymethyl methacrylate, maleic anhydride/methyl vinyl ether copolymers, polyvinyl alcohol, sodium polyacrylate and calcium polyacrylate, Polyacrylic acid, acidic carboxyl polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymers, methyl vinyl ether-maleic anhydride copolymers, carboxymethylamide, potassium methacrylate divinylbenzene copolymers, polyoxyethylene glycols, polyethylene oxide and derivatives, salts and combinations thereof.
In a particular embodiment, the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).
The saccharide can be any saccharide, provided that it comprises or is made to comprise a carbonyl group having an alpha leaving group. For example, the saccharide can be a saccharide derivatized to include a carbonyl group having an alpha leaving group. In this regard, the saccharide can be a derivatized form of a monosaccharide (e.g., glucose, galactose, fructose), disaccharide (e.g., sucrose, lactose, maltose), oligosaccharide (e.g., raffinose, stachyose), polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.
The lipid can be any lipid comprising a carbonyl group having an alpha leaving group. For example, the lipid can be a lipid derivatized to include a carbonyl group. In this aspect, the lipid can be a derivative of a fatty acid (e.g., a C4-C30 fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, an N-acylethanolamine), a glycerolipid (e.g., a mono-, di-, tri-substituted glycerol), a glycerophospholipid (e.g., a phosphatidylcholine, a phosphatidylinositol, a phosphatidylethanolamine, a phosphatidylserine), a sphingolipid (e.g., a sphingosine, a ceramide), a sterol lipid (e.g., a steroid, a cholesterol), an isopentenol lipid, a sugar lipid or polyketide, an oil, a wax, a cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, a phospholipid.
In some embodiments, R7 has a molecular weight of about 100kDa or less, e.g., about 90kDa or less, about 80kDa or less, about 70kDa or less, about 60kDa or less, about 50kDa or less, about 40kDa or less. Thus, the molecular weight of R7 may be about 35kDa or less, about 30kDa or less, about 25kDa or less, about 20kDa or less, about 15kDa or less, about 10kDa or less, about 5kDa or less, or about 1 kDa.
In an alternative embodiment, the peptide comprising the general structure a-B-C comprises as a peptide of 2-6 amino acids, wherein 2 consecutive amino acids of the a peptide are connected by an ester or ether bond. Ester or ether linkages may be between, for example, amino acids 2 and 3, 3 and 4, 4 and 5, or 5 and 6. Optionally, the a peptide may be further modified by covalent attachment to another chemical moiety, including attachment to a polymer (e.g., a hydrophilic polymer), alkylation, or acylation.
In a specific embodiment, the above class 5 peptide comprising PLA is modified to comprise an oxy derivative of PLA, such as an ester of PLA or an ether of PLA. For example, class 5 peptides may comprise SEQ ID NO: 1402. 1405-1420, 1422-1425, 1432-1436, 1438, 1439, 1445, 1446 and 1451, wherein the PLA is linked to the amino acid, peptide, polymer, acyl or alkyl group via an ester or ether bond. The amino acid, peptide, polymer, acyl, or alkyl group can be any of those described herein. In the case where PLA is linked to an amino acid or peptide by an ester bond, the class 5 peptide may be considered an depsipeptide.
Furthermore, in another specific embodiment, the above class 5 peptide without PLA is modified to contain at least one ester or ether bond between 2 consecutive amino acids N-terminal to amino acid position 7 (according to numbering of native glucagon). In a specific embodiment, the class 5 peptide comprises at least one ester or ether bond between 2 consecutive amino acids. In a more specific embodiment, the class 5 peptide comprises SEQ ID NO: 1401N-terminal 6 amino acids, and 2 consecutive amino acids among the N-terminal 6 amino acids are linked by an ester bond or an ether bond.
The a peptide may comprise any synthetic or naturally occurring amino acid, provided that at least 2 consecutive amino acids are linked by an ester or ether bond. In a specific embodiment, the a peptide comprises the amino acids of native glucagon. The amino acid at position 1 and/or 2 can be an amino acid that decreases susceptibility to cleavage by dipeptidyl peptidase IV. For example, the a peptide may comprise an amino acid at position 1 selected from: d-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine. More specifically, in some embodiments, position 2 of the a peptide is an amino acid selected from the group consisting of: d-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB). Further, for example, the amino acid at position 3 of the a peptide may be glutamic acid, as opposed to the native glutamine residue of native glucagon. Thus, a peptide of the general structure a-B-C may comprise the following amino acid sequence:
Xaa1-Xaa2-Xaa3-Thr-Gly-Phe(SEQ ID NO：1507)；
Xaa2-Xaa3-Thr-Gly-Phe (SEQ ID NO: 1508); or
Xaa3-Thr-Gly-Phe(SEQ ID NO：1509)；
Wherein Xaa1Selected from: his, D-histidine, α -dimethylimidazolium acetate (DMIA), N-methylhistidine, α -methylhistidine, imidazolium acetate, deaminated histidine, hydroxy-histidine, acetyl-histidine and homohistidine; xaa2Selected from: ser, D-serine, D-alanine, valine, glycine, N-methylserine, N-methylalanine and aminoisobutyric Acid (AIB); xaa3Is Gln or Glu.
In some embodiments, B is modified by up to 3 amino acid modifications. For example, the sequences shown in SEQ ID NO: 1401 is modified by one or more conservative amino acid modifications.
In another embodiment, B comprises one or more amino acid modifications selected from (iv) - (x) described herein. In a specific embodiment, B comprises one or both of amino acid modifications (v) and (vi). In a further specific embodiment, B comprises, in addition to (v) and (vi), one or a combination of amino acid modifications selected from (iv), (vii), (viii), (ix), and (x).
As described herein, a peptide comprising the general structure a-B-C may comprise one or more charged amino acids on the C-terminus, e.g., as Y and/or Z as described herein. Alternatively or additionally, if C comprises X-Y-Z, a peptide comprising the general structure A-B-C may additionally comprise 1-2 charged amino acids C-terminal to Z. The charged amino acid can be, for example, one of Lys, Arg, His, Asp, and Glu. In a particular embodiment, Y is Asp.
In some embodiments, the peptide comprising the general structure A-B-C comprises a hydrophilic moiety covalently bound to an amino acid residue at position 1, 16, 20, 21, or 24 (amino acid numbering according to SEQ ID NO: 1401) or an N-or C-terminal residue of the peptide comprising the general structure A-B-C. In a specific embodiment, the hydrophilic moiety is linked to a Cys residue of a peptide comprising the general structure A-B-C. In this aspect, the amino acid at position 16, 21, 24 or 29 of native glucagon (SEQ ID NO: 1401) may be substituted with a Cys residue. Alternatively, for example, if the peptide comprising the general structure A-B-C comprises a C-terminal overhang, a Cys residue comprising a hydrophilic moiety may be added to the C-terminal of the peptide comprising the general structure A-B-C as position 30 or as position 40 (position according to amino acid numbering of SEQ ID NO: 1401). Alternatively, the hydrophilic moiety can be linked to the PLA comprising the peptide of the general structure A-B-C through the hydroxyl moiety of the PLA. The hydrophilic moiety can be any hydrophilic moiety described herein, including, for example, polyethylene glycol.
In a particular aspect, a peptide comprising the general structure a-B-C comprises an alpha helix stabilized by incorporation of an intramolecular bridge. In some embodiments, the intramolecular bridge is a lactam bridge. The lactam bridge may be between amino acids 9 and 12, 12 and 16, 16 and 20, 20 and 24 or 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1401). In a specific embodiment, the amino acids at positions 12 and 16 or 16 and 20 (amino acid numbering according to SEQ ID NO: 1401) are linked via a lactam bridge. Other positions of the lactam bridge are contemplated.
Additionally or alternatively, a peptide comprising the general structure A-B-C may comprise an alpha, alpha disubstituted amino acid at, for example, any of positions 16, 20, 21 or 24 (according to the amino acid numbering of SEQ ID NO: 1401). In some embodiments, the α, α -disubstituted amino acid is AIB. In a specific aspect, the AIB is located at position 16 (numbering according to SEQ ID NO: 1401).
Alternatively or additionally, peptides comprising the general structure A-B-C may be modified to include an acidic amino acid at position 16 (according to the numbering of SEQ ID NO: 1401), which modification increases the stability of the alpha helix. In some embodiments, the acidic amino acid is an amino acid comprising a side chain sulfonic acid or a side chain carboxylic acid. In a more specific embodiment, the acidic amino acid is selected from the group consisting of Glu, Asp, homoglutamic acid, a sulfonic acid derivative of Cys, cysteic acid, homocysteic acid, Asp, and alkylated derivatives of Cys having the structure:
wherein X5Is C1-C4Alkyl radical, C2-C4Alkenyl or C2-C4Alkynyl.
In a specific embodiment, the class 5 peptide may comprise SEQ ID NO: an amino acid sequence of any one of 1460-1470, 1473-1478, 1480-1488, 1490-1496, 1503, 1504, 1506 and 1514-1518, or an amino acid sequence comprising any one of: peptides 2-6 of table 13, peptides 1-8 of table 14, and peptides 2-6, 8, and 9 of table 15.
In some embodiments, the peptide comprising the general structure a-B-C is a class 5 peptide. In a specific embodiment, the peptide has at least about 50% of the maximal agonism achieved by native GLP-1 at the GLP-1 receptor and at least about 50% inhibition of the maximal response achieved by native glucagon at the glucagon receptor. In another specific embodiment, the peptide has at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the maximal agonism obtained by native GLP-1 at the GLP-1 receptor. Alternatively or additionally, the peptide may have at least about 55%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% inhibition of the maximal response obtained for the glucagon receptor to native glucagon.
In some embodiments, there is provided a peptide having a class 5 peptide or a conjugate thereof comprising:
(1) modifications that confer glucagon antagonist activity include, but are not limited to:
(a) phe at position 6 is replaced with PLA (numbered according to amino acids of wild-type glucagon), optionally with 1-5 amino acids deleted from the N-terminus of wild-type glucagon; or
(b) Deletion of 2-5 amino acids from the N-terminus of wild-type glucagon; optionally substituting Asp at position 9 of wild-type glucagon with a sulfonic acid derivative of glutamic acid, homoglutamic acid or cysteine (numbering according to amino acids of wild-type glucagon); and
(2) modifications that confer GLP-1 agonist activity include, but are not limited to:
(a) insertion or substitution of an α, α -disubstituted amino acid within amino acids 12-29 of wild type glucagon, for example at 1, 2, 3, 4 or more of positions 16, 17, 18, 19, 20, 21, 24 or 29 (numbering according to amino acids of wild type glucagon); or
(b) Introducing an intramolecular bridge, such as a salt bridge or a lactam bridge or another type of covalent bond, within amino acids 12-29 of wild-type glucagon; or
(c) Substitution of an amino acid at one or more of positions 2, 3, 17, 18, 21, 23 or 24 (according to the amino acid numbering of native glucagon) with the corresponding amino acid of GLP-1, e.g. substitution of Ser2 with Ala, substitution of Gln3 with Glu, substitution of Arg17 with Gln, substitution of Arg at position 18 with Ala, substitution of Asp at position 21 with Glu, substitution of Val at position 23 with Ile and/or substitution of Gln at position 24 with Ala; or
(d) Additional modifications of the alpha helical structure near positions 12-29 of the stabilizing amino acid (numbered according to amino acids of wild-type glucagon);
And
(3) other modifications to enhance GLP-1 agonist activity, e.g.
(a) C-terminal amide or ester instead of C-terminal carboxylic acid; and optionally
(4) One or more of the following modifications:
(a) covalently linked to a hydrophilic moiety (e.g. polyethylene glycol), e.g. at the N-terminus, or at position 6, 16, 17, 20, 21, 24, 29, 40 or at the C-terminal amino acid; and/or
(b) Acylation or alkylation; and optionally
(5) One or more of the following additional modifications:
(a) covalent attachment of an amino acid to the N-terminus (e.g., 1-5 amino acids to the N-terminus), optionally via an ester linkage to PLA at position 6 (according to numbering of wild-type glucagon), optionally together with, for example, a modification at position 1 or 2 that improves resistance to DPP-IV cleavage as described herein;
(b) amino acid deletions at positions 29 and/or 28 and optionally 27 (according to numbering of wild-type glucagon);
(c) the amino acid is covalently linked to the C-terminus;
(d) non-conservative substitutions, additions or deletions while retaining the desired activity, e.g. conservative substitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28 or 29, substitution of Tyr at position 10 by Val or Phe, substitution of Lys at position 12 by Arg, substitution of one or more of these positions by Ala;
(e) Modification of the aspartic acid at position 15, for example by substitution with glutamic acid, homoglutamic acid, cysteic acid, or homocysteic acid, which can reduce degradation; or serine at position 16, for example by substitution of threonine, AIB, glutamic acid or another negatively charged amino acid with a side chain of 4 atoms in length, or with any of glutamine, homoglutamic acid or homocysteine, which likewise may also reduce degradation due to cleavage of the Asp15-Ser16 bond;
(f) modification of the methionine at position 27, for example by substitution with leucine or norleucine, to reduce oxidative degradation;
(g) modification of Gln at positions 20 or 24, e.g. by substitution with Ala or AIB to reduce degradation by deamidation of Gln;
(h) modification of the Asp at position 21, for example by substitution with Glu, to reduce degradation due to dehydration of the Asp to form a cyclic succinimide intermediate followed by isomerization to isoaspartic acid;
(j) homo-or heterodimerization as described herein; and
(k) combinations of the above.
It is to be understood that any of the modifications in the same class may be combined together and/or combinations of modifications from different classes. For example, the modification of (1) (a) can be combined with (2) (a) and (3); (1) (a) may be combined with (2) (b) (e.g., a lactam bridge or a salt bridge) and (3); (1) (a) may be combined with (2) (c) and (3); (1) (b) may be combined with (2) (a) and (3); (1) (b) may be combined with (2) (b) (e.g., a lactam bridge or a salt bridge) and (3); (1) (b) may be combined with (2) (c) and (3); any of the above may be combined with (4) (a) and/or (4) (b); and any of the foregoing may be combined with any of (5) (a) - (5) (k).
In exemplary embodiments, the α, α -disubstituted amino acid AIB is substituted at position 16, 20, 21 or 24 (according to the amino acid numbering of wild-type glucagon) at position 1, 2, 3 or all.
In exemplary embodiments, the intramolecular bridge is a salt bridge.
In other exemplary embodiments, the intramolecular bridge is a covalent bond, such as a lactam bridge. In some embodiments, the lactam bridge is located between amino acids 9 and 12, amino acids 12 and 16, amino acids 16 and 20, amino acids 20 and 24, or amino acids 24 and 28 (according to the amino acid numbering of SEQ ID NO: 1401).
In exemplary embodiments, acylation or alkylation (according to amino acid numbering of wild-type glucagon SEQ ID NO: 1401) at position 6, 10, 20 or 24, or at the N-or C-terminus.
In exemplary embodiments, the modifications include:
(i) asp at position 15 (numbering according to SEQ ID NO: 1401) is substituted with cysteic acid, glutamic acid, homoglutamic acid and homocysteic acid;
(ii) ser at position 16 (numbering according to SEQ ID NO: 1401) is substituted with cysteic acid, glutamic acid, homoglutamic acid, and homocysteine;
(iii) (ii) Asn at position 28 is substituted with a charged amino acid;
(iv) Asn at position 28 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine;
(v) (xiii) substitution at position 28 with Asn, Asp or Glu;
(vi) asp at position 28;
(vii) substituted at position 28 with Glu;
(viii) thr at position 29 is substituted with a charged amino acid;
(ix) thr at position 29 is substituted with a charged amino acid selected from Lys, Arg, His, Asp, Glu, cysteic acid and homocysteine;
(x) Asp, Glu or Lys at position 29;
(xi) Substituted at position 29 with Glu;
(xii) 1-3 charged amino acids are inserted after position 29;
(xiii) Glu or Lys after position 29;
(xiv) Gly-Lys or Lys-Lys after position 29;
or a combination thereof.
Any of the modifications described above to enhance GLP-1 receptor agonist activity, glucagon receptor antagonist activity, peptide solubility and/or peptide stability may be used alone or in combination.
Modifications to improve stability
According to some embodiments, the class 5 peptides disclosed herein may be further modified to include a peptide of SEQ ID NO: 1421(GPSSGAPPPS) or SEQ ID NO: 1450 and administered to the individual to cause weight loss or to help maintain weight. More specifically, class 5 peptides comprise a sequence selected from the group consisting of: SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407. SEQ ID NO: 1408. SEQ ID NO: 1409. SEQ ID NO: 1412. SEQ ID NO: 1413. SEQ ID NO: 1414. SEQ ID NO: 1416. SEQ ID NO: 1417. SEQ ID NO: 1418. SEQ ID NO: 1419. SEQ ID NO: 1422. SEQ ID NO: 1423. SEQ ID NO: 1424 and SEQ ID NO: 1425 and further comprises the amino acid sequence of SEQ ID NO: 1421(GPSSGAPPPS) or SEQ ID NO: 1450 for suppressing appetite and inducing weight loss/maintenance. In some embodiments, the administered peptide or class 5 peptide comprises a sequence selected from SEQ ID NO: 1416. SEQ ID NO: 1417. SEQ ID NO: 1418 and SEQ ID NO: 1419, further comprising the sequence of SEQ ID NO: 1421 (GPSSGAPPPS). In some embodiments, the method comprises administering a peptide comprising SEQ ID NO: 1445 or SEQ ID NO: 1446 or a class 5 peptide.
Thus, it is expected that similar modifications can be made to the class 5 peptides disclosed herein to reduce their susceptibility to premature chemical cleavage in aqueous buffers. According to some embodiments, the class 5 peptides described herein may be further modified to increase their stability in aqueous solution by replacing the native aspartic acid at the corresponding 15-position of native glucagon with an amino acid selected from the group consisting of cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid. According to some embodiments, the nucleic acid sequence of SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408 the aspartic acid residue at position 10 of the class 5 peptide may be substituted with an amino acid selected from the group consisting of cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid, and in some embodiments, the amino acid sequence of SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408 replacement of the native aspartic acid at position 10 with glutamic acid. According to some embodiments, there is provided a class 5 peptide having improved stability in aqueous solution, wherein the antagonist comprises SEQ ID NO: 1409, wherein the modification comprises substitution of Glu for SEQ ID NO: asp at position 1409. In some embodiments, there is provided a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1422. SEQ ID NO: 1423. SEQ ID NO: 1424 and SEQ ID NO: 1425 sequence of the peptide of class 5. In some embodiments, the class 5 peptide is amidated.
The Asp-Ser sequence at positions 15-16 of native glucagon is identified as the only unstable dipeptide, which leads to early chemical cleavage of the native hormone in aqueous buffer. For example, more than 50% of native glucagon can be cleaved into fragments when kept in 0.01N HCl for 2 weeks at 37 ℃. The 2 strips of released cleaved peptides 1-15 and 16-29 have no glucagon-like biological activity and thus represent a limitation of aqueous pre-formulations of glucagon and related analogs. Selective chemical substitution of Asp by Glu at position 15 of native glucagon is observed to almost eliminate chemical cleavage of the 15-16 peptide bond.
In yet further exemplary embodiments, any of the foregoing compounds may be further modified to improve stability by modifying the amino acid corresponding to position 15 or 16 of native glucagon to reduce degradation of the peptide over time, particularly in acidic or basic buffers.
Modifications to improve solubility
In certain aspects, class 5 peptides can be further modified to improve the solubility of the peptide in aqueous solution at physiological pH while maintaining glucagon antagonist and GLP-1 agonist activity. In a sequence corresponding to SEQ ID NO: 1405 at positions 12, 15, 16, 19 and 24 of the peptide of SEQ ID NO: 1406, introducing a hydrophilic group at position 12, 16, 19 or 24 of the peptide, can improve the solubility of the resulting peptide in solutions having physiological pH while maintaining the parent compound glucagon antagonist and GLP agonist activity. Thus, in some embodiments, the presently disclosed class 5 peptides are further modified to comprise an amino acid sequence corresponding to SEQ ID NO: 1405 or SEQ ID NO: 1406 amino acid side chains at amino acids 12, 15, 16, 19 and 24 of the peptide are covalently attached to one or more hydrophilic groups. In yet another embodiment, the polypeptide corresponding to SEQ ID NO: 1405 or SEQ ID NO: the amino acid side chains at amino acids 16 and 19 of 1406 are covalently bound to a hydrophilic group, and in some embodiments, the hydrophilic group is polyethylene glycol (PEG).
The class 5 glucagon-related peptides can be modified by introducing a charge at their carboxy terminus to increase the solubility of the peptide while maintaining the agonist properties of the peptide. The increased solubility allows the glucagon solution to be prepared and stored at near neutral pH. Formulating glucagon solutions at a relatively neutral pH (e.g., a pH of about 6.0 to about 8.0) increases the long-term stability of class 5 peptides.
Applicants contemplate that, in certain instances, similar modifications may be made to the class 5 peptides disclosed herein to increase their solubility in aqueous solution at relatively neutral pH (e.g., pH of about 6.0 to about 8.0) while maintaining glucagon antagonist and GLP-1 activity. Thus, some embodiments relate to SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408 glucagon antagonist/GLP-1 which has further modified the natural amino acids present at positions 6-29 relative to wild type glucagon (SEQ ID NO: 1401) by substituting the natural uncharged amino acid with a charged amino acid, or adding a charged amino acid to the carboxy terminus, to add charge to the peptide. According to some embodiments, 1-3 uncharged natural amino acids of a class 5 peptide disclosed herein are substituted with charged amino acids. In some embodiments, the charged amino acid is selected from the group consisting of lysine, arginine, histidine, aspartic acid, and glutamic acid. More specifically, applicants have discovered that substitution of the normally occurring amino acids corresponding to positions 28 and/or 29 (relative to native glucagon) with charged amino acids, and/or addition of 1-2 charged amino acids at the carboxy terminus of the peptide, improves the solubility and stability of class 5 peptides in aqueous solutions at physiologically relevant phs (i.e., pH of about 6.5 to about 7.5). Thus, such modifications of class 5 peptides are expected to have a similar effect on solubility in aqueous solutions, particularly at a pH in the range of about 5.5 to about 8.0, while maintaining the biological activity of the parent peptide.
According to some embodiments, the nucleic acid sequence of SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408, modified with a peptide of class 5: by substituting those natural amino acids at positions 23 and/or 24 of the sequence with negatively charged amino acids (e.g., aspartic acid or glutamic acid), and optionally adding a negatively charged amino acid (e.g., aspartic acid or glutamic acid) to the carboxy terminus of the peptide. In an alternative embodiment, the amino acid sequence of SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408, and optionally one or two positively charged amino acids (e.g., lysine, arginine, or histidine) are added to the carboxy terminus of the peptide to react the amino acid sequence comprising SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407 or SEQ ID NO: 1408, modified. According to some embodiments, there is provided a class 5 peptide with improved solubility and stability, wherein the analogue comprises SEQ ID NO: 1415 or SEQ ID NO: 1451 with the proviso that SEQ ID NO: 1415 or SEQ ID NO: at least one amino acid at position 23 or 24 of 1451 is substituted with an acidic amino acid and/or a further acidic amino acid is added to the amino acid sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451 carboxyl terminal. In some embodiments, the acidic amino acids are independently selected from Asp, Glu, cysteic acid, and homocysteine.
According to some embodiments, there is provided a class 5 peptide with improved solubility and stability, wherein the antagonist comprises SEQ ID NO: 1416. SEQ ID NO: 1417. SEQ ID NO: 1418 or SEQ ID NO: 1419. According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 1416 or SEQ ID NO: a glucagon agonist of sequence 1417. In some embodiments, the class 5 peptide comprises SEQ ID NO: 1420.
According to some embodiments, there is provided a polypeptide comprising SEQ ID NO: 1415 or SEQ ID NO: 1451. In some embodiments, the nucleic acid sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451 is aspartic acid, glutamic acid, homoglutamic acid, cysteic acid, or homocysteic acid, and in some embodiments, position 4 is aspartic acid, glutamic acid, cysteic acid, or homocysteic acid, and in yet another embodiment, SEQ ID NO: 1415 or SEQ ID NO: position 4 of 1451 is aspartic acid or glutamic acid, and in some embodiments, SEQ ID NO: 1415 or SEQ ID NO: aspartic acid at position 4 of 1451. In some embodiments, there is provided a nucleic acid comprising SEQ ID NO: 1415 or SEQ ID NO: 1451, wherein the sequence of SEQ ID NO: position 4 of 1415 is aspartic acid, SEQ id no: glutamic acid at position 10 of 1415. In yet another embodiment, the nucleic acid sequence of SEQ ID NO: 1415 or SEQ ID NO: 1451 the C-terminal amino acid is modified to replace the native carboxylic acid group with a charge neutral group (e.g., amide or ester).
Class 5 peptide fusions
In yet another embodiment, the carboxy-terminal amino acid of a class 5 peptide described herein is identical to a carboxy-terminal amino acid comprising a sequence selected from SEQ ID NOs: 1421. a second peptide of the sequence of 1426, 1427, and 1450. For example, in some embodiments, SEQ ID NO: 1415. SEQ ID NO: 1451. SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407. SEQ ID NO: 1408. SEQ ID NO: 1412. SEQ ID NO: 1413. SEQ ID NO: 1414. SEQ ID NO: 1416. SEQ ID NO: 1417. SEQ ID NO: 1418. SEQ ID NO: 1419. SEQ ID NO: 1422. SEQ ID NO: 1423. SEQ ID NO: 1424 and SEQ ID NO: 1425 with a peptide comprising a sequence selected from seq id NO: 1421(GPSSGAPPPS), SEQ ID NO: 1426(KRNRNNIA), SEQID NO: 1427(KRNR) and SEQ ID NO: 1450 (GPSSGAPPPSX).
In some embodiments, a class 5 peptide dimer is provided comprising 2 sequences independently selected from: SEQ ID NO: 1405. SEQ ID NO: 1406. SEQ ID NO: 1407. SEQ ID NO: 1408. SEQ ID NO: 1409. SEQ ID NO: 1422. SEQ ID NO: 1423. SEQ ID NO: 1424 and SEQ ID NO: 1425, further comprising the amino acid sequence of SEQ ID NO: 1421 (GPSSGAPPPS).
In some embodiments, the class 5 peptide is further modified by truncation or deletion of one or two amino acids from the C-terminus of the peptide (i.e., truncation of the amino acids at position 29 or 28 and 29 of native glucagon). Preferably, the truncation does not affect the activity of the class 5 peptide (e.g., glucagon antagonism/GLP-1 agonism).
Class 5 peptide conjugates
Also provided are conjugates of a class 5 peptide, wherein the glucagon peptide is optionally bound by a covalent bond and optionally linked to the conjugate moiety by a linker.
In those embodiments in which the class 5 peptide comprises a polyethylene glycol chain, the polyethylene glycol chain may be in the form of a straight chain, or may be branched. According to some embodiments, the polyethylene glycol chains have an average molecular weight selected from about 500 to about 10,000 daltons. In some embodiments, the polyethylene glycol chains have an average molecular weight selected from about 1,000 to about 5,000 daltons. In some embodiments, the polyethylene glycol chains have an average molecular weight selected from about 1,000 to about 5,000 daltons. In some embodiments, the average molecular weight of the polyethylene glycol chains is selected from about 1,000 to about 2,000 daltons. In some embodiments, the polyethylene glycol chains have an average molecular weight of about 1,000 daltons.
In some embodiments, the pegylated class 5 peptide comprises a peptide consisting of SEQ ID NO: 1415 or SEQ ID NO: 1451, wherein the polyethylene glycol chain is identical to a sequence selected from SEQ ID NO: 1415 or SEQ ID NO: 1451 amino acid positions 11, 12, 15, 16, 19 and 24, and the PEG chain has a molecular weight of about 1,000 to about 5,000 daltons. In some embodiments, the pegylated class 5 peptide comprises a peptide consisting of SEQ ID NO: 1415 or SEQ ID NO: 1451, wherein the polyethylene glycol chain is substantially identical to SEQ ID NO: 1415 or SEQ ID NO: 1451 amino acid at position 16 or 19, and the molecular weight of the PEG chain is about 1,000 to about 5,000 daltons. In yet another embodiment, the modified class 5 peptide comprises two or more polyethylene glycol chains covalently attached to the peptide, wherein the total molecular weight of the glucagon chain is from about 1,000 to about 5,000 daltons. In some embodiments, the class 5 peptide comprises SEQ ID NO: 1415 or SEQ ID NO: 1451, wherein the polyethylene glycol chain is identical to SEQ ID NO: 1415 or SEQ ID NO: the amino acids at positions 16 and 19 of 1451 are linked, and the total molecular weight of the 2 PEG chains is from about 1,000 to about 5,000 daltons.
The glucagon-like-5-like peptide of claim 5 may comprise SEQ ID NO: 1401, 1518, optionally with up to 1, 2, 3, 4, or 5 additional modifications that maintain glucagon antagonist and GLP-1 agonist activity.
Effect of dipeptide prodrug element Structure on cleavage Rate
As previously described herein, the rate of cleavage of the dipeptide prodrug element a-B from the glucagon superfamily peptide and thus prodrug activation depends on the structure of the amino acids of the dipeptide prodrug element, including N-alkylation, number of substituents, length or bulkiness (bulkiness), and stereochemistry. The rate of cleavage of dipeptide prodrug elements a-B from glucagon superfamily peptides also depends on the steric hindrance, nucleophilicity, stability of the leaving group of Q during diketopiperazine formation. Some of these structural features are described in class I, class II and class III below, which form part of the present invention. To the extent that they fall completely within and/or overlap a portion of any of the subclasses described herein, and only if necessary to confer novelty to the claimed subject matter, specifically excluded from any of these categories are the peptide sequences disclosed in international application No. PCT/US2009/68745 filed 12/18, 2009 or its sequence listing, and the subclasses of (1) dipeptide prodrug element (2) amino acid a and/or (3) amino acid B disclosed in international application No. PCT/US2009/68745 filed 12/18, 2009.
Class I: composition of dipeptide prodrug element amino acid B
In some embodiments, the half-life of the prodrug is, for example, the chemical cleavage half-life (t) of a-B from Q in PBS under physiological conditions for at least about 1 hour to about 1 week1/2) Depending on the presence and length of the N-alkyl substituent on the amino acid B. For example, prodrugs having a shorter N-alkyl substituent on amino acid B (e.g., Gly (N-methyl)) may experience a slower A-B cleavage rate and have a longer half-life than prodrugs having a longer N-alkyl substituent on amino acid B (e.g., Gly (N-hexyl)).
In some embodiments, the half-life of the prodrug is dependent on the degree of substitution at the beta position of amino acid B of the dipeptide prodrug element. For example, a prodrug having an amino acid B that is disubstituted in the beta position (e.g., an N-alkylated isoleucine) will undergo slower A-B cleavage and have a longer half-life than a prodrug having an amino acid B that is monosubstituted in the beta position (e.g., an N-alkylated leucine). In addition, prodrugs with amino acid B that is monosubstituted at the beta position (e.g., N-alkylated leucine) will undergo slower A-B cleavage and have a longer half-life than prodrugs with amino acid B that is not substituted at the beta position (e.g., N-alkylated alanine). Still further, prodrugs with amino acid B having a carbon at the beta position (e.g., N-alkylated alanine) may undergo slower A-B cleavage and longer half-life than prodrugs with glycine as amino acid B
In some embodiments, the half-life of the prodrug is dependent on the bulkiness of the amino acid B side chain. For example, a prodrug having a more bulky side chain (e.g., N-alkylated phenylalanine) on amino acid B will undergo slower A-B cleavage and have a longer half-life than a prodrug having a less bulky side chain (e.g., N-alkylated alanine) on amino acid B.
The composition of amino acid B of the dipeptide prodrug element can be classified into the following subclasses IA, IB and IC. In general, the dipeptide prodrug element at subclass IA undergoes the fastest cleavage and the dipeptide prodrug element at subclass IC undergoes the slowest cleavage.
Subclass IA: amino acid B of the dipeptide prodrug element is N-alkylated glycine
In some embodiments, the prodrug comprises the structure:
A-B-Q；
wherein Q is a glucagon superfamily peptide;
wherein A-B comprises the following structure:
wherein
R1And R2Independently selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group;
R3is C1-C18An alkyl group;
R4and R8Are all H;
R5is NHR6；
R6Is H or C1-C4Alkyl, or R5And R2Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7selected from H, OH, halogen, (C)1-C7Alkyl group), (C)2-C7Alkenyl), OCF3、NO2、CN、NC、O(C1-C7Alkyl), CO2H、CO2(C1-C7Alkyl), NHR6Aryl and heteroaryl.
In some embodiments, amino acid B is selected from glycine (N-methyl), glycine (N-ethyl), glycine (N-propyl), glycine (N-butyl), glycine (N-pentyl), glycine (N-hexyl), glycine (N-heptyl), and glycine (N-octyl). For example, amino acid B may be glycine (N-methyl) or glycine (N-hexyl).
In some embodiments, when R1And R2When both are hydrogen, R3Is C1-C4Alkyl, for example, when a-B is conjugated to an aliphatic amine. In some embodiments, when R1And R2When both are hydrogen, R3Is C5-C8Alkyl, for example, when a-B is conjugated to an aliphatic amine. In some embodiments, when R1Or R2When at least one of (A) is not hydrogen, R3Is C1-C4Alkyl, for example, when a-B is conjugated to an aliphatic amine. In some embodiments, when R1Or R2When at least one of (A) is not hydrogen, R3Is C5-C8Alkyl radicals, e.g. when When a-B is conjugated to an aliphatic amine.
In some embodiments, when R1And R2Are all hydrogen, and R3When methyl, A-B is not conjugated to F7Alpha amino group of GLP-1 (8-37).
Subclass IB: amino acid B of the dipeptide prodrug element is unsubstituted or monosubstituted at the beta-position
In some embodiments, the prodrug comprises the structure:
A-B-Q；
wherein Q is a glucagon superfamily peptide;
wherein A-B comprises the following structure:
wherein
R1And R2Independently selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group;
R3is C1-C18An alkyl group;
R4is selected from CH3、CH2(C1-C10Alkyl), CH2(C2-C10Alkenyl), CH2(C0-C10Alkyl) OH, CH2(C0-C10Alkyl) SH, CH2(C0-C3Alkyl) SCH3、CH2(C0-C3Alkyl) CONH2、CH2(C0-C3Alkyl) COOH, CH2(C0-C3Alkyl) NH2、CH2(C0-C3Alkyl) NHC (NH)2+)NH2、CH2(C0-C3Alkyl) (C3-C6Cycloalkyl), CH2(C0-C3Alkyl) (C2-C5Heterocyclyl), CH2(C0-C3Alkyl) (C6-C10Aryl) R7、CH2(C1-C3Alkyl) (C3-C9Heteroaryl) and CH2(C0-C12Alkyl) (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R4And R3Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring;
R8is a compound of formula (I) in the formula (H),
R5is NHR6Or R is5And R2Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring;
R6is H or C1-C4An alkyl group; and
R7selected from H, OH, halogen, (C)1-C7Alkyl group), (C)2-C7Alkenyl), OCF3、NO2、CN、NC、O(C1-C7Alkyl), CO2H、CO2(C1-C7Alkyl), NHR6Aryl and heteroaryl.
In some embodiments, R4Is selected from CH3、CH2(C1-C4Alkyl), CH2(C1-C4) Alkenyl, CH2(C0-C4Alkyl) OH, CH2(C0-C4Alkyl) SH, CH2(C0-C3Alkyl) SCH3、CH2(C0-C3Alkyl) CONH2、CH2(C0-C3Alkyl) COOH, CH2(C0-C4Alkyl) NH2And CH2(C0-C3Alkyl) NHC (NH)2+)NH2。
Non-limiting examples of amino acid B include alanine (N-C) in these embodiments1-C10Alkyl), leucine (N-C)1-C10Alkyl), methionine (N-C)1-C10Alkyl), asparagine (N-C)1-C10Alkyl), glutamic acid (N-C)1-C10Alkyl), aspartic acid (N-C)1-C10Alkyl), glutamine (N-C)1-C10Alkyl), histidine (N-C)1-C10Alkyl), lysine (N-C)1-C10Alkyl), arginine (N-C)1-C10Alkyl), serine (N-C)1-C10Alkyl) and cysteine (N-C)1-C10Alkyl groups).
In some embodiments, amino acid B is selected from alanine (N-C)1-C6Alkyl), leucine (N-C)1-C6Alkyl), methionine (N-C)1-C6Alkyl), asparagine (N-C)1-C6Alkyl), glutamic acid (N-C)1-C6Alkyl), aspartic acid (N-C)1-C6Alkyl), glutamine (N-C)1-C6Alkyl), histidineAcid (N-C)1-C6Alkyl), lysine (N-C)1-C6Alkyl), arginine (N-C)1-C6Alkyl), serine (N-C)1-C6Alkyl) and cysteine (N-C)1-C6Alkyl groups).
For example, amino acid B may include alanine (N-methyl), leucine (N-methyl), methionine (N-methyl), asparagine (N-methyl), glutamic acid (N-methyl), aspartic acid (N-methyl), glutamine (N-methyl), histidine (N-methyl), lysine (N-methyl), arginine (N-methyl), serine (N-methyl), and cysteine (N-methyl).
In some embodiments, R4Is selected from CH2(C0-C3Alkyl) (C3-C6Cycloalkyl), CH2(C0-C3Alkyl) (C2-C5Heterocyclyl), CH2(C0-C3Alkyl) (C6-C10Aryl) R7、CH2(C1-C3Alkyl) (C3-C9Heteroaryl) and CH2(C0-C12Alkyl) (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, and wherein R7Selected from H and OH.
Non-limiting examples of amino acid B include phenylalanine (N-C) in these embodiments1-C10Alkyl), tyrosine (N-C)1-C10Alkyl) and tryptophan (N-C)1-C10Alkyl groups). In some embodiments, amino acid B is selected from phenylalanine (N-C)1-C6Alkyl), tyrosine (N-C)1-C6Alkyl) and tryptophan (N-C)1-C6Alkyl groups). For example, amino acid B may include phenylalanine (N-methyl), tyrosine (N-methyl), and tryptophan (N-methyl).
In some embodiments, amino acid B is proline. In some embodiments, proline is excluded from subclass IB.
Subclass IC: amino acid B of a dipeptide prodrug element disubstituted in the beta position
In some embodiments, the prodrug comprises the structure:
A-B-Q；
wherein Q is a glucagon superfamily peptide;
wherein A-B comprises the following structure:
wherein
R1And R2Independently selected from H, C1-C18Alkyl radical, C2-C18Alkenyl, (C)1-C18Alkyl) OH, (C)1-C18Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group; or R1And R2Together with the atom to which they are attached form C3-C12A cycloalkyl group;
R3is C1-C18An alkyl group;
R4is independently selected from CH (C)1-C8Alkyl radical)2、CH(C2-C8Alkenyl)2、CH(C1-C8Alkyl) (OH), CH (C)1-C8Alkyl) ((C)1-C8Alkyl) SH), CH (C)1-C3Alkyl) ((C)1-C8Alkyl) (NH)2))；
R8Is H;
R5is NHR6Or R is5And R2Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring;
R6is H or C1-C4Alkyl, and
R7selected from H, OH, halogen, (C)1-C7Alkyl group), (C)2-C7Alkenyl), OCF3、NO2、CN、NC、O(C1-C7Alkyl), CO2H、CO2(C1-C7Alkyl), NHR6Aryl and heteroaryl.
In some embodiments, R4Is CH (C)1-C8Alkyl radical)2Or CH (C)1-C8Alkyl) OH. Non-limiting examples of amino acid B include isoleucine (N-C)1-C10Alkyl), valine (N-C)1-C10Alkyl) and threonine (N-C)1-C10Alkyl groups). In some embodiments, amino acid B is selected from isoleucine (N-C)1-C6Alkyl), valine (N-C)1-C6Alkyl) and threonine (N-C)1-C6Alkyl groups). For example, amino acid B can include isoleucine (N-methyl), valine (N-methyl), and threonine (N-methyl).
Class II: composition of dipeptide prodrug element amino acid A
In some embodiments, the half-life of the prodrug depends on the number of substituents alpha to amino acid a. For example, a prodrug comprising amino acid a (e.g., Ala) as an α -monosubstituted amino acid will undergo slower cleavage and have a longer half-life than a prodrug comprising amino acid a (e.g., Aib) as an α, α -disubstituted amino acid.
In some embodiments, the half-life of the prodrug depends on the degree of alkylation of the alpha amino group of amino acid a. In general, the higher the degree of alkylation, the slower the cleavage rate and the longer the half-life of the prodrug. For example, a dipeptide prodrug element with N-alkylated Ala will cleave at a slower rate and have a longer half-life than a dipeptide prodrug element with Ala.
The composition of amino acid A of the dipeptide prodrug element can be classified into subclasses IIA and IIB below, and in general, the dipeptide prodrug element of subclass IIA cleaves faster than the dipeptide prodrug element of subclass IIB.
Subclass IIA: amino acid A of the dipeptide prodrug element is disubstituted in the alpha position
In some embodiments, the amino acid a of the dipeptide prodrug element is disubstituted in the alpha position. In these embodiments, R of the structures described in subclasses IA, IB and IC1And R2Is independently selected from C1-C10Alkyl radical, C2-C10Alkenyl, (C)1-C10Alkyl) OH, (C)1-C10Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atoms to which they are attached form C3-C12Cycloalkyl, and wherein R7Selected from H and OH.
For example, amino acid a can include aminoisobutyric acid (Aib).
Subclass IIB: amino acid A of the dipeptide prodrug element is unsubstituted or monosubstituted at the alpha position
In some embodiments, amino acid a of the dipeptide prodrug element is unsubstituted or mono-substituted at the alpha position. In these embodiments, R of the structures described in subclasses IA, IB and IC1Is H, and R of the structure described in subclasses IA, IB and IC2Selected from H, C1-C10Alkyl radical, C2-C10Alkenyl, (C)1-C10Alkyl) OH, (C)1-C10Alkyl) SH, (C)2-C3Alkyl) SCH3、(C1-C4Alkyl) CONH2、(C1-C4Alkyl group) COOH, (C)1-C4Alkyl) NH2、(C1-C4Alkyl) NHC (NH)2+)NH2、(C0-C4Alkyl) (C3-C6Cycloalkyl group), (C)0-C4Alkyl) (C2-C5Heterocyclic group), (C)0-C4Alkyl) (C6-C10Aryl) R7、(C1-C4Alkyl) (C3-C9Heteroaryl) and C1-C12Alkyl (W)1)C1-C12Alkyl radical, wherein R7Is selected from H and OH, and wherein W1Is a heteroatom selected from N, S and O, or R1And R2Together with the atom to which they are attached form C3-C12Cycloalkyl, or R2And R5Together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring.
In some embodiments, amino acid a of the dipeptide prodrug element has a'd' -stereochemistry. Non-limiting examples of amino acid A in these embodiments include: lysine, cysteine and alanine. For example, d-lysine, d-cysteine and d-alanine. In some embodiments, the d-stereochemistry may increase half-life by reducing proteolytic degradation of the prodrug peptide.
In some embodiments, amino acid A is N-alkylated with a group having 1-4 carbon atoms, such as Ala (N-C)1-C4Alkyl), Lys (N-C)1-C4Alkyl) and Cys (N-C)1-C4Alkyl groups). For example, amino acid A can be Ala (N-methyl), Lys (N-methyl), and Cys (N-methyl). N-alkylation of amino acid a reduces the rate of cleavage of the dipeptide prodrug element from Q and provides a longer half-life.
Class III: conjugation site of dipeptide prodrug element (A-B) to glucagon superfamily peptide (Q)
In some embodiments, the half-life of the prodrug depends on the steric hindrance, nucleophilicity, and stability of the leaving group on Q during diketopiperazine formation. The less steric hindrance hindering the leaving group, the lower the nucleophilicity of the leaving group or the higher the stability of the leaving group after cleavage, the shorter the half-life of the prodrug. The type of leaving group on Q can be determined by the type of linkage between A-B and the amino group of Q, as described in subclasses IIIA and IIIB below. In general, the dipeptide prodrug elements of subclass IIIA cleave slower from Q and have a longer half-life than the dipeptide prodrug elements of subclass IIIB.
Subclass IIIA: A-B linked to the aliphatic amino group of Q
In some embodiments, as described previously herein, a-B is linked to Q through an amide bond between a-B and the aliphatic amino group of Q, resulting in a polymer having a chemical cleavage half-life (t) from Q in PBS under physiological conditions1/2) Is a prodrug for at least about 1 hour to about 1 week.
In some embodiments, a-B is linked to Q through an amide bond between a-B and the alpha amino group of the N-terminal amino acid of Q. For example, having a chemical formula fromDipeptide prodrug elements of amino acid B of any of classes IA, IB and IC and amino acid A from any of subclasses IIA and IIB can be attached to the N-terminal amino acid of Q, resulting in a dipeptide prodrug having a chemically cleavable half-life (t) of A-B from Q in PBS under physiological conditions1/2) Is a prodrug for at least about 1 hour to about 1 week.
In some embodiments, A-B is linked to Q through an amide bond between A-B and an aliphatic amino group on the amino acid side chain of Q. For example, a dipeptide prodrug element having amino acid B from any of subclasses IA, IB, and IC, and amino acid A from any of subclasses IIA and IIB, can be attached to the aliphatic amino group of the Q amino acid side chain, resulting in a dipeptide prodrug element having a chemically cleavable half-life (t) of A-B from Q in PBS under physiological conditions (T)1/2) Is a prodrug for at least about 1 hour to about 1 week.
In some embodiments, when a-B is linked to Q through an amide bond between a-B and the aliphatic amino group of Q, a should be an α, α -disubstituted amino acid (subclass IIA) or B should be N-alkylated (any of subclasses IA, IB, or IC), or both. For example, when A is an alpha-monosubstituted amino acid (e.g., Ala), B is non-N-alkylated, and A-B is linked to Q through the aliphatic amino group of Q, there is no significant cleavage of A-B.
In other embodiments, when A-B is attached to F7When the alpha amino group of GLP-1(8-37) is present, A-B is not Gly-Gly (N-Me).
In other optional embodiments, when A-B is linked to Q through an amide linkage between A-B and the aliphatic amino group of Q, and A is an amino acid unsubstituted at the alpha position (e.g., glycine) and B is an amino acid from subclass IA (N-alkylated glycine), the N-alkyl substituent of the B amino acid has a length of at least 5 carbon atoms (e.g., N-C)5-C8Alkyl groups).
In yet other embodiments, when A-B is linked to Q through an amide bond between A-B and the aliphatic amino group of Q, and amino acid A is unsubstituted or monosubstituted in the alpha position (subclass IIB), amino acid B is not proline. In some embodiments, when A-B is linked to Q through an amide bond between A-B and the aliphatic amino group of Q, A-B is not Gly-Pro. In some embodiments, when amino acid B is proline, amino acid a is from subclass IIA.
Subclass IIIB: A-B of an aromatic amino group bound to Q
In some embodiments, as described previously herein, a-B is linked to Q through an amide bond between a-B and an aromatic amino group of an amino acid side chain of Q, resulting in a polymer having a chemical cleavage half-life (t) of a-B from Q in PBS under physiological conditions (t;)1/2) Is a prodrug for at least about 1 hour to about 1 week. For example, a dipeptide prodrug element having amino acid B from any of subclasses IA, IB, and IC, and amino acid A from any of subclasses IIA and IIB, can be attached to the aromatic amino group of the amino acid side chain of Q, resulting in a dipeptide prodrug element having a chemically cleavable half-life (t) from Q of A-B in PBS under physiological conditions (t) and a prodrug having a prodrug structure that is chemically cleavable from Q under physiological conditions1/2) Is a prodrug for at least about 1 hour to about 1 week.
Any amino acid B defined by class I can be combined with any amino acid a defined by class II to form a dipeptide prodrug element. The dipeptide prodrug element may be attached to any position described by class III. The half-life of the prodrug can be adjusted by selecting:
(i) The number of substituents in position alpha to amino acid A;
(ii) the degree of N-alkylation of amino acid A and amino acid B;
(iii) the number of substituents at position β of amino acid B;
(iv) the looseness of the side chain of the amino acid B; and
(iii) steric hindrance, nucleophilicity and stability of the leaving group on Q during diketopiperazine formation.
Modification of dipeptide prodrug elements A-B
The dipeptide prodrug elements described above may be further modified to include a hydrophilic moiety, an acyl group, or an alkyl group, as described previously herein. In some embodiments, the dipeptide prodrug element comprises a lysine conjugated to an acyl or alkyl group through its side chain amino group. In some embodiments, the dipeptide prodrug element comprises a cysteine conjugated to a hydrophilic moiety (e.g., 40kD PEG) through a side chain thiol group. The hydrophilic moiety, acyl group, or alkyl group can be conjugated to the dipeptide prodrug element directly or through a spacer. In some exemplary embodiments, a hydrophilic group, alkyl group, and/or acyl group is conjugated to amino acid a of the dipeptide prodrug element.
In some embodiments, the following dipeptide prodrug elements are pegylated: dCys-Gly (N-hexyl), dCys-Gly (N-methyl) and dCys-Phe (N-methyl). In some embodiments, the following dipeptide prodrug elements comprise acyl groups: dLys-Gly (N-hexyl), dLys-Gly (N-methyl) and dLys-Phe (N-methyl). In some embodiments, the following dipeptide prodrug elements comprise alkyl groups: dLys-Gly (N-hexyl), dLys-Gly (N-methyl) and dLys-Phe (N-methyl).
Exemplary embodiments
The dipeptide prodrug element of the invention may comprise any combination of amino acid B from class I and any amino acid a from class II. Non-limiting examples of suitable amino acids for amino acid A and amino acid B of the dipeptide prodrug element are listed below.
| Amino acid # | Amino acid "A" | Amino acid "B" |
| 1 | Aib | Gly(N-C1-C8Alkyl radical) |
| 2 | Gly | Ala(N-C1-C8Alkyl radical) |
| 3 | Ala | Leu(N-C1-C8Alkyl radical) |
| 4 | Leu | Met(N-C1-C8Alkyl radical) |
| 5 | Met | Asn(N-C1-C8Alkyl radical) |
| 6 | Asn | Glu(N-C1-C8Alkyl radical) |
| 7 | Glu | Asp(N-C1-C8Alkyl radical) |
| 8 | Asp | Gln(N-C1-C8Alkyl radical) |
| 9 | Gln | His(N-C1-C8Alkyl radical) |
| 10 | His | Lys(N-C1-C8Alkyl radical) |
| 11 | Lys | Arg(N-C1-C8Alkyl radical) |
| 12 | Arg | Ser(N-C1-C8Alkyl radical) |
| 13 | Ser | Cys(N-C1-C8Alkyl radical) |
| 14 | Cys | Pro |
| 15 | Pro | Phe(N-C1-C8Alkyl radical) |
| 16 | Phe | Tyr(N-C1-C8Alkyl radical) |
| 17 | Tyr | Trp(N-C1-C8Alkyl radical) |
| 18 | Trp | Ile(N-C1-C8Alkyl radical) |
| 19 | Ile | Val(N-C1-C8Alkyl radical) |
| 20 | Val | Thr(N-C1-C8Alkyl radical) |
| 21 | Thr | d-Ala(N-C1-C8Alkyl radical) |
| 22 | d-Ala | d-Leu(N-C1-C8Alkyl radical) |
| 23 | d-Leu | d-Met(N-C1-C8Alkyl radical) |
| 24 | d-Met | d-Asn(N-C1-C8Alkyl radical) |
| 25 | d-Asn | d-Glu(N-C1-C8Alkyl radical) |
| 26 | d-Glu | d-Asp(N-C1-C8Alkyl radical) |
| 27 | d-Asp | d-Gln(N-C1-C8Alkyl radical) |
| 28 | d-Gln | d-His(N-C1-C8Alkyl radical) |
| 29 | d-His | d-Lys(N-C1-C8Alkyl radical) |
| 30 | d-Lys | d-Arg(N-C1-C8Alkyl radical) |
| 31 | d-Arg | d-Ser(N-C1-C8Alkyl radical) |
| 32 | d-Ser | d-Cys(N-C1-C8Alkyl radical) |
| 33 | d-Cys | d-Pro |
| 34 | d-Pro | d-Phe(N-C1-C8Alkyl radical) |
| 35 | d-Phe | d-Tyr(N-C1-C8Alkyl radical) |
| 36 | d-Tyr | d-Trp(N-C1-C8Alkyl radical) |
| 37 | d-Trp | d-Ile(N-C1-C8Alkyl radical) |
| 38 | d-Ile | d-Val(N-C1-C8Alkyl radical) |
| 39 | d-Val | d-Thr(N-C1-C8Alkyl radical) |
| 40 | d-Thr | Gly (N-methyl) |
| 41 | Gly (N-methyl) | Ala (N-methyl) |
| 42 | Ala (N-methyl) | Leu (N-methyl) |
| 43 | Leu (N-methyl) | Met (N-methyl) |
| 44 | Met (N-methyl) | Asn (N-methyl) |
| 45 | Asn (N-methyl) | Glu (N-methyl) |
| 46 | Glu (N-methyl) | Asp (N-methyl) |
| 47 | Asp (N-methyl) | Gln (N-methyl) |
| 48 | Gln (N-methyl) | His (N-methyl) |
| 49 | His (N-methyl) | Lys (N-methyl) |
| 50 | Lys (N-methyl) | Arg (N-methyl) |
| 51 | Arg (N-methyl) | Ser (N-methyl) |
| 52 | Ser (N-methyl) | Cys (N-methyl) |
| 53 | Cys (N-methyl) | Phe (N-methyl) |
| 54 | Phe (N-methyl) | Tyr (N-methyl) |
| 55 | Tyr (N-methyl) | Trp (N-methyl) |
| 56 | Trp (N-methyl) | Ile (N-methyl) |
| 57 | Ile (N-methyl) | Val (N-methyl) |
| 58 | Val (N-methyl) | Thr (N-methyl) |
| 59 | Thr (N-methyl) | d-Ala (N-methyl) |
| 60 | d-Ala (N-methyl) | d-Leu (N-methyl) |
| 61 | d-Leu (N-methyl) | d-Met (N-methyl) |
| 62 | d-Met (N-methyl) | d-Asn (N-methyl) |
| 63 | d-Asn (N-methyl) | d-Glu (N-methyl) |
| 64 | d-Glu (N-methyl) | d-Asp (N-methyl) |
| 65 | d-Asp (N-methyl) | d-Gln (N-methyl) |
| 66 | d-Gln (N-methyl) | d-His (N-methyl) |
| 67 | d-His (N-methyl) | d-Lys (N-methyl) |
| 68 | d-Lys (N-methyl) | d-Arg (N-methyl) |
| 69 | d-Arg (N-methyl) | d-Ser (N-methyl) |
| 70 | d-Ser (N-methyl) | d-Cys (N-methyl) |
| 71 | d-Cys (N-methyl) | d-Phe (N-methyl) |
| 72 | d-Phe (N-methyl) | d-Tyr (N-methyl) |
| 73 | d-Tyr (N-methyl) | d-Trp (N-methyl) |
| 74 | d-Trp (N-methyl) | d-Ile (N-methyl) |
| 75 | d-Ile (N-methyl) | d-Val (N-methyl) |
| 76 | d-Val (N-methyl) | d-Thr (N-methyl) |
| 77 | d-Thr (N-methyl) | Gly (N-hexyl) |
| 78 | | Ala (N-hexyl) |
| 79 | | Leu (N-hexyl) |
| 80 | | Met (N-hexyl) |
| 81 | | Asn (N-hexyl) |
| 82 | | Glu (N-hexyl) |
| 83 | | Asp (N-hexyl) |
| 84 | | Gln (N-hexyl) |
| 85 | | His (N-hexyl) |
| 86 | | Lys (N-hexyl) |
| 87 | | Arg (N-hexyl) |
| 88 | | Ser (N-hexyl) |
| 89 | | Cys (N-hexyl) |
| 90 | | Phe (N-hexyl) |
| 91 | | Tyr (N-hexyl) |
| 92 | | Trp (N-hexyl) |
| 93 | | Ile (N-hexyl) |
| 94 | | Val (N-hexyl) |
| 95 | | Thr (N-hexyl) |
| 96 | | d-Ala (N-hexyl) |
| 97 | | d-Leu (N-hexyl) |
| 98 | | d-Met (N-hexyl) |
| 99 | | d-Asn (N-hexyl) |
| 100 | | d-Glu (N-hexyl) |
| 101 | | d-Asp (N-hexyl) |
| 102 | | d-Gln (N-hexyl) |
| 103 | | d-His (N-hexyl) |
| 104 | | d-Lys (N-hexyl) |
| 105 | | d-Arg (N-hexyl) |
| 106 | | d-Ser (N-hexyl) |
| 107 | | d-Cys (N-hexyl) |
| 108 | | d-Phe (N-hexyl) |
| 109 | | d-Tyr (N-hexyl) |
| 110 | | d-Trp (N-hexyl) |
| 111 | | d-Ile (N-hexyl) |
| 112 | | d-Val (N-hexyl) |
| 113 | | d-Thr (N-hexyl) |
In some embodiments, the dipeptide prodrug element comprises a combination of any one of a1-a77 and any one of B1-B113. For example, the combination of amino acid a and amino acid B of the dipeptide prodrug element may include: A1-B1; A1-B2; A1-B3; A1-B4; A1-B5; A1-B6; A1-B7; A1-B8; A1-B9; A1-B10; A1-B11; A1-B12; A1-B13; A1-B14; A1-B15; A1-B16; A1-B17; A1-B18; A1-B19; A1-B20; A1-B21; A1-B22; A1-B23; A1-B24; A1-B25; A1-B26; A1-B27; A1-B28; A1-B29; A1-B30; A1-B31; A1-B32; A1-B33; A1-B34; A1-B35; A1-B36; A1-B37; A1-B38; A1-B39; A1-B40; A1-B41; A1-B42; A1-B43; A1-B44; A1-B45; A1-B46; A1-B47; A1-B48; A1-B49; A1-B50; A1-B51; A1-B52; A1-B53; A1-B54; A1-B55; A1-B56; A1-B57; A1-B58; A1-B59; A1-B60; A1-B61; A1-B62; A1-B63; A1-B64; A1-B65; A1-B66; A1-B67; A1-B68; A1-B69; A1-B70; A1-B71; A1-B72; A1-B73; A1-B74; A1-B75; A1-B76; A1-B77; A1-B78; A1-B79; A1-B80; A1-B81; A1-B82; A1-B83; A1-B84; A1-B85; A1-B86; A1-B87; A1-B88; A1-B89; A1-B90; A1-B91; A1-B92; A1-B93; A1-B94; A1-B95; A1-B96; A1-B97; A1-B98; A1-B99; A1-B100; A1-B101; A1-B102; A1-B103; A1-B104; A1-B105; A1-B106; A1-B107; A1-B108; A1-B109; A1-B110; A1-B111; A1-B112; A1-B113;
A2-B1；A2-B2；A2-B3；A2-B4；A2-B5；A2-B6；A2-B7；A2-B8；A2-B9；A2-B10；A2-B11；A2-B12；A2-B13；A2-B14；A2-B15；A2-B16；A2-B17；A2-B18；A2-B19；A2-B20；A2-B21；A2-B22；A2-B23；A2-B24；A2-B25；A2-B26；A2-B27；A2-B28；A2-B29；A2-B30；A2-B31；A2-B32；A2-B33；A2-B34；A2-B35；A2-B36；A2-B37；A2-B38；A2-B39；A2-B40；A2-B41；A2-B42；A2-B43；A2-B44；A2-B45；A2-B46；A2-B47；A2-B48；A2-B49；A2-B50；A2-B51；A2-B52；A2-B53；A2-B54；A2-B55；A2-B56；A2-B57；A2-B58；A2-B59；A2-B60；A2-B61；A2-B62；A2-B63；A2-B64；A2-B65；A2-B66；A2-B67；A2-B68；A2-B69；A2-B70；A2-B71；A2-B72；A2-B73；A2-B74；A2-B75；A2-B76；A2-B77；A2-B78；A2-B79；A2-B80；A2-B81；A2-B82；A2-B83；A2-B84；A2-B85；A2-B86；A2-B87；A2-B88；A2-B89；A2-B90；A2-B91；A2-B92；A2-B93；A2-B94；A2-B95；A2-B96；A2-B97；A2-B98；A2-B99；A2-B100；A2-B101；A2-B102；A2-B103；A2-B104；A2-B105；A2-B106；A2-B107；A2-B108；A2-B109；A2-B110；A2-B111；A2-B112；A2-B113；
A3-B1；A3-B2；A3-B3；A3-B4；A3-B5；A3-B6；A3-B7；A3-B8；A3-B9；A3-B10；A3-B11；A3-B12；A3-B13；A3-B14；A3-B15；A3-B16；A3-B17；A3-B18；A3-B19；A3-B20；A3-B21；A3-B22；A3-B23；A3-B24；A3-B25；A3-B26；A3-B27；A3-B28；A3-B29；A3-B30；A3-B31；A3-B32；A3-B33；A3-B34；A3-B35；A3-B36；A3-B37；A3-B38；A3-B39；A3-B40；A3-B41；A3-B42；A3-B43；A3-B44；A3-B45；A3-B46；A3-B47；A3-B48；A3-B49；A3-B50；A3-B51；A3-B52；A3-B53；A3-B54；A3-B55；A3-B56；A3-B57；A3-B58；A3-B59；A3-B60；A3-B61；A3-B62；A3-B63；A3-B64；A3-B65；A3-B66；A3-B67；A3-B68；A3-B69；A3-B70；A3-B71；A3-B72；A3-B73；A3-B74；A3-B75；A3-B76；A3-B77；A3-B78；A3-B79；A3-B80；A3-B81；A3-B82；A3-B83；A3-B84；A3-B85；A3-B86；A3-B87；A3-B88；A3-B89；A3-B90；A3-B91；A3-B92；A3-B93；A3-B94；A3-B95；A3-B96；A3-B97；A3-B98；A3-B99；A3-B100；A3-B101；A3-B102；A3-B103；A3-B104；A3-B105；A3-B106；A3-B107；A3-B108；A3-B109；A3-B110；A3-B111；A3-B112；A3-B113；
A4-B1；A4-B2；A4-B3；A4-B4；A4-B5；A4-B6；A4-B7；A4-B8；A4-B9；A4-B10；A4-B11；A4-B12；A4-B13；A4-B14；A4-B15；A4-B16；A4-B17；A4-B18；A4-B19；A4-B20；A4-B21；A4-B22；A4-B23；A4-B24；A4-B25；A4-B26；A4-B27；A4-B28；A4-B29；A4-B30；A4-B31；A4-B32；A4-B33；A4-B34；A4-B35；A4-B36；A4-B37；A4-B38；A4-B39；A4-B40；A4-B41；A4-B42；A4-B43；A4-B44；A4-B45；A4-B46；A4-B47；A4-B48；A4-B49；A4-B50；A4-B51；A4-B52；A4-B53；A4-B54；A4-B55；A4-B56；A4-B57；A4-B58；A4-B59；A4-B60；A4-B61；A4-B62；A4-B63；A4-B64；A4-B65；A4-B66；A4-B67；A4-B68；A4-B69；A4-B70；A4-B71；A4-B72；A4-B73；A4-B74；A4-B75；A4-B76；A4-B77；A4-B78；A4-B79；A4-B80；A4-B81；A4-B82；A4-B83；A4-B84；A4-B85；A4-B86；A4-B87；A4-B88；A4-B89；A4-B90；A4-B91；A4-B92；A4-B93；A4-B94；A4-B95；A4-B96；A4-B97；A4-B98；A4-B99；A4-B100；A4-B101；A4-B102；A4-B103；A4-B104；A4-B105；A4-B106；A4-B107；A4-B108；A4-B109；A4-B110；A4-B111；A4-B112；A4-B113；
A5-B1；A5-B2；A5-B3；A5-B4；A5-B5；A5-B6；A5-B7；A5-B8；A5-B9；A5-B10；A5-B11；A5-B12；A5-B13；A5-B14；A5-B15；A5-B16；A5-B17；A5-B18；A5-B19；A5-B20；A5-B21；A5-B22；A5-B23；A5-B24；A5-B25；A5-B26；A5-B27；A5-B28；A5-B29；A5-B30；A5-B31；A5-B32；A5-B33；A5-B34；A5-B35；A5-B36；A5-B37；A5-B38；A5-B39；A5-B40；A5-B41；A5-B42；A5-B43；A5-B44；A5-B45；A5-B46；A5-B47；A5-B48；A5-B49；A5-B50；A5-B51；A5-B52；A5-B53；A5-B54；A5-B55；A5-B56；A5-B57；A5-B58；A5-B59；A5-B60；A5-B61；A5-B62；A5-B63；A5-B64；A5-B65；A5-B66；A5-B67；A5-B68；A5-B69；A5-B70；A5-B71；A5-B72；A5-B73；A5-B74；A5-B75；A5-B76；A5-B77；A5-B78；A5-B79；A5-B80；A5-B81；A5-B82；A5-B83；A5-B84；A5-B85；A5-B86；A5-B87；A5-B88；A5-B89；A5-B90；A5-B91；A5-B92；A5-B93；A5-B94；A5-B95；A5-B96；A5-B97；A5-B98；A5-B99；A5-B100；A5-B101；A5-B102；A5-B103；A5-B104；A5-B105；A5-B106；A5-B107；A5-B108；A5-B109；A5-B110；A5-B111；A5-B112；A5-B113；
A6-B1；A6-B2；A6-B3；A6-B4；A6-B5；A6-B6；A6-B7；A6-B8；A6-B9；A6-B10；A6-B11；A6-B12；A6-B13；A6-B14；A6-B15；A6-B16；A6-B17；A6-B18；A6-B19；A6-B20；A6-B21；A6-B22；A6-B23；A6-B24；A6-B25；A6-B26；A6-B27；A6-B28；A6-B29；A6-B30；A6-B31；A6-B32；A6-B33；A6-B34；A6-B35；A6-B36；A6-B37；A6-B38；A6-B39；A6-B40；A6-B41；A6-B42；A6-B43；A6-B44；A6-B45；A6-B46；A6-B47；A6-B48；A6-B49；A6-B50；A6-B51；A6-B52；A6-B53；A6-B54；A6-B55；A6-B56；A6-B57；A6-B58；A6-B59；A6-B60；A6-B61；A6-B62；A6-B63；A6-B64；A6-B65；A6-B66；A6-B67；A6-B68；A6-B69；A6-B70；A6-B71；A6-B72；A6-B73；A6-B74；A6-B75；A6-B76；A6-B77；A6-B78；A6-B79；A6-B80；A6-B81；A6-B82；A6-B83；A6-B84；A6-B85；A6-B86；A6-B87；A6-B88；A6-B89；A6-B90；A6-B91；A6-B92；A6-B93；A6-B94；A6-B95；A6-B96；A6-B97；A6-B98；A6-B99；A6-B100；A6-B101；A6-B102；A6-B103；A6-B104；A6-B105；A6-B106；A6-B107；A6-B108；A6-B109；A6-B110；A6-B111；A6-B112；A6-B113；
A7-B1；A7-B2；A7-B3；A7-B4；A7-B5；A7-B6；A7-B7；A7-B8；A7-B9；A7-B10；A7-B11；A7-B12；A7-B13；A7-B14；A7-B15；A7-B16；A7-B17；A7-B18；A7-B19；A7-B20；A7-B21；A7-B22；A7-B23；A7-B24；A7-B25；A7-B26；A7-B27；A7-B28；A7-B29；A7-B30；A7-B31；A7-B32；A7-B33；A7-B34；A7-B35；A7-B36；A7-B37；A7-B38；A7-B39；A7-B40；A7-B41；A7-B42；A7-B43；A7-B44；A7-B45；A7-B46；A7-B47；A7-B48；A7-B49；A7-B50；A7-B51；A7-B52；A7-B53；A7-B54；A7-B55；A7-B56；A7-B57；A7-B58；A7-B59；A7-B60；A7-B61；A7-B62；A7-B63；A7-B64；A7-B65；A7-B66；A7-B67；A7-B68；A7-B69；A7-B70；A7-B71；A7-B72；A7-B73；A7-B74；A7-B75；A7-B76；A7-B77；A7-B78；A7-B79；A7-B80；A7-B81；A7-B82；A7-B83；A7-B84；A7-B85；A7-B86；A7-B87；A7-B88；A7-B89；A7-B90；A7-B91；A7-B92；A7-B93；A7-B94；A7-B95；A7-B96；A7-B97；A7-B98；A7-B99；A7-B100；A7-B101；A7-B102；A7-B103；A7-B104；A7-B105；A7-B106；A7-B107；A7-B108；A7-B109；A7-B110；A7-B111；A7-B112；A7-B113；
A8-B1；A8-B2；A8-B3；A8-B4；A8-B5；A8-B6；A8-B7；A8-B8；A8-B9；A8-B10；A8-B11；A8-B12；A8-B13；A8-B14；A8-B15；A8-B16；A8-B17；A8-B18；A8-B19；A8-B20；A8-B21；A8-B22；A8-B23；A8-B24；A8-B25；A8-B26；A8-B27；A8-B28；A8-B29；A8-B30；A8-B31；A8-B32；A8-B33；A8-B34；A8-B35；A8-B36；A8-B37；A8-B38；A8-B39；A8-B40；A8-B41；A8-B42；A8-B43；A8-B44；A8-B45；A8-B46；A8-B47；A8-B48；A8-B49；A8-B50；A8-B51；A8-B52；A8-B53；A8-B54；A8-B55；A8-B56；A8-B57；A8-B58；A8-B59；A8-B60；A8-B61；A8-B62；A8-B63；A8-B64；A8-B65；A8-B66；A8-B67；A8-B68；A8-B69；A8-B70；A8-B71；A8-B72；A8-B73；A8-B74；A8-B75；A8-B76；A8-B77；A8-B78；A8-B79；A8-B80；A8-B81；A8-B82；A8-B83；A8-B84；A8-B85；A8-B86；A8-B87；A8-B88；A8-B89；A8-B90；A8-B91；A8-B92；A8-B93；A8-B94；A8-B95；A8-B96；A8-B97；A8-B98；A8-B99；A8-B100；A8-B101；A8-B102；A8-B103；A8-B104；A8-B105；A8-B106；A8-B107；A8-B108；A8-B109；A8-B110；A8-B111；A8-B112；A8-B113；
A9-B1；A9-B2；A9-B3；A9-B4；A9-B5；A9-B6；A9-B7；A9-B8；A9-B9；A9-B10；A9-B11；A9-B12；A9-B13；A9-B14；A9-B15；A9-B16；A9-B17；A9-B18；A9-B19；A9-B20；A9-B21；A9-B22；A9-B23；A9-B24；A9-B25；A9-B26；A9-B27；A9-B28；A9-B29；A9-B30；A9-B31；A9-B32；A9-B33；A9-B34；A9-B35；A9-B36；A9-B37；A9-B38；A9-B39；A9-B40；A9-B41；A9-B42；A9-B43；A9-B44；A9-B45；A9-B46；A9-B47；A9-B48；A9-B49；A9-B50；A9-B51；A9-B52；A9-B53；A9-B54；A9-B55；A9-B56；A9-B57；A9-B58；A9-B59；A9-B60；A9-B61；A9-B62；A9-B63；A9-B64；A9-B65；A9-B66；A9-B67；A9-B68；A9-B69；A9-B70；A9-B71；A9-B72；A9-B73；A9-B74；A9-B75；A9-B76；A9-B77；A9-B78；A9-B79；A9-B80；A9-B81；A9-B82；A9-B83；A9-B84；A9-B85；A9-B86；A9-B87；A9-B88；A9-B89；A9-B90；A9-B91；A9-B92；A9-B93；A9-B94；A9-B95；A9-B96；A9-B97；A9-B98；A9-B99；A9-B100；A9-B101；A9-B102；A9-B103；A9-B104；A9-B105；A9-B106；A9-B107；A9-B108；A9-B109；A9-B110；A9-B111；A9-B112；A9-B113；
A10-B1；A10-B2；A10-B3；A10-B4；A10-B5；A10-B6；A10-B7；A10-B8；A10-B9；A10-B10；A10-B11；A10-B12；A10-B13；A10-B14；A10-B15；A10-B16；A10-B17；A10-B18；A10-B19；A10-B20；A10-B21；A10-B22；A10-B23；A10-B24；A10-B25；A10-B26；A10-B27；A10-B28；A10-B29；A10-B30；A10-B31；A10-B32；A10-B33；A10-B34；A10-B35；A10-B36；A10-B37；A10-B38；A10-B39；A10-B40；A10-B41；A10-B42；A10-B43；A10-B44；A10-B45；A10-B46；A10-B47；A10-B48；A10-B49；A10-B50；A10-B51；A10-B52；A10-B53；A10-B54；A10-B55；A10-B56；A10-B57；A10-B58；A10-B59；A10-B60；A10-B61；A10-B62；A10-B63；A10-B64；A10-B65；A10-B66；A10-B67；A10-B68；A10-B69；A10-B70；A10-B71；A10-B72；A10-B73；A10-B74；A10-B75；A10-B76；A10-B77；A10-B78；A10-B79；A10-B80；A10-B81；A10-B82；A10-B83；A10-B84；A10-B85；A10-B86；A10-B87；A10-B88；A10-B89；A10-B90；A10-B91；A10-B92；A10-B93；A10-B94；A10-B95；A10-B96；A10-B97；A10-B98；A10-B99；A10-B100；A10-B101；A10-B102；A10-B103；A10-B104；A10-B105；A10-B106；A10-B107；A10-B108；A10-B109；A10-B110；A10-B111；A10-B112；A10-B113；
A11-B1；A11-B2；A11-B3；A11-B4；A11-B5；A11-B6；A11-B7；A11-B8；A11-B9；A11-B10；A11-B11；A11-B12；A11-B13；A11-B14；A11-B15；A11-B16；A11-B17；A11-B18；A11-B19；A11-B20；A11-B21；A11-B22；A11-B23；A11-B24；A11-B25；A11-B26；A11-B27；A11-B28；A11-B29；A11-B30；A11-B31；A11-B32；A11-B33；A11-B34；A11-B35；A11-B36；A11-B37；A11-B38；A11-B39；A11-B40；A11-B41；A11-B42；A11-B43；A11-B44；A11-B45；A11-B46；A11-B47；A11-B48；A11-B49；A11-B50；A11-B51；A11-B52；A11-B53；A11-B54；A11-B55；A11-B56；A11-B57；A11-B58；A11-B59；A11-B60；A11-B61；A11-B62；A11-B63；A11-B64；A11-B65；A11-B66；A11-B67；A11-B68；A11-B69；A11-B70；A11-B71；A11-B72；A11-B73；A11-B74；A11-B75；A11-B76；A11-B77；A11-B78；A11-B79；A11-B80；A11-B81；A11-B82；A11-B83；A11-B84；A11-B85；A11-B86；A11-B87；A11-B88；A11-B89；A11-B90；A11-B91；A11-B92；A11-B93；A11-B94；A11-B95；A11-B96；A11-B97；A11-B98；A11-B99；A11-B100；A11-B101；A11-B102；A11-B103；A11-B104；A11-B105；A11-B106；A11-B107；A11-B108；A11-B109；A11-B110；A11-B111；A11-B112；A11-B113；
A12-B1；A12-B2；A12-B3；A12-B4；A12-B5；A12-B6；A12-B7；A12-B8；A12-B9；A12-B10；A12-B11；A12-B12；A12-B13；A12-B14；A12-B15；A12-B16；A12-B17；A12-B18；A12-B19；A12-B20；A12-B21；A12-B22；A12-B23；A12-B24；A12-B25；A12-B26；A12-B27；A12-B28；A12-B29；A12-B30；A12-B31；A12-B32；A12-B33；A12-B34；A12-B35；A12-B36；A12-B37；A12-B38；A12-B39；A12-B40；A12-B41；A12-B42；A12-B43；A12-B44；A12-B45；A12-B46；A12-B47；A12-B48；A12-B49；A12-B50；A12-B51；A12-B52；A12-B53；A12-B54；A12-B55；A12-B56；A12-B57；A12-B58；A12-B59；A12-B60；A12-B61；A12-B62；A12-B63；A12-B64；A12-B65；A12-B66；A12-B67；A12-B68；A12-B69；A12-B70；A12-B71；A12-B72；A12-B73；A12-B74；A12-B75；A12-B76；A12-B77；A12-B78；A12-B79；A12-B80；A12-B81；A12-B82；A12-B83；A12-B84；A12-B85；A12-B86；A12-B87；A12-B88；A12-B89；A12-B90；A12-B91；A12-B92；A12-B93；A12-B94；A12-B95；A12-B96；A12-B97；A12-B98；A12-B99；A12-B100；A12-B101；A12-B102；A12-B103；A12-B104；A12-B105；A12-B106；A12-B107；A12-B108；A12-B109；A12-B110；A12-B111；A12-B112；A12-B113；
A13-B1；A13-B2；A13-B3；A13-B4；A13-B5；A13-B6；A13-B7；A13-B8；A13-B9；A13-B10；A13-B11；A13-B12；A13-B13；A13-B14；A13-B15；A13-B16；A13-B17；A13-B18；A13-B19；A13-B20；A13-B21；A13-B22；A13-B23；A13-B24；A13-B25；A13-B26；A13-B27；A13-B28；A13-B29；A13-B30；A13-B31；A13-B32；A13-B33；A13-B34；A13-B35；A13-B36；A13-B37；A13-B38；A13-B39；A13-B40；A13-B41；A13-B42；A13-B43；A13-B44；A13-B45；A13-B46；A13-B47；A13-B48；A13-B49；A13-B50；A13-B51；A13-B52；A13-B53；A13-B54；A13-B55；A13-B56；A13-B57；A13-B58；A13-B59；A13-B60；A13-B61；A13-B62；A13-B63；A13-B64；A13-B65；A13-B66；A13-B67；A13-B68；A13-B69；A13-B70；A13-B71；A13-B72；A13-B73；A13-B74；A13-B75；A13-B76；A13-B77；A13-B78；A13-B79；A13-B80；A13-B81；A13-B82；A13-B83；A13-B84；A13-B85；A13-B86；A13-B87；A13-B88；A13-B89；A13-B90；A13-B91；A13-B92；A13-B93；A13-B94；A13-B95；A13-B96；A13-B97；A13-B98；A13-B99；A13-B100；A13-B101；A13-B102；A13-B103；A13-B104；A13-B105；A13-B106；A13-B107；A13-B108；A13-B109；A13-B110；A13-B111；A13-B112；A13-B113；
A14-B1；A14-B2；A14-B3；A14-B4；A14-B5；A14-B6；A14-B7；A14-B8；A14-B9；A14-B10；A14-B11；A14-B12；A14-B13；A14-B14；A14-B15；A14-B16；A14-B17；A14-B18；A14-B19；A14-B20；A14-B21；A14-B22；A14-B23；A14-B24；A14-B25；A14-B26；A14-B27；A14-B28；A14-B29；A14-B30；A14-B31；A14-B32；A14-B33；A14-B34；A14-B35；A14-B36；A14-B37；A14-B38；A14-B39；A14-B40；A14-B41；A14-B42；A14-B43；A14-B44；A14-B45；A14-B46；A14-B47；A14-B48；A14-B49；A14-B50；A14-B51；A14-B52；A14-B53；A14-B54；A14-B55；A14-B56；A14-B57；A14-B58；A14-B59；A14-B60；A14-B61；A14-B62；A14-B63；A14-B64；A14-B65；A14-B66；A14-B67；A14-B68；A14-B69；A14-B70；A14-B71；A14-B72；A14-B73；A14-B74；A14-B75；A14-B76；A14-B77；A14-B78；A14-B79；A14-B80；A14-B81；A14-B82；A14-B83；A14-B84；A14-B85；A14-B86；A14-B87；A14-B88；A14-B89；A14-B90；A14-B91；A14-B92；A14-B93；A14-B94；A14-B95；A14-B96；A14-B97；A14-B98；A14-B99；A14-B100；A14-B101；A14-B102；A14-B103；A14-B104；A14-B105；A14-B106；A14-B107；A14-B108；A14-B109；A14-B110；A14-B111；A14-B112；A14-B113；
A15-B1；A15-B2；A15-B3；A15-B4；A15-B5；A15-B6；A15-B7；A15-B8；A15-B9；A15-B10；A15-B11；A15-B12；A15-B13；A15-B14；A15-B15；A15-B16；A15-B17；A15-B18；A15-B19；A15-B20；A15-B21；A15-B22；A15-B23；A15-B24；A15-B25；A15-B26；A15-B27；A15-B28；A15-B29；A15-B30；A15-B31；A15-B32；A15-B33；A15-B34；A15-B35；A15-B36；A15-B37；A15-B38；A15-B39；A15-B40；A15-B41；A15-B42；A15-B43；A15-B44；A15-B45；A15-B46；A15-B47；A15-B48；A15-B49；A15-B50；A15-B51；A15-B52；A15-B53；A15-B54；A15-B55；A15-B56；A15-B57；A15-B58；A15-B59；A15-B60；A15-B61；A15-B62；A15-B63；A15-B64；A15-B65；A15-B66；A15-B67；A15-B68；A15-B69；A15-B70；A15-B71；A15-B72；A15-B73；A15-B74；A15-B75；A15-B76；A15-B77；A15-B78；A15-B79；A15-B80；A15-B81；A15-B82；A15-B83；A15-B84；A15-B85；A15-B86；A15-B87；A15-B88；A15-B89；A15-B90；A15-B91；A15-B92；A15-B93；A15-B94；A15-B95；A15-B96；A15-B97；A15-B98；A15-B99；A15-B100；A15-B101；A15-B102；A15-B103；A15-B104；A15-B105；A15-B106；A15-B107；A15-B108；A15-B109；A15-B110；A15-B111；A15-B112；A15-B113；
A16-B1；A16-B2；A16-B3；A16-B4；A16-B5；A16-B6；A16-B7；A16-B8；A16-B9；A16-B10；A16-B11；A16-B12；A16-B13；A16-B14；A16-B15；A16-B16；A16-B17；A16-B18；A16-B19；A16-B20；A16-B21；A16-B22；A16-B23；A16-B24；A16-B25；A16-B26；A16-B27；A16-B28；A16-B29；A16-B30；A16-B31；A16-B32；A16-B33；A16-B34；A16-B35；A16-B36；A16-B37；A16-B38；A16-B39；A16-B40；A16-B41；A16-B42；A16-B43；A16-B44；A16-B45；A16-B46；A16-B47；A16-B48；A16-B49；A16-B50；A16-B51；A16-B52；A16-B53；A16-B54；A16-B55；A16-B56；A16-B57；A16-B58；A16-B59；A16-B60；A16-B61；A16-B62；A16-B63；A16-B64；A16-B65；A16-B66；A16-B67；A16-B68；A16-B69；A16-B70；A16-B71；A16-B72；A16-B73；A16-B74；A16-B75；A16-B76；A16-B77；A16-B78；A16-B79；A16-B80；A16-B81；A16-B82；A16-B83；A16-B84；A16-B85；A16-B86；A16-B87；A16-B88；A16-B89；A16-B90；A16-B91；A16-B92；A16-B93；A16-B94；A16-B95；A16-B96；A16-B97；A16-B98；A16-B99；A16-B100；A16-B101；A16-B102；A16-B103；A16-B104；A16-B105；A16-B106；A16-B107；A16-B108；A16-B109；A16-B110；A16-B111；A16-B112；A16-B113；
A17-B1；A17-B2；A17-B3；A17-B4；A17-B5；A17-B6；A17-B7；A17-B8；A17-B9；A17-B10；A17-B11；A17-B12；A17-B13；A17-B14；A17-B15；A17-B16；A17-B17；A17-B18；A17-B19；A17-B20；A17-B21；A17-B22；A17-B23；A17-B24；A17-B25；A17-B26；A17-B27；A17-B28；A17-B29；A17-B30；A17-B31；A17-B32；A17-B33；A17-B34；A17-B35；A17-B36；A17-B37；A17-B38；A17-B39；A17-B40；A17-B41；A17-B42；A17-B43；A17-B44；A17-B45；A17-B46；A17-B47；A17-B48；A17-B49；A17-B50；A17-B51；A17-B52；A17-B53；A17-B54；A17-B55；A17-B56；A17-B57；A17-B58；A17-B59；A17-B60；A17-B61；A17-B62；A17-B63；A17-B64；A17-B65；A17-B66；A17-B67；A17-B68；A17-B69；A17-B70；A17-B71；A17-B72；A17-B73；A17-B74；A17-B75；A17-B76；A17-B77；A17-B78；A17-B79；A17-B80；A17-B81；A17-B82；A17-B83；A17-B84；A17-B85；A17-B86；A17-B87；A17-B88；A17-B89；A17-B90；A17-B91；A17-B92；A17-B93；A17-B94；A17-B95；A17-B96；A17-B97；A17-B98；A17-B99；A17-B100；A17-B101；A17-B102；A17-B103；A17-B104；A17-B105；A17-B106；A17-B107；A17-B108；A17-B109；A17-B110；A17-B111；A17-B112；A17-B113；
A18-B1；A18-B2；A18-B3；A18-B4；A18-B5；A18-B6；A18-B7；A18-B8；A18-B9；A18-B10；A18-B11；A18-B12；A18-B13；A18-B14；A18-B15；A18-B16；A18-B17；A18-B18；A18-B19；A18-B20；A18-B21；A18-B22；A18-B23；A18-B24；A18-B25；A18-B26；A18-B27；A18-B28；A18-B29；A18-B30；A18-B31；A18-B32；A18-B33；A18-B34；A18-B35；A18-B36；A18-B37；A18-B38；A18-B39；A18-B40；A18-B41；A18-B42；A18-B43；A18-B44；A18-B45；A18-B46；A18-B47；A18-B48；A18-B49；A18-B50；A18-B51；A18-B52；A18-B53；A18-B54；A18-B55；A18-B56；A18-B57；A18-B58；A18-B59；A18-B60；A18-B61；A18-B62；A18-B63；A18-B64；A18-B65；A18-B66；A18-B67；A18-B68；A18-B69；A18-B70；A18-B71；A18-B72；A18-B73；A18-B74；A18-B75；A18-B76；A18-B77；A18-B78；A18-B79；A18-B80；A18-B81；A18-B82；A18-B83；A18-B84；A18-B85；A18-B86；A18-B87；A18-B88；A18-B89；A18-B90；A18-B91；A18-B92；A18-B93；A18-B94；A18-B95；A18-B96；A18-B97；A18-B98；A18-B99；A18-B100；A18-B101；A18-B102；A18-B103；A18-B104；A18-B105；A18-B106；A18-B107；A18-B108；A18-B109；A18-B110；A18-B111；A18-B112；A18-B113；
A19-B1；A19-B2；A19-B3；A19-B4；A19-B5；A19-B6；A19-B7；A19-B8；A19-B9；A19-B10；A19-B11；A19-B12；A19-B13；A19-B14；A19-B15；A19-B16；A19-B17；A19-B18；A19-B19；A19-B20；A19-B21；A19-B22；A19-B23；A19-B24；A19-B25；A19-B26；A19-B27；A19-B28；A19-B29；A19-B30；A19-B31；A19-B32；A19-B33；A19-B34；A19-B35；A19-B36；A19-B37；A19-B38；A19-B39；A19-B40；A19-B41；A19-B42；A19-B43；A19-B44；A19-B45；A19-B46；A19-B47；A19-B48；A19-B49；A19-B50；A19-B51；A19-B52；A19-B53；A19-B54；A19-B55；A19-B56；A19-B57；A19-B58；A19-B59；A19-B60；A19-B61；A19-B62；A19-B63；A19-B64；A19-B65；A19-B66；A19-B67；A19-B68；A19-B69；A19-B70；A19-B71；A19-B72；A19-B73；A19-B74；A19-B75；A19-B76；A19-B77；A19-B78；A19-B79；A19-B80；A19-B81；A19-B82；A19-B83；A19-B84；A19-B85；A19-B86；A19-B87；A19-B88；A19-B89；A19-B90；A19-B91；A19-B92；A19-B93；A19-B94；A19-B95；A19-B96；A19-B97；A19-B98；A19-B99；A19-B100；A19-B101；A19-B102；A19-B103；A19-B104；A19-B105；A19-B106；A19-B107；A19-B108；A19-B109；A19-B110；A19-B111；A19-B112；A19-B113；
A20-B1；A20-B2；A20-B3；A20-B4；A20-B5；A20-B6；A20-B7；A20-B8；A20-B9；A20-B10；A20-B11；A20-B12；A20-B13；A20-B14；A20-B15；A20-B16；A20-B17；A20-B18；A20-B19；A20-B20；A20-B21；A20-B22；A20-B23；A20-B24；A20-B25；A20-B26；A20-B27；A20-B28；A20-B29；A20-B30；A20-B31；A20-B32；A20-B33；A20-B34；A20-B35；A20-B36；A20-B37；A20-B38；A20-B39；A20-B40；A20-B41；A20-B42；A20-B43；A20-B44；A20-B45；A20-B46；A20-B47；A20-B48；A20-B49；A20-B50；A20-B51；A20-B52；A20-B53；A20-B54；A20-B55；A20-B56；A20-B57；A20-B58；A20-B59；A20-B60；A20-B61；A20-B62；A20-B63；A20-B64；A20-B65；A20-B66；A20-B67；A20-B68；A20-B69；A20-B70；A20-B71；A20-B72；A20-B73；A20-B74；A20-B75；A20-B76；A20-B77；A20-B78；A20-B79；A20-B80；A20-B81；A20-B82；A20-B83；A20-B84；A20-B85；A20-B86；A20-B87；A20-B88；A20-B89；A20-B90；A20-B91；A20-B92；A20-B93；A20-B94；A20-B95；A20-B96；A20-B97；A20-B98；A20-B99；A20-B100；A20-B101；A20-B102；A20-B103；A20-B104；A20-B105；A20-B106；A20-B107；A20-B108；A20-B109；A20-B110；A20-B111；A20-B112；A20-B113；
A21-B1；A21-B2；A21-B3；A21-B4；A21-B5；A21-B6；A21-B7；A21-B8；A21-B9；A21-B10；A21-B11；A21-B12；A21-B13；A21-B14；A21-B15；A21-B16；A21-B17；A21-B18；A21-B19；A21-B20；A21-B21；A21-B22；A21-B23；A21-B24；A21-B25；A21-B26；A21-B27；A21-B28；A21-B29；A21-B30；A21-B31；A21-B32；A21-B33；A21-B34；A21-B35；A21-B36；A21-B37；A21-B38；A21-B39；A21-B40；A21-B41；A21-B42；A21-B43；A21-B44；A21-B45；A21-B46；A21-B47；A21-B48；A21-B49；A21-B50；A21-B51；A21-B52；A21-B53；A21-B54；A21-B55；A21-B56；A21-B57；A21-B58；A21-B59；A21-B60；A21-B61；A21-B62；A21-B63；A21-B64；A21-B65；A21-B66；A21-B67；A21-B68；A21-B69；A21-B70；A21-B71；A21-B72；A21-B73；A21-B74；A21-B75；A21-B76；A21-B77；A21-B78；A21-B79；A21-B80；A21-B81；A21-B82；A21-B83；A21-B84；A21-B85；A21-B86；A21-B87；A21-B88；A21-B89；A21-B90；A21-B91；A21-B92；A21-B93；A21-B94；A21-B95；A21-B96；A21-B97；A21-B98；A21-B99；A21-B100；A21-B101；A21-B102；A21-B103；A21-B104；A21-B105；A21-B106；A21-B107；A21-B108；A21-B109；A21-B110；A21-B111；A21-B112；A21-B113；
A22-B1；A22-B2；A22-B3；A22-B4；A22-B5；A22-B6；A22-B7；A22-B8；A22-B9；A22-B10；A22-B11；A22-B12；A22-B13；A22-B14；A22-B15；A22-B16；A22-B17；A22-B18；A22-B19；A22-B20；A22-B21；A22-B22；A22-B23；A22-B24；A22-B25；A22-B26；A22-B27；A22-B28；A22-B29；A22-B30；A22-B31；A22-B32；A22-B33；A22-B34；A22-B35；A22-B36；A22-B37；A22-B38；A22-B39；A22-B40；A22-B41；A22-B42；A22-B43；A22-B44；A22-B45；A22-B46；A22-B47；A22-B48；A22-B49；A22-B50；A22-B51；A22-B52；A22-B53；A22-B54；A22-B55；A22-B56；A22-B57；A22-B58；A22-B59；A22-B60；A22-B61；A22-B62；A22-B63；A22-B64；A22-B65；A22-B66；A22-B67；A22-B68；A22-B69；A22-B70；A22-B71；A22-B72；A22-B73；A22-B74；A22-B75；A22-B76；A22-B77；A22-B78；A22-B79；A22-B80；A22-B81；A22-B82；A22-B83；A22-B84；A22-B85；A22-B86；A22-B87；A22-B88；A22-B89；A22-B90；A22-B91；A22-B92；A22-B93；A22-B94；A22-B95；A22-B96；A22-B97；A22-B98；A22-B99；A22-B100；A22-B101；A22-B102；A22-B103；A22-B104；A22-B105；A22-B106；A22-B107；A22-B108；A22-B109；A22-B110；A22-B111；A22-B112；A22-B113；
A23-B1；A23-B2；A23-B3；A23-B4；A23-B5；A23-B6；A23-B7；A23-B8；A23-B9；A23-B10；A23-B11；A23-B12；A23-B13；A23-B14；A23-B15；A23-B16；A23-B17；A23-B18；A23-B19；A23-B20；A23-B21；A23-B22；A23-B23；A23-B24；A23-B25；A23-B26；A23-B27；A23-B28；A23-B29；A23-B30；A23-B31；A23-B32；A23-B33；A23-B34；A23-B35；A23-B36；A23-B37；A23-B38；A23-B39；A23-B40；A23-B41；A23-B42；A23-B43；A23-B44；A23-B45；A23-B46；A23-B47；A23-B48；A23-B49；A23-B50；A23-B51；A23-B52；A23-B53；A23-B54；A23-B55；A23-B56；A23-B57；A23-B58；A23-B59；A23-B60；A23-B61；A23-B62；A23-B63；A23-B64；A23-B65；A23-B66；A23-B67；A23-B68；A23-B69；A23-B70；A23-B71；A23-B72；A23-B73；A23-B74；A23-B75；A23-B76；A23-B77；A23-B78；A23-B79；A23-B80；A23-B81；A23-B82；A23-B83；A23-B84；A23-B85；A23-B86；A23-B87；A23-B88；A23-B89；A23-B90；A23-B91；A23-B92；A23-B93；A23-B94；A23-B95；A23-B96；A23-B97；A23-B98；A23-B99；A23-B100；A23-B101；A23-B102；A23-B103；A23-B104；A23-B105；A23-B106；A23-B107；A23-B108；A23-B109；A23-B110；A23-B111；A23-B112；A23-B113；
A24-B1；A24-B2；A24-B3；A24-B4；A24-B5；A24-B6；A24-B7；A24-B8；A24-B9；A24-B10；A24-B11；A24-B12；A24-B13；A24-B14；A24-B15；A24-B16；A24-B17；A24-B18；A24-B19；A24-B20；A24-B21；A24-B22；A24-B23；A24-B24；A24-B25；A24-B26；A24-B27；A24-B28；A24-B29；A24-B30；A24-B31；A24-B32；A24-B33；A24-B34；A24-B35；A24-B36；A24-B37；A24-B38；A24-B39；A24-B40；A24-B41；A24-B42；A24-B43；A24-B44；A24-B45；A24-B46；A24-B47；A24-B48；A24-B49；A24-B50；A24-B51；A24-B52；A24-B53；A24-B54；A24-B55；A24-B56；A24-B57；A24-B58；A24-B59；A24-B60；A24-B61；A24-B62；A24-B63；A24-B64；A24-B65；A24-B66；A24-B67；A24-B68；A24-B69；A24-B70；A24-B71；A24-B72；A24-B73；A24-B74；A24-B75；A24-B76；A24-B77；A24-B78；A24-B79；A24-B80；A24-B81；A24-B82；A24-B83；A24-B84；A24-B85；A24-B86；A24-B87；A24-B88；A24-B89；A24-B90；A24-B91；A24-B92；A24-B93；A24-B94；A24-B95；A24-B96；A24-B97；A24-B98；A24-B99；A24-B100；A24-B101；A24-B102；A24-B103；A24-B104；A24-B105；A24-B106；A24-B107；A24-B108；A24-B109；A24-B110；A24-B111；A24-B112；A24-B113；
A25-B1；A25-B2；A25-B3；A25-B4；A25-B5；A25-B6；A25-B7；A25-B8；A25-B9；A25-B10；A25-B11；A25-B12；A25-B13；A25-B14；A25-B15；A25-B16；A25-B17；A25-B18；A25-B19；A25-B20；A25-B21；A25-B22；A25-B23；A25-B24；A25-B25；A25-B26；A25-B27；A25-B28；A25-B29；A25-B30；A25-B31；A25-B32；A25-B33；A25-B34；A25-B35；A25-B36；A25-B37；A25-B38；A25-B39；A25-B40；A25-B41；A25-B42；A25-B43；A25-B44；A25-B45；A25-B46；A25-B47；A25-B48；A25-B49；A25-B50；A25-B51；A25-B52；A25-B53；A25-B54；A25-B55；A25-B56；A25-B57；A25-B58；A25-B59；A25-B60；A25-B61；A25-B62；A25-B63；A25-B64；A25-B65；A25-B66；A25-B67；A25-B68；A25-B69；A25-B70；A25-B71；A25-B72；A25-B73；A25-B74；A25-B75；A25-B76；A25-B77；A25-B78；A25-B79；A25-B80；A25-B81；A25-B82；A25-B83；A25-B84；A25-B85；A25-B86；A25-B87；A25-B88；A25-B89；A25-B90；A25-B91；A25-B92；A25-B93；A25-B94；A25-B95；A25-B96；A25-B97；A25-B98；A25-B99；A25-B100；A25-B101；A25-B102；A25-B103；A25-B104；A25-B105；A25-B106；A25-B107；A25-B108；A25-B109；A25-B110；A25-B111；A25-B112；A25-B113；
A26-B1；A26-B2；A26-B3；A26-B4；A26-B5；A26-B6；A26-B7；A26-B8；A26-B9；A26-B10；A26-B11；A26-B12；A26-B13；A26-B14；A26-B15；A26-B16；A26-B17；A26-B18；A26-B19；A26-B20；A26-B21；A26-B22；A26-B23；A26-B24；A26-B25；A26-B26；A26-B27；A26-B28；A26-B29；A26-B30；A26-B31；A26-B32；A26-B33；A26-B34；A26-B35；A26-B36；A26-B37；A26-B38；A26-B39；A26-B40；A26-B41；A26-B42；A26-B43；A26-B44；A26-B45；A26-B46；A26-B47；A26-B48；A26-B49；A26-B50；A26-B51；A26-B52；A26-B53；A26-B54；A26-B55；A26-B56；A26-B57；A26-B58；A26-B59；A26-B60；A26-B61；A26-B62；A26-B63；A26-B64；A26-B65；A26-B66；A26-B67；A26-B68；A26-B69；A26-B70；A26-B71；A26-B72；A26-B73；A26-B74；A26-B75；A26-B76；A26-B77；A26-B78；A26-B79；A26-B80；A26-B81；A26-B82；A26-B83；A26-B84；A26-B85；A26-B86；A26-B87；A26-B88；A26-B89；A26-B90；A26-B91；A26-B92；A26-B93；A26-B94；A26-B95；A26-B96；A26-B97；A26-B98；A26-B99；A26-B100；A26-B101；A26-B102；A26-B103；A26-B104；A26-B105；A26-B106；A26-B107；A26-B108；A26-B109；A26-B110；A26-B111；A26-B112；A26-B113；
A27-B1；A27-B2；A27-B3；A27-B4；A27-B5；A27-B6；A27-B7；A27-B8；A27-B9；A27-B10；A27-B11；A27-B12；A27-B13；A27-B14；A27-B15；A27-B16；A27-B17；A27-B18；A27-B19；A27-B20；A27-B21；A27-B22；A27-B23；A27-B24；A27-B25；A27-B26；A27-B27；A27-B28；A27-B29；A27-B30；A27-B31；A27-B32；A27-B33；A27-B34；A27-B35；A27-B36；A27-B37；A27-B38；A27-B39；A27-B40；A27-B41；A27-B42；A27-B43；A27-B44；A27-B45；A27-B46；A27-B47；A27-B48；A27-B49；A27-B50；A27-B51；A27-B52；A27-B53；A27-B54；A27-B55；A27-B56；A27-B57；A27-B58；A27-B59；A27-B60；A27-B61；A27-B62；A27-B63；A27-B64；A27-B65；A27-B66；A27-B67；A27-B68；A27-B69；A27-B70；A27-B71；A27-B72；A27-B73；A27-B74；A27-B75；A27-B76；A27-B77；A27-B78；A27-B79；A27-B80；A27-B81；A27-B82；A27-B83；A27-B84；A27-B85；A27-B86；A27-B87；A27-B88；A27-B89；A27-B90；A27-B91；A27-B92；A27-B93；A27-B94；A27-B95；A27-B96；A27-B97；A27-B98；A27-B99；A27-B100；A27-B101；A27-B102；A27-B103；A27-B104；A27-B105；A27-B106；A27-B107；A27-B108；A27-B109；A27-B110；A27-B111；A27-B112；A27-B113；
A28-B1；A28-B2；A28-B3；A28-B4；A28-B5；A28-B6；A28-B7；A28-B8；A28-B9；A28-B10；A28-B11；A28-B12；A28-B13；A28-B14；A28-B15；A28-B16；A28-B17；A28-B18；A28-B19；A28-B20；A28-B21；A28-B22；A28-B23；A28-B24；A28-B25；A28-B26；A28-B27；A28-B28；A28-B29；A28-B30；A28-B31；A28-B32；A28-B33；A28-B34；A28-B35；A28-B36；A28-B37；A28-B38；A28-B39；A28-B40；A28-B41；A28-B42；A28-B43；A28-B44；A28-B45；A28-B46；A28-B47；A28-B48；A28-B49；A28-B50；A28-B51；A28-B52；A28-B53；A28-B54；A28-B55；A28-B56；A28-B57；A28-B58；A28-B59；A28-B60；A28-B61；A28-B62；A28-B63；A28-B64；A28-B65；A28-B66；A28-B67；A28-B68；A28-B69；A28-B70；A28-B71；A28-B72；A28-B73；A28-B74；A28-B75；A28-B76；A28-B77；A28-B78；A28-B79；A28-B80；A28-B81；A28-B82；A28-B83；A28-B84；A28-B85；A28-B86；A28-B87；A28-B88；A28-B89；A28-B90；A28-B91；A28-B92；A28-B93；A28-B94；A28-B95；A28-B96；A28-B97；A28-B98；A28-B99；A28-B100；A28-B101；A28-B102；A28-B103；A28-B104；A28-B105；A28-B106；A28-B107；A28-B108；A28-B109；A28-B110；A28-B111；A28-B112；A28-B113；
A29-B1；A29-B2；A29-B3；A29-B4；A29-B5；A29-B6；A29-B7；A29-B8；A29-B9；A29-B10；A29-B11；A29-B12；A29-B13；A29-B14；A29-B15；A29-B16；A29-B17；A29-B18；A29-B19；A29-B20；A29-B21；A29-B22；A29-B23；A29-B24；A29-B25；A29-B26；A29-B27；A29-B28；A29-B29；A29-B30；A29-B31；A29-B32；A29-B33；A29-B34；A29-B35；A29-B36；A29-B37；A29-B38；A29-B39；A29-B40；A29-B41；A29-B42；A29-B43；A29-B44；A29-B45；A29-B46；A29-B47；A29-B48；A29-B49；A29-B50；A29-B51；A29-B52；A29-B53；A29-B54；A29-B55；A29-B56；A29-B57；A29-B58；A29-B59；A29-B60；A29-B61；A29-B62；A29-B63；A29-B64；A29-B65；A29-B66；A29-B67；A29-B68；A29-B69；A29-B70；A29-B71；A29-B72；A29-B73；A29-B74；A29-B75；A29-B76；A29-B77；A29-B78；A29-B79；A29-B80；A29-B81；A29-B82；A29-B83；A29-B84；A29-B85；A29-B86；A29-B87；A29-B88；A29-B89；A29-B90；A29-B91；A29-B92；A29-B93；A29-B94；A29-B95；A29-B96；A29-B97；A29-B98；A29-B99；A29-B100；A29-B101；A29-B102；A29-B103；A29-B104；A29-B105；A29-B106；A29-B107；A29-B108；A29-B109；A29-B110；A29-B111；A29-B112；A29-B113；
A30-B1；A30-B2；A30-B3；A30-B4；A30-B5；A30-B6；A30-B7；A30-B8；A30-B9；A30-B10；A30-B11；A30-B12；A30-B13；A30-B14；A30-B15；A30-B16；A30-B17；A30-B18；A30-B19；A30-B20；A30-B21；A30-B22；A30-B23；A30-B24；A30-B25；A30-B26；A30-B27；A30-B28；A30-B29；A30-B30；A30-B31；A30-B32；A30-B33；A30-B34；A30-B35；A30-B36；A30-B37；A30-B38；A30-B39；A30-B40；A30-B41；A30-B42；A30-B43；A30-B44；A30-B45；A30-B46；A30-B47；A30-B48；A30-B49；A30-B50；A30-B51；A30-B52；A30-B53；A30-B54；A30-B55；A30-B56；A30-B57；A30-B58；A30-B59；A30-B60；A30-B61；A30-B62；A30-B63；A30-B64；A30-B65；A30-B66；A30-B67；A30-B68；A30-B69；A30-B70；A30-B71；A30-B72；A30-B73；A30-B74；A30-B75；A30-B76；A30-B77；A30-B78；A30-B79；A30-B80；A30-B81；A30-B82；A30-B83；A30-B84；A30-B85；A30-B86；A30-B87；A30-B88；A30-B89；A30-B90；A30-B91；A30-B92；A30-B93；A30-B94；A30-B95；A30-B96；A30-B97；A30-B98；A30-B99；A30-B100；A30-B101；A30-B102；A30-B103；A30-B104；A30-B105；A30-B106；A30-B107；A30-B108；A30-B109；A30-B110；A30-B111；A30-B112；A30-B113；
A31-B1；A31-B2；A31-B3；A31-B4；A31-B5；A31-B6；A31-B7；A31-B8；A31-B9；A31-B10；A31-B11；A31-B12；A31-B13；A31-B14；A31-B15；A31-B16；A31-B17；A31-B18；A31-B19；A31-B20；A31-B21；A31-B22；A31-B23；A31-B24；A31-B25；A31-B26；A31-B27；A31-B28；A31-B29；A31-B30；A31-B31；A31-B32；A31-B33；A31-B34；A31-B35；A31-B36；A31-B37；A31-B38；A31-B39；A31-B40；A31-B41；A31-B42；A31-B43；A31-B44；A31-B45；A31-B46；A31-B47；A31-B48；A31-B49；A31-B50；A31-B51；A31-B52；A31-B53；A31-B54；A31-B55；A31-B56；A31-B57；A31-B58；A31-B59；A31-B60；A31-B61；A31-B62；A31-B63；A31-B64；A31-B65；A31-B66；A31-B67；A31-B68；A31-B69；A31-B70；A31-B71；A31-B72；A31-B73；A31-B74；A31-B75；A31-B76；A31-B77；A31-B78；A31-B79；A31-B80；A31-B81；A31-B82；A31-B83；A31-B84；A31-B85；A31-B86；A31-B87；A31-B88；A31-B89；A31-B90；A31-B91；A31-B92；A31-B93；A31-B94；A31-B95；A31-B96；A31-B97；A31-B98；A31-B99；A31-B100；A31-B101；A31-B102；A31-B103；A31-B104；A31-B105；A31-B106；A31-B107；A31-B108；A31-B109；A31-B110；A31-B111；A31-B112；A31-B113；
A32-B1；A32-B2；A32-B3；A32-B4；A32-B5；A32-B6；A32-B7；A32-B8；A32-B9；A32-B10；A32-B11；A32-B12；A32-B13；A32-B14；A32-B15；A32-B16；A32-B17；A32-B18；A32-B19；A32-B20；A32-B21；A32-B22；A32-B23；A32-B24；A32-B25；A32-B26；A32-B27；A32-B28；A32-B29；A32-B30；A32-B31；A32-B32；A32-B33；A32-B34；A32-B35；A32-B36；A32-B37；A32-B38；A32-B39；A32-B40；A32-B41；A32-B42；A32-B43；A32-B44；A32-B45；A32-B46；A32-B47；A32-B48；A32-B49；A32-B50；A32-B51；A32-B52；A32-B53；A32-B54；A32-B55；A32-B56；A32-B57；A32-B58；A32-B59；A32-B60；A32-B61；A32-B62；A32-B63；A32-B64；A32-B65；A32-B66；A32-B67；A32-B68；A32-B69；A32-B70；A32-B71；A32-B72；A32-B73；A32-B74；A32-B75；A32-B76；A32-B77；A32-B78；A32-B79；A32-B80；A32-B81；A32-B82；A32-B83；A32-B84；A32-B85；A32-B86；A32-B87；A32-B88；A32-B89；A32-B90；A32-B91；A32-B92；A32-B93；A32-B94；A32-B95；A32-B96；A32-B97；A32-B98；A32-B99；A32-B100；A32-B101；A32-B102；A32-B103；A32-B104；A32-B105；A32-B106；A32-B107；A32-B108；A32-B109；A32-B110；A32-B111；A32-B112；A32-B113；
A33-B1；A33-B2；A33-B3；A33-B4；A33-B5；A33-B6；A33-B7；A33-B8；A33-B9；A33-B10；A33-B11；A33-B12；A33-B13；A33-B14；A33-B15；A33-B16；A33-B17；A33-B18；A33-B19；A33-B20；A33-B21；A33-B22；A33-B23；A33-B24；A33-B25；A33-B26；A33-B27；A33-B28；A33-B29；A33-B30；A33-B31；A33-B32；A33-B33；A33-B34；A33-B35；A33-B36；A33-B37；A33-B38；A33-B39；A33-B40；A33-B41；A33-B42；A33-B43；A33-B44；A33-B45；A33-B46；A33-B47；A33-B48；A33-B49；A33-B50；A33-B51；A33-B52；A33-B53；A33-B54；A33-B55；A33-B56；A33-B57；A33-B58；A33-B59；A33-B60；A33-B61；A33-B62；A33-B63；A33-B64；A33-B65；A33-B66；A33-B67；A33-B68；A33-B69；A33-B70；A33-B71；A33-B72；A33-B73；A33-B74；A33-B75；A33-B76；A33-B77；A33-B78；A33-B79；A33-B80；A33-B81；A33-B82；A33-B83；A33-B84；A33-B85；A33-B86；A33-B87；A33-B88；A33-B89；A33-B90；A33-B91；A33-B92；A33-B93；A33-B94；A33-B95；A33-B96；A33-B97；A33-B98；A33-B99；A33-B100；A33-B101；A33-B102；A33-B103；A33-B104；A33-B105；A33-B106；A33-B107；A33-B108；A33-B109；A33-B110；A33-B111；A33-B112；A33-B113；
A34-B1；A34-B2；A34-B3；A34-B4；A34-B5；A34-B6；A34-B7；A34-B8；A34-B9；A34-B10；A34-B11；A34-B12；A34-B13；A34-B14；A34-B15；A34-B16；A34-B17；A34-B18；A34-B19；A34-B20；A34-B21；A34-B22；A34-B23；A34-B24；A34-B25；A34-B26；A34-B27；A34-B28；A34-B29；A34-B30；A34-B31；A34-B32；A34-B33；A34-B34；A34-B35；A34-B36；A34-B37；A34-B38；A34-B39；A34-B40；A34-B41；A34-B42；A34-B43；A34-B44；A34-B45；A34-B46；A34-B47；A34-B48；A34-B49；A34-B50；A34-B51；A34-B52；A34-B53；A34-B54；A34-B55；A34-B56；A34-B57；A34-B58；A34-B59；A34-B60；A34-B61；A34-B62；A34-B63；A34-B64；A34-B65；A34-B66；A34-B67；A34-B68；A34-B69；A34-B70；A34-B71；A34-B72；A34-B73；A34-B74；A34-B75；A34-B76；A34-B77；A34-B78；A34-B79；A34-B80；A34-B81；A34-B82；A34-B83；A34-B84；A34-B85；A34-B86；A34-B87；A34-B88；A34-B89；A34-B90；A34-B91；A34-B92；A34-B93；A34-B94；A34-B95；A34-B96；A34-B97；A34-B98；A34-B99；A34-B100；A34-B101；A34-B102；A34-B103；A34-B104；A34-B105；A34-B106；A34-B107；A34-B108；A34-B109；A34-B110；A34-B111；A34-B112；A34-B113；
A35-B1；A35-B2；A35-B3；A35-B4；A35-B5；A35-B6；A35-B7；A35-B8；A35-B9；A35-B10；A35-B11；A35-B12；A35-B13；A35-B14；A35-B15；A35-B16；A35-B17；A35-B18；A35-B19；A35-B20；A35-B21；A35-B22；A35-B23；A35-B24；A35-B25；A35-B26；A35-B27；A35-B28；A35-B29；A35-B30；A35-B31；A35-B32；A35-B33；A35-B34；A35-B35；A35-B36；A35-B37；A35-B38；A35-B39；A35-B40；A35-B41；A35-B42；A35-B43；A35-B44；A35-B45；A35-B46；A35-B47；A35-B48；A35-B49；A35-B50；A35-B51；A35-B52；A35-B53；A35-B54；A35-B55；A35-B56；A35-B57；A35-B58；A35-B59；A35-B60；A35-B61；A35-B62；A35-B63；A35-B64；A35-B65；A35-B66；A35-B67；A35-B68；A35-B69；A35-B70；A35-B71；A35-B72；A35-B73；A35-B74；A35-B75；A35-B76；A35-B77；A35-B78；A35-B79；A35-B80；A35-B81；A35-B82；A35-B83；A35-B84；A35-B85；A35-B86；A35-B87；A35-B88；A35-B89；A35-B90；A35-B91；A35-B92；A35-B93；A35-B94；A35-B95；A35-B96；A35-B97；A35-B98；A35-B99；A35-B100；A35-B101；A35-B102；A35-B103；A35-B104；A35-B105；A35-B106；A35-B107；A35-B108；A35-B109；A35-B110；A35-B111；A35-B112；A35-B113；
A36-B1；A36-B2；A36-B3；A36-B4；A36-B5；A36-B6；A36-B7；A36-B8；A36-B9；A36-B10；A36-B11；A36-B12；A36-B13；A36-B14；A36-B15；A36-B16；A36-B17；A36-B18；A36-B19；A36-B20；A36-B21；A36-B22；A36-B23；A36-B24；A36-B25；A36-B26；A36-B27；A36-B28；A36-B29；A36-B30；A36-B31；A36-B32；A36-B33；A36-B34；A36-B35；A36-B36；A36-B37；A36-B38；A36-B39；A36-B40；A36-B41；A36-B42；A36-B43；A36-B44；A36-B45；A36-B46；A36-B47；A36-B48；A36-B49；A36-B50；A36-B51；A36-B52；A36-B53；A36-B54；A36-B55；A36-B56；A36-B57；A36-B58；A36-B59；A36-B60；A36-B61；A36-B62；A36-B63；A36-B64；A36-B65；A36-B66；A36-B67；A36-B68；A36-B69；A36-B70；A36-B71；A36-B72；A36-B73；A36-B74；A36-B75；A36-B76；A36-B77；A36-B78；A36-B79；A36-B80；A36-B81；A36-B82；A36-B83；A36-B84；A36-B85；A36-B86；A36-B87；A36-B88；A36-B89；A36-B90；A36-B91；A36-B92；A36-B93；A36-B94；A36-B95；A36-B96；A36-B97；A36-B98；A36-B99；A36-B100；A36-B101；A36-B102；A36-B103；A36-B104；A36-B105；A36-B106；A36-B107；A36-B108；A36-B109；A36-B110；A36-B111；A36-B112；A36-B113；
A37-B1；A37-B2；A37-B3；A37-B4；A37-B5；A37-B6；A37-B7；A37-B8；A37-B9；A37-B10；A37-B11；A37-B12；A37-B13；A37-B14；A37-B15；A37-B16；A37-B17；A37-B18；A37-B19；A37-B20；A37-B21；A37-B22；A37-B23；A37-B24；A37-B25；A37-B26；A37-B27；A37-B28；A37-B29；A37-B30；A37-B31；A37-B32；A37-B33；A37-B34；A37-B35；A37-B36；A37-B37；A37-B38；A37-B39；A37-B40；A37-B41；A37-B42；A37-B43；A37-B44；A37-B45；A37-B46；A37-B47；A37-B48；A37-B49；A37-B50；A37-B51；A37-B52；A37-B53；A37-B54；A37-B55；A37-B56；A37-B57；A37-B58；A37-B59；A37-B60；A37-B61；A37-B62；A37-B63；A37-B64；A37-B65；A37-B66；A37-B67；A37-B68；A37-B69；A37-B70；A37-B71；A37-B72；A37-B73；A37-B74；A37-B75；A37-B76；A37-B77；A37-B78；A37-B79；A37-B80；A37-B81；A37-B82；A37-B83；A37-B84；A37-B85；A37-B86；A37-B87；A37-B88；A37-B89；A37-B90；A37-B91；A37-B92；A37-B93；A37-B94；A37-B95；A37-B96；A37-B97；A37-B98；A37-B99；A37-B100；A37-B101；A37-B102；A37-B103；A37-B104；A37-B105；A37-B106；A37-B107；A37-B108；A37-B109；A37-B110；A37-B111；A37-B112；A37-B113；
A38-B1；A38-B2；A38-B3；A38-B4；A38-B5；A38-B6；A38-B7；A38-B8；A38-B9；A38-B10；A38-B11；A38-B12；A38-B13；A38-B14；A38-B15；A38-B16；A38-B17；A38-B18；A38-B19；A38-B20；A38-B21；A38-B22；A38-B23；A38-B24；A38-B25；A38-B26；A38-B27；A38-B28；A38-B29；A38-B30；A38-B31；A38-B32；A38-B33；A38-B34；A38-B35；A38-B36；A38-B37；A38-B38；A38-B39；A38-B40；A38-B41；A38-B42；A38-B43；A38-B44；A38-B45；A38-B46；A38-B47；A38-B48；A38-B49；A38-B50；A38-B51；A38-B52；A38-B53；A38-B54；A38-B55；A38-B56；A38-B57；A38-B58；A38-B59；A38-B60；A38-B61；A38-B62；A38-B63；A38-B64；A38-B65；A38-B66；A38-B67；A38-B68；A38-B69；A38-B70；A38-B71；A38-B72；A38-B73；A38-B74；A38-B75；A38-B76；A38-B77；A38-B78；A38-B79；A38-B80；A38-B81；A38-B82；A38-B83；A38-B84；A38-B85；A38-B86；A38-B87；A38-B88；A38-B89；A38-B90；A38-B91；A38-B92；A38-B93；A38-B94；A38-B95；A38-B96；A38-B97；A38-B98；A38-B99；A38-B100；A38-B101；A38-B102；A38-B103；A38-B104；A38-B105；A38-B106；A38-B107；A38-B108；A38-B109；A38-B110；A38-B111；A38-B112；A38-B113；
A39-B1；A39-B2；A39-B3；A39-B4；A39-B5；A39-B6；A39-B7；A39-B8；A39-B9；A39-B10；A39-B11；A39-B12；A39-B13；A39-B14；A39-B15；A39-B16；A39-B17；A39-B18；A39-B19；A39-B20；A39-B21；A39-B22；A39-B23；A39-B24；A39-B25；A39-B26；A39-B27；A39-B28；A39-B29；A39-B30；A39-B31；A39-B32；A39-B33；A39-B34；A39-B35；A39-B36；A39-B37；A39-B38；A39-B39；A39-B40；A39-B41；A39-B42；A39-B43；A39-B44；A39-B45；A39-B46；A39-B47；A39-B48；A39-B49；A39-B50；A39-B51；A39-B52；A39-B53；A39-B54；A39-B55；A39-B56；A39-B57；A39-B58；A39-B59；A39-B60；A39-B61；A39-B62；A39-B63；A39-B64；A39-B65；A39-B66；A39-B67；A39-B68；A39-B69；A39-B70；A39-B71；A39-B72；A39-B73；A39-B74；A39-B75；A39-B76；A39-B77；A39-B78；A39-B79；A39-B80；A39-B81；A39-B82；A39-B83；A39-B84；A39-B85；A39-B86；A39-B87；A39-B88；A39-B89；A39-B90；A39-B91；A39-B92；A39-B93；A39-B94；A39-B95；A39-B96；A39-B97；A39-B98；A39-B99；A39-B100；A39-B101；A39-B102；A39-B103；A39-B104；A39-B105；A39-B106；A39-B107；A39-B108；A39-B109；A39-B110；A39-B111；A39-B112；A39-B113；
A40-B1；A40-B2；A40-B3；A40-B4；A40-B5；A40-B6；A40-B7；A40-B8；A40-B9；A40-B10；A40-B11；A40-B12；A40-B13；A40-B14；A40-B15；A40-B16；A40-B17；A40-B18；A40-B19；A40-B20；A40-B21；A40-B22；A40-B23；A40-B24；A40-B25；A40-B26；A40-B27；A40-B28；A40-B29；A40-B30；A40-B31；A40-B32；A40-B33；A40-B34；A40-B35；A40-B36；A40-B37；A40-B38；A40-B39；A40-B40；A40-B41；A40-B42；A40-B43；A40-B44；A40-B45；A40-B46；A40-B47；A40-B48；A40-B49；A40-B50；A40-B51；A40-B52；A40-B53；A40-B54；A40-B55；A40-B56；A40-B57；A40-B58；A40-B59；A40-B60；A40-B61；A40-B62；A40-B63；A40-B64；A40-B65；A40-B66；A40-B67；A40-B68；A40-B69；A40-B70；A40-B71；A40-B72；A40-B73；A40-B74；A40-B75；A40-B76；A40-B77；A40-B78；A40-B79；A40-B80；A40-B81；A40-B82；A40-B83；A40-B84；A40-B85；A40-B86；A40-B87；A40-B88；A40-B89；A40-B90；A40-B91；A40-B92；A40-B93；A40-B94；A40-B95；A40-B96；A40-B97；A40-B98；A40-B99；A40-B100；A40-B101；A40-B102；A40-B103；A40-B104；A40-B105；A40-B106；A40-B107；A40-B108；A40-B109；A40-B110；A40-B111；A40-B112；A40-B113；
A41-B1；A41-B2；A41-B3；A41-B4；A41-B5；A41-B6；A41-B7；A41-B8；A41-B9；A41-B10；A41-B11；A41-B12；A41-B13；A41-B14；A41-B15；A41-B16；A41-B17；A41-B18；A41-B19；A41-B20；A41-B21；A41-B22；A41-B23；A41-B24；A41-B25；A41-B26；A41-B27；A41-B28；A41-B29；A41-B30；A41-B31；A41-B32；A41-B33；A41-B34；A41-B35；A41-B36；A41-B37；A41-B38；A41-B39；A41-B40；A41-B41；A41-B42；A41-B43；A41-B44；A41-B45；A41-B46；A41-B47；A41-B48；A41-B49；A41-B50；A41-B51；A41-B52；A41-B53；A41-B54；A41-B55；A41-B56；A41-B57；A41-B58；A41-B59；A41-B60；A41-B61；A41-B62；A41-B63；A41-B64；A41-B65；A41-B66；A41-B67；A41-B68；A41-B69；A41-B70；A41-B71；A41-B72；A41-B73；A41-B74；A41-B75；A41-B76；A41-B77；A41-B78；A41-B79；A41-B80；A41-B81；A41-B82；A41-B83；A41-B84；A41-B85；A41-B86；A41-B87；A41-B88；A41-B89；A41-B90；A41-B91；A41-B92；A41-B93；A41-B94；A41-B95；A41-B96；A41-B97；A41-B98；A41-B99；A41-B100；A41-B101；A41-B102；A41-B103；A41-B104；A41-B105；A41-B106；A41-B107；A41-B108；A41-B109；A41-B110；A41-B111；A41-B112；A41-B113；
A42-B1；A42-B2；A42-B3；A42-B4；A42-B5；A42-B6；A42-B7；A42-B8；A42-B9；A42-B10；A42-B11；A42-B12；A42-B13；A42-B14；A42-B15；A42-B16；A42-B17；A42-B18；A42-B19；A42-B20；A42-B21；A42-B22；A42-B23；A42-B24；A42-B25；A42-B26；A42-B27；A42-B28；A42-B29；A42-B30；A42-B31；A42-B32；A42-B33；A42-B34；A42-B35；A42-B36；A42-B37；A42-B38；A42-B39；A42-B40；A42-B41；A42-B42；A42-B43；A42-B44；A42-B45；A42-B46；A42-B47；A42-B48；A42-B49；A42-B50；A42-B51；A42-B52；A42-B53；A42-B54；A42-B55；A42-B56；A42-B57；A42-B58；A42-B59；A42-B60；A42-B61；A42-B62；A42-B63；A42-B64；A42-B65；A42-B66；A42-B67；A42-B68；A42-B69；A42-B70；A42-B71；A42-B72；A42-B73；A42-B74；A42-B75；A42-B76；A42-B77；A42-B78；A42-B79；A42-B80；A42-B81；A42-B82；A42-B83；A42-B84；A42-B85；A42-B86；A42-B87；A42-B88；A42-B89；A42-B90；A42-B91；A42-B92；A42-B93；A42-B94；A42-B95；A42-B96；A42-B97；A42-B98；A42-B99；A42-B100；A42-B101；A42-B102；A42-B103；A42-B104；A42-B105；A42-B106；A42-B107；A42-B108；A42-B109；A42-B110；A42-B111；A42-B112；A42-B113；
A43-B1；A43-B2；A43-B3；A43-B4；A43-B5；A43-B6；A43-B7；A43-B8；A43-B9；A43-B10；A43-B11；A43-B12；A43-B13；A43-B14；A43-B15；A43-B16；A43-B17；A43-B18；A43-B19；A43-B20；A43-B21；A43-B22；A43-B23；A43-B24；A43-B25；A43-B26；A43-B27；A43-B28；A43-B29；A43-B30；A43-B31；A43-B32；A43-B33；A43-B34；A43-B35；A43-B36；A43-B37；A43-B38；A43-B39；A43-B40；A43-B41；A43-B42；A43-B43；A43-B44；A43-B45；A43-B46；A43-B47；A43-B48；A43-B49；A43-B50；A43-B51；A43-B52；A43-B53；A43-B54；A43-B55；A43-B56；A43-B57；A43-B58；A43-B59；A43-B60；A43-B61；A43-B62；A43-B63；A43-B64；A43-B65；A43-B66；A43-B67；A43-B68；A43-B69；A43-B70；A43-B71；A43-B72；A43-B73；A43-B74；A43-B75；A43-B76；A43-B77；A43-B78；A43-B79；A43-B80；A43-B81；A43-B82；A43-B83；A43-B84；A43-B85；A43-B86；A43-B87；A43-B88；A43-B89；A43-B90；A43-B91；A43-B92；A43-B93；A43-B94；A43-B95；A43-B96；A43-B97；A43-B98；A43-B99；A43-B100；A43-B101；A43-B102；A43-B103；A43-B104；A43-B105；A43-B106；A43-B107；A43-B108；A43-B109；A43-B110；A43-B111；A43-B112；A43-B113；
A44-B1；A44-B2；A44-B3；A44-B4；A44-B5；A44-B6；A44-B7；A44-B8；A44-B9；A44-B10；A44-B11；A44-B12；A44-B13；A44-B14；A44-B15；A44-B16；A44-B17；A44-B18；A44-B19；A44-B20；A44-B21；A44-B22；A44-B23；A44-B24；A44-B25；A44-B26；A44-B27；A44-B28；A44-B29；A44-B30；A44-B31；A44-B32；A44-B33；A44-B34；A44-B35；A44-B36；A44-B37；A44-B38；A44-B39；A44-B40；A44-B41；A44-B42；A44-B43；A44-B44；A44-B45；A44-B46；A44-B47；A44-B48；A44-B49；A44-B50；A44-B51；A44-B52；A44-B53；A44-B54；A44-B55；A44-B56；A44-B57；A44-B58；A44-B59；A44-B60；A44-B61；A44-B62；A44-B63；A44-B64；A44-B65；A44-B66；A44-B67；A44-B68；A44-B69；A44-B70；A44-B71；A44-B72；A44-B73；A44-B74；A44-B75；A44-B76；A44-B77；A44-B78；A44-B79；A44-B80；A44-B81；A44-B82；A44-B83；A44-B84；A44-B85；A44-B86；A44-B87；A44-B88；A44-B89；A44-B90；A44-B91；A44-B92；A44-B93；A44-B94；A44-B95；A44-B96；A44-B97；A44-B98；A44-B99；A44-B100；A44-B101；A44-B102；A44-B103；A44-B104；A44-B105；A44-B106；A44-B107；A44-B108；A44-B109；A44-B110；A44-B111；A44-B112；A44-B113；
A45-B1；A45-B2；A45-B3；A45-B4；A45-B5；A45-B6；A45-B7；A45-B8；A45-B9；A45-B10；A45-B11；A45-B12；A45-B13；A45-B14；A45-B15；A45-B16；A45-B17；A45-B18；A45-B19；A45-B20；A45-B21；A45-B22；A45-B23；A45-B24；A45-B25；A45-B26；A45-B27；A45-B28；A45-B29；A45-B30；A45-B31；A45-B32；A45-B33；A45-B34；A45-B35；A45-B36；A45-B37；A45-B38；A45-B39；A45-B40；A45-B41；A45-B42；A45-B43；A45-B44；A45-B45；A45-B46；A45-B47；A45-B48；A45-B49；A45-B50；A45-B51；A45-B52；A45-B53；A45-B54；A45-B55；A45-B56；A45-B57；A45-B58；A45-B59；A45-B60；A45-B61；A45-B62；A45-B63；A45-B64；A45-B65；A45-B66；A45-B67；A45-B68；A45-B69；A45-B70；A45-B71；A45-B72；A45-B73；A45-B74；A45-B75；A45-B76；A45-B77；A45-B78；A45-B79；A45-B80；A45-B81；A45-B82；A45-B83；A45-B84；A45-B85；A45-B86；A45-B87；A45-B88；A45-B89；A45-B90；A45-B91；A45-B92；A45-B93；A45-B94；A45-B95；A45-B96；A45-B97；A45-B98；A45-B99；A45-B100；A45-B101；A45-B102；A45-B103；A45-B104；A45-B105；A45-B106；A45-B107；A45-B108；A45-B109；A45-B110；A45-B111；A45-B112；A45-B113；
A46-B1；A46-B2；A46-B3；A46-B4；A46-B5；A46-B6；A46-B7；A46-B8；A46-B9；A46-B10；A46-B11；A46-B12；A46-B13；A46-B14；A46-B15；A46-B16；A46-B17；A46-B18；A46-B19；A46-B20；A46-B21；A46-B22；A46-B23；A46-B24；A46-B25；A46-B26；A46-B27；A46-B28；A46-B29；A46-B30；A46-B31；A46-B32；A46-B33；A46-B34；A46-B35；A46-B36；A46-B37；A46-B38；A46-B39；A46-B40；A46-B41；A46-B42；A46-B43；A46-B44；A46-B45；A46-B46；A46-B47；A46-B48；A46-B49；A46-B50；A46-B51；A46-B52；A46-B53；A46-B54；A46-B55；A46-B56；A46-B57；A46-B58；A46-B59；A46-B60；A46-B61；A46-B62；A46-B63；A46-B64；A46-B65；A46-B66；A46-B67；A46-B68；A46-B69；A46-B70；A46-B71；A46-B72；A46-B73；A46-B74；A46-B75；A46-B76；A46-B77；A46-B78；A46-B79；A46-B80；A46-B81；A46-B82；A46-B83；A46-B84；A46-B85；A46-B86；A46-B87；A46-B88；A46-B89；A46-B90；A46-B91；A46-B92；A46-B93；A46-B94；A46-B95；A46-B96；A46-B97；A46-B98；A46-B99；A46-B100；A46-B101；A46-B102；A46-B103；A46-B104；A46-B105；A46-B106；A46-B107；A46-B108；A46-B109；A46-B110；A46-B111；A46-B112；A46-B113；
A47-B1；A47-B2；A47-B3；A47-B4；A47-B5；A47-B6；A47-B7；A47-B8；A47-B9；A47-B10；A47-B11；A47-B12；A47-B13；A47-B14；A47-B15；A47-B16；A47-B17；A47-B18；A47-B19；A47-B20；A47-B21；A47-B22；A47-B23；A47-B24；A47-B25；A47-B26；A47-B27；A47-B28；A47-B29；A47-B30；A47-B31；A47-B32；A47-B33；A47-B34；A47-B35；A47-B36；A47-B37；A47-B38；A47-B39；A47-B40；A47-B41；A47-B42；A47-B43；A47-B44；A47-B45；A47-B46；A47-B47；A47-B48；A47-B49；A47-B50；A47-B51；A47-B52；A47-B53；A47-B54；A47-B55；A47-B56；A47-B57；A47-B58；A47-B59；A47-B60；A47-B61；A47-B62；A47-B63；A47-B64；A47-B65；A47-B66；A47-B67；A47-B68；A47-B69；A47-B70；A47-B71；A47-B72；A47-B73；A47-B74；A47-B75；A47-B76；A47-B77；A47-B78；A47-B79；A47-B80；A47-B81；A47-B82；A47-B83；A47-B84；A47-B85；A47-B86；A47-B87；A47-B88；A47-B89；A47-B90；A47-B91；A47-B92；A47-B93；A47-B94；A47-B95；A47-B96；A47-B97；A47-B98；A47-B99；A47-B100；A47-B101；A47-B102；A47-B103；A47-B104；A47-B105；A47-B106；A47-B107；A47-B108；A47-B109；A47-B110；A47-B111；A47-B112；A47-B113；
A48-B1；A48-B2；A48-B3；A48-B4；A48-B5；A48-B6；A48-B7；A48-B8；A48-B9；A48-B10；A48-B11；A48-B12；A48-B13；A48-B14；A48-B15；A48-B16；A48-B17；A48-B18；A48-B19；A48-B20；A48-B21；A48-B22；A48-B23；A48-B24；A48-B25；A48-B26；A48-B27；A48-B28；A48-B29；A48-B30；A48-B31；A48-B32；A48-B33；A48-B34；A48-B35；A48-B36；A48-B37；A48-B38；A48-B39；A48-B40；A48-B41；A48-B42；A48-B43；A48-B44；A48-B45；A48-B46；A48-B47；A48-B48；A48-B49；A48-B50；A48-B51；A48-B52；A48-B53；A48-B54；A48-B55；A48-B56；A48-B57；A48-B58；A48-B59；A48-B60；A48-B61；A48-B62；A48-B63；A48-B64；A48-B65；A48-B66；A48-B67；A48-B68；A48-B69；A48-B70；A48-B71；A48-B72；A48-B73；A48-B74；A48-B75；A48-B76；A48-B77；A48-B78；A48-B79；A48-B80；A48-B81；A48-B82；A48-B83；A48-B84；A48-B85；A48-B86；A48-B87；A48-B88；A48-B89；A48-B90；A48-B91；A48-B92；A48-B93；A48-B94；A48-B95；A48-B96；A48-B97；A48-B98；A48-B99；A48-B100；A48-B101；A48-B102；A48-B103；A48-B104；A48-B105；A48-B106；A48-B107；A48-B108；A48-B109；A48-B110；A48-B111；A48-B112；A48-B113；
A49-B1；A49-B2；A49-B3；A49-B4；A49-B5；A49-B6；A49-B7；A49-B8；A49-B9；A49-B10；A49-B11；A49-B12；A49-B13；A49-B14；A49-B15；A49-B16；A49-B17；A49-B18；A49-B19；A49-B20；A49-B21；A49-B22；A49-B23；A49-B24；A49-B25；A49-B26；A49-B27；A49-B28；A49-B29；A49-B30；A49-B31；A49-B32；A49-B33；A49-B34；A49-B35；A49-B36；A49-B37；A49-B38；A49-B39；A49-B40；A49-B41；A49-B42；A49-B43；A49-B44；A49-B45；A49-B46；A49-B47；A49-B48；A49-B49；A49-B50；A49-B51；A49-B52；A49-B53；A49-B54；A49-B55；A49-B56；A49-B57；A49-B58；A49-B59；A49-B60；A49-B61；A49-B62；A49-B63；A49-B64；A49-B65；A49-B66；A49-B67；A49-B68；A49-B69；A49-B70；A49-B71；A49-B72；A49-B73；A49-B74；A49-B75；A49-B76；A49-B77；A49-B78；A49-B79；A49-B80；A49-B81；A49-B82；A49-B83；A49-B84；A49-B85；A49-B86；A49-B87；A49-B88；A49-B89；A49-B90；A49-B91；A49-B92；A49-B93；A49-B94；A49-B95；A49-B96；A49-B97；A49-B98；A49-B99；A49-B100；A49-B101；A49-B102；A49-B103；A49-B104；A49-B105；A49-B106；A49-B107；A49-B108；A49-B109；A49-B110；A49-B111；A49-B112；A49-B113；
A50-B1；A50-B2；A50-B3；A50-B4；A50-B5；A50-B6；A50-B7；A50-B8；A50-B9；A50-B10；A50-B11；A50-B12；A50-B13；A50-B14；A50-B15；A50-B16；A50-B17；A50-B18；A50-B19；A50-B20；A50-B21；A50-B22；A50-B23；A50-B24；A50-B25；A50-B26；A50-B27；A50-B28；A50-B29；A50-B30；A50-B31；A50-B32；A50-B33；A50-B34；A50-B35；A50-B36；A50-B37；A50-B38；A50-B39；A50-B40；A50-B41；A50-B42；A50-B43；A50-B44；A50-B45；A50-B46；A50-B47；A50-B48；A50-B49；A50-B50；A50-B51；A50-B52；A50-B53；A50-B54；A50-B55；A50-B56；A50-B57；A50-B58；A50-B59；A50-B60；A50-B61；A50-B62；A50-B63；A50-B64；A50-B65；A50-B66；A50-B67；A50-B68；A50-B69；A50-B70；A50-B71；A50-B72；A50-B73；A50-B74；A50-B75；A50-B76；A50-B77；A50-B78；A50-B79；A50-B80；A50-B81；A50-B82；A50-B83；A50-B84；A50-B85；A50-B86；A50-B87；A50-B88；A50-B89；A50-B90；A50-B91；A50-B92；A50-B93；A50-B94；A50-B95；A50-B96；A50-B97；A50-B98；A50-B99；A50-B100；A50-B101；A50-B102；A50-B103；A50-B104；A50-B105；A50-B106；A50-B107；A50-B108；A50-B109；A50-B110；A50-B111；A50-B112；A50-B113；
A51-B1；A51-B2；A51-B3；A51-B4；A51-B5；A51-B6；A51-B7；A51-B8；A51-B9；A51-B10；A51-B11；A51-B12；A51-B13；A51-B14；A51-B15；A51-B16；A51-B17；A51-B18；A51-B19；A51-B20；A51-B21；A51-B22；A51-B23；A51-B24；A51-B25；A51-B26；A51-B27；A51-B28；A51-B29；A51-B30；A51-B31；A51-B32；A51-B33；A51-B34；A51-B35；A51-B36；A51-B37；A51-B38；A51-B39；A51-B40；A51-B41；A51-B42；A51-B43；A51-B44；A51-B45；A51-B46；A51-B47；A51-B48；A51-B49；A51-B50；A51-B51；A51-B52；A51-B53；A51-B54；A51-B55；A51-B56；A51-B57；A51-B58；A51-B59；A51-B60；A51-B61；A51-B62；A51-B63；A51-B64；A51-B65；A51-B66；A51-B67；A51-B68；A51-B69；A51-B70；A51-B71；A51-B72；A51-B73；A51-B74；A51-B75；A51-B76；A51-B77；A51-B78；A51-B79；A51-B80；A51-B81；A51-B82；A51-B83；A51-B84；A51-B85；A51-B86；A51-B87；A51-B88；A51-B89；A51-B90；A51-B91；A51-B92；A51-B93；A51-B94；A51-B95；A51-B96；A51-B97；A51-B98；A51-B99；A51-B100；A51-B101；A51-B102；A51-B103；A51-B104；A51-B105；A51-B106；A51-B107；A51-B108；A51-B109；A51-B110；A51-B111；A51-B112；A51-B113；
A52-B1；A52-B2；A52-B3；A52-B4；A52-B5；A52-B6；A52-B7；A52-B8；A52-B9；A52-B10；A52-B11；A52-B12；A52-B13；A52-B14；A52-B15；A52-B16；A52-B17；A52-B18；A52-B19；A52-B20；A52-B21；A52-B22；A52-B23；A52-B24；A52-B25；A52-B26；A52-B27；A52-B28；A52-B29；A52-B30；A52-B31；A52-B32；A52-B33；A52-B34；A52-B35；A52-B36；A52-B37；A52-B38；A52-B39；A52-B40；A52-B41；A52-B42；A52-B43；A52-B44；A52-B45；A52-B46；A52-B47；A52-B48；A52-B49；A52-B50；A52-B51；A52-B52；A52-B53；A52-B54；A52-B55；A52-B56；A52-B57；A52-B58；A52-B59；A52-B60；A52-B61；A52-B62；A52-B63；A52-B64；A52-B65；A52-B66；A52-B67；A52-B68；A52-B69；A52-B70；A52-B71；A52-B72；A52-B73；A52-B74；A52-B75；A52-B76；A52-B77；A52-B78；A52-B79；A52-B80；A52-B81；A52-B82；A52-B83；A52-B84；A52-B85；A52-B86；A52-B87；A52-B88；A52-B89；A52-B90；A52-B91；A52-B92；A52-B93；A52-B94；A52-B95；A52-B96；A52-B97；A52-B98；A52-B99；A52-B100；A52-B101；A52-B102；A52-B103；A52-B104；A52-B105；A52-B106；A52-B107；A52-B108；A52-B109；A52-B110；A52-B111；A52-B112；A52-B113；
A53-B1；A53-B2；A53-B3；A53-B4；A53-B5；A53-B6；A53-B7；A53-B8；A53-B9；A53-B10；A53-B11；A53-B12；A53-B13；A53-B14；A53-B15；A53-B16；A53-B17；A53-B18；A53-B19；A53-B20；A53-B21；A53-B22；A53-B23；A53-B24；A53-B25；A53-B26；A53-B27；A53-B28；A53-B29；A53-B30；A53-B31；A53-B32；A53-B33；A53-B34；A53-B35；A53-B36；A53-B37；A53-B38；A53-B39；A53-B40；A53-B41；A53-B42；A53-B43；A53-B44；A53-B45；A53-B46；A53-B47；A53-B48；A53-B49；A53-B50；A53-B51；A53-B52；A53-B53；A53-B54；A53-B55；A53-B56；A53-B57；A53-B58；A53-B59；A53-B60；A53-B61；A53-B62；A53-B63；A53-B64；A53-B65；A53-B66；A53-B67；A53-B68；A53-B69；A53-B70；A53-B71；A53-B72；A53-B73；A53-B74；A53-B75；A53-B76；A53-B77；A53-B78；A53-B79；A53-B80；A53-B81；A53-B82；A53-B83；A53-B84；A53-B85；A53-B86；A53-B87；A53-B88；A53-B89；A53-B90；A53-B91；A53-B92；A53-B93；A53-B94；A53-B95；A53-B96；A53-B97；A53-B98；A53-B99；A53-B100；A53-B101；A53-B102；A53-B103；A53-B104；A53-B105；A53-B106；A53-B107；A53-B108；A53-B109；A53-B110；A53-B111；A53-B112；A53-B113；
A54-B1；A54-B2；A54-B3；A54-B4；A54-B5；A54-B6；A54-B7；A54-B8；A54-B9；A54-B10；A54-B11；A54-B12；A54-B13；A54-B14；A54-B15；A54-B16；A54-B17；A54-B18；A54-B19；A54-B20；A54-B21；A54-B22；A54-B23；A54-B24；A54-B25；A54-B26；A54-B27；A54-B28；A54-B29；A54-B30；A54-B31；A54-B32；A54-B33；A54-B34；A54-B35；A54-B36；A54-B37；A54-B38；A54-B39；A54-B40；A54-B41；A54-B42；A54-B43；A54-B44；A54-B45；A54-B46；A54-B47；A54-B48；A54-B49；A54-B50；A54-B51；A54-B52；A54-B53；A54-B54；A54-B55；A54-B56；A54-B57；A54-B58；A54-B59；A54-B60；A54-B61；A54-B62；A54-B63；A54-B64；A54-B65；A54-B66；A54-B67；A54-B68；A54-B69；A54-B70；A54-B71；A54-B72；A54-B73；A54-B74；A54-B75；A54-B76；A54-B77；A54-B78；A54-B79；A54-B80；A54-B81；A54-B82；A54-B83；A54-B84；A54-B85；A54-B86；A54-B87；A54-B88；A54-B89；A54-B90；A54-B91；A54-B92；A54-B93；A54-B94；A54-B95；A54-B96；A54-B97；A54-B98；A54-B99；A54-B100；A54-B101；A54-B102；A54-B103；A54-B104；A54-B105；A54-B106；A54-B107；A54-B108；A54-B109；A54-B110；A54-B111；A54-B112；A54-B113；
A55-B1；A55-B2；A55-B3；A55-B4；A55-B5；A55-B6；A55-B7；A55-B8；A55-B9；A55-B10；A55-B11；A55-B12；A55-B13；A55-B14；A55-B15；A55-B16；A55-B17；A55-B18；A55-B19；A55-B20；A55-B21；A55-B22；A55-B23；A55-B24；A55-B25；A55-B26；A55-B27；A55-B28；A55-B29；A55-B30；A55-B31；A55-B32；A55-B33；A55-B34；A55-B35；A55-B36；A55-B37；A55-B38；A55-B39；A55-B40；A55-B41；A55-B42；A55-B43；A55-B44；A55-B45；A55-B46；A55-B47；A55-B48；A55-B49；A55-B50；A55-B51；A55-B52；A55-B53；A55-B54；A55-B55；A55-B56；A55-B57；A55-B58；A55-B59；A55-B60；A55-B61；A55-B62；A55-B63；A55-B64；A55-B65；A55-B66；A55-B67；A55-B68；A55-B69；A55-B70；A55-B71；A55-B72；A55-B73；A55-B74；A55-B75；A55-B76；A55-B77；A55-B78；A55-B79；A55-B80；A55-B81；A55-B82；A55-B83；A55-B84；A55-B85；A55-B86；A55-B87；A55-B88；A55-B89；A55-B90；A55-B91；A55-B92；A55-B93；A55-B94；A55-B95；A55-B96；A55-B97；A55-B98；A55-B99；A55-B100；A55-B101；A55-B102；A55-B103；A55-B104；A55-B105；A55-B106；A55-B107；A55-B108；A55-B109；A55-B110；A55-B111；A55-B112；A55-B113；
A56-B1；A56-B2；A56-B3；A56-B4；A56-B5；A56-B6；A56-B7；A56-B8；A56-B9；A56-B10；A56-B11；A56-B12；A56-B13；A56-B14；A56-B15；A56-B16；A56-B17；A56-B18；A56-B19；A56-B20；A56-B21；A56-B22；A56-B23；A56-B24；A56-B25；A56-B26；A56-B27；A56-B28；A56-B29；A56-B30；A56-B31；A56-B32；A56-B33；A56-B34；A56-B35；A56-B36；A56-B37；A56-B38；A56-B39；A56-B40；A56-B41；A56-B42；A56-B43；A56-B44；A56-B45；A56-B46；A56-B47；A56-B48；A56-B49；A56-B50；A56-B51；A56-B52；A56-B53；A56-B54；A56-B55；A56-B56；A56-B57；A56-B58；A56-B59；A56-B60；A56-B61；A56-B62；A56-B63；A56-B64；A56-B65；A56-B66；A56-B67；A56-B68；A56-B69；A56-B70；A56-B71；A56-B72；A56-B73；A56-B74；A56-B75；A56-B76；A56-B77；A56-B78；A56-B79；A56-B80；A56-B81；A56-B82；A56-B83；A56-B84；A56-B85；A56-B86；A56-B87；A56-B88；A56-B89；A56-B90；A56-B91；A56-B92；A56-B93；A56-B94；A56-B95；A56-B96；A56-B97；A56-B98；A56-B99；A56-B100；A56-B101；A56-B102；A56-B103；A56-B104；A56-B105；A56-B106；A56-B107；A56-B108；A56-B109；A56-B110；A56-B111；A56-B112；A56-B113；
A57-B1；A57-B2；A57-B3；A57-B4；A57-B5；A57-B6；A57-B7；A57-B8；A57-B9；A57-B10；A57-B11；A57-B12；A57-B13；A57-B14；A57-B15；A57-B16；A57-B17；A57-B18；A57-B19；A57-B20；A57-B21；A57-B22；A57-B23；A57-B24；A57-B25；A57-B26；A57-B27；A57-B28；A57-B29；A57-B30；A57-B31；A57-B32；A57-B33；A57-B34；A57-B35；A57-B36；A57-B37；A57-B38；A57-B39；A57-B40；A57-B41；A57-B42；A57-B43；A57-B44；A57-B45；A57-B46；A57-B47；A57-B48；A57-B49；A57-B50；A57-B51；A57-B52；A57-B53；A57-B54；A57-B55；A57-B56；A57-B57；A57-B58；A57-B59；A57-B60；A57-B61；A57-B62；A57-B63；A57-B64；A57-B65；A57-B66；A57-B67；A57-B68；A57-B69；A57-B70；A57-B71；A57-B72；A57-B73；A57-B74；A57-B75；A57-B76；A57-B77；A57-B78；A57-B79；A57-B80；A57-B81；A57-B82；A57-B83；A57-B84；A57-B85；A57-B86；A57-B87；A57-B88；A57-B89；A57-B90；A57-B91；A57-B92；A57-B93；A57-B94；A57-B95；A57-B96；A57-B97；A57-B98；A57-B99；A57-B100；A57-B101；A57-B102；A57-B103；A57-B104；A57-B105；A57-B106；A57-B107；A57-B108；A57-B109；A57-B110；A57-B111；A57-B112；A57-B113；
A58-B1；A58-B2；A58-B3；A58-B4；A58-B5；A58-B6；A58-B7；A58-B8；A58-B9；A58-B10；A58-B11；A58-B12；A58-B13；A58-B14；A58-B15；A58-B16；A58-B17；A58-B18；A58-B19；A58-B20；A58-B21；A58-B22；A58-B23；A58-B24；A58-B25；A58-B26；A58-B27；A58-B28；A58-B29；A58-B30；A58-B31；A58-B32；A58-B33；A58-B34；A58-B35；A58-B36；A58-B37；A58-B38；A58-B39；A58-B40；A58-B41；A58-B42；A58-B43；A58-B44；A58-B45；A58-B46；A58-B47；A58-B48；A58-B49；A58-B50；A58-B51；A58-B52；A58-B53；A58-B54；A58-B55；A58-B56；A58-B57；A58-B58；A58-B59；A58-B60；A58-B61；A58-B62；A58-B63；A58-B64；A58-B65；A58-B66；A58-B67；A58-B68；A58-B69；A58-B70；A58-B71；A58-B72；A58-B73；A58-B74；A58-B75；A58-B76；A58-B77；A58-B78；A58-B79；A58-B80；A58-B81；A58-B82；A58-B83；A58-B84；A58-B85；A58-B86；A58-B87；A58-B88；A58-B89；A58-B90；A58-B91；A58-B92；A58-B93；A58-B94；A58-B95；A58-B96；A58-B97；A58-B98；A58-B99；A58-B100；A58-B101；A58-B102；A58-B103；A58-B104；A58-B105；A58-B106；A58-B107；A58-B108；A58-B109；A58-B110；A58-B111；A58-B112；A58-B113；
A59-B1；A59-B2；A59-B3；A59-B4；A59-B5；A59-B6；A59-B7；A59-B8；A59-B9；A59-B10；A59-B11；A59-B12；A59-B13；A59-B14；A59-B15；A59-B16；A59-B17；A59-B18；A59-B19；A59-B20；A59-B21；A59-B22；A59-B23；A59-B24；A59-B25；A59-B26；A59-B27；A59-B28；A59-B29；A59-B30；A59-B31；A59-B32；A59-B33；A59-B34；A59-B35；A59-B36；A59-B37；A59-B38；A59-B39；A59-B40；A59-B41；A59-B42；A59-B43；A59-B44；A59-B45；A59-B46；A59-B47；A59-B48；A59-B49；A59-B50；A59-B51；A59-B52；A59-B53；A59-B54；A59-B55；A59-B56；A59-B57；A59-B58；A59-B59；A59-B60；A59-B61；A59-B62；A59-B63；A59-B64；A59-B65；A59-B66；A59-B67；A59-B68；A59-B69；A59-B70；A59-B71；A59-B72；A59-B73；A59-B74；A59-B75；A59-B76；A59-B77；A59-B78；A59-B79；A59-B80；A59-B81；A59-B82；A59-B83；A59-B84；A59-B85；A59-B86；A59-B87；A59-B88；A59-B89；A59-B90；A59-B91；A59-B92；A59-B93；A59-B94；A59-B95；A59-B96；A59-B97；A59-B98；A59-B99；A59-B100；A59-B101；A59-B102；A59-B103；A59-B104；A59-B105；A59-B106；A59-B107；A59-B108；A59-B109；A59-B110；A59-B111；A59-B112；A59-B113；
A60-B1；A60-B2；A60-B3；A60-B4；A60-B5；A60-B6；A60-B7；A60-B8；A60-B9；A60-B10；A60-B11；A60-B12；A60-B13；A60-B14；A60-B15；A60-B16；A60-B17；A60-B18；A60-B19；A60-B20；A60-B21；A60-B22；A60-B23；A60-B24；A60-B25；A60-B26；A60-B27；A60-B28；A60-B29；A60-B30；A60-B31；A60-B32；A60-B33；A60-B34；A60-B35；A60-B36；A60-B37；A60-B38；A60-B39；A60-B40；A60-B41；A60-B42；A60-B43；A60-B44；A60-B45；A60-B46；A60-B47；A60-B48；A60-B49；A60-B50；A60-B51；A60-B52；A60-B53；A60-B54；A60-B55；A60-B56；A60-B57；A60-B58；A60-B59；A60-B60；A60-B61；A60-B62；A60-B63；A60-B64；A60-B65；A60-B66；A60-B67；A60-B68；A60-B69；A60-B70；A60-B71；A60-B72；A60-B73；A60-B74；A60-B75；A60-B76；A60-B77；A60-B78；A60-B79；A60-B80；A60-B81；A60-B82；A60-B83；A60-B84；A60-B85；A60-B86；A60-B87；A60-B88；A60-B89；A60-B90；A60-B91；A60-B92；A60-B93；A60-B94；A60-B95；A60-B96；A60-B97；A60-B98；A60-B99；A60-B100；A60-B101；A60-B102；A60-B103；A60-B104；A60-B105；A60-B106；A60-B107；A60-B108；A60-B109；A60-B110；A60-B111；A60-B112；A60-B113；
A61-B1；A61-B2；A61-B3；A61-B4；A61-B5；A61-B6；A61-B7；A61-B8；A61-B9；A61-B10；A61-B11；A61-B12；A61-B13；A61-B14；A61-B15；A61-B16；A61-B17；A61-B18；A61-B19；A61-B20；A61-B21；A61-B22；A61-B23；A61-B24；A61-B25；A61-B26；A61-B27；A61-B28；A61-B29；A61-B30；A61-B31；A61-B32；A61-B33；A61-B34；A61-B35；A61-B36；A61-B37；A61-B38；A61-B39；A61-B40；A61-B41；A61-B42；A61-B43；A61-B44；A61-B45；A61-B46；A61-B47；A61-B48；A61-B49；A61-B50；A61-B51；A61-B52；A61-B53；A61-B54；A61-B55；A61-B56；A61-B57；A61-B58；A61-B59；A61-B60；A61-B61；A61-B62；A61-B63；A61-B64；A61-B65；A61-B66；A61-B67；A61-B68；A61-B69；A61-B70；A61-B71；A61-B72；A61-B73；A61-B74；A61-B75；A61-B76；A61-B77；A61-B78；A61-B79；A61-B80；A61-B81；A61-B82；A61-B83；A61-B84；A61-B85；A61-B86；A61-B87；A61-B88；A61-B89；A61-B90；A61-B91；A61-B92；A61-B93；A61-B94；A61-B95；A61-B96；A61-B97；A61-B98；A61-B99；A61-B100；A61-B101；A61-B102；A61-B103；A61-B104；A61-B105；A61-B106；A61-B107；A61-B108；A61-B109；A61-B110；A61-B111；A61-B112；A61-B113；
A62-B1；A62-B2；A62-B3；A62-B4；A62-B5；A62-B6；A62-B7；A62-B8；A62-B9；A62-B10；A62-B11；A62-B12；A62-B13；A62-B14；A62-B15；A62-B16；A62-B17；A62-B18；A62-B19；A62-B20；A62-B21；A62-B22；A62-B23；A62-B24；A62-B25；A62-B26；A62-B27；A62-B28；A62-B29；A62-B30；A62-B31；A62-B32；A62-B33；A62-B34；A62-B35；A62-B36；A62-B37；A62-B38；A62-B39；A62-B40；A62-B41；A62-B42；A62-B43；A62-B44；A62-B45；A62-B46；A62-B47；A62-B48；A62-B49；A62-B50；A62-B51；A62-B52；A62-B53；A62-B54；A62-B55；A62-B56；A62-B57；A62-B58；A62-B59；A62-B60；A62-B61；A62-B62；A62-B63；A62-B64；A62-B65；A62-B66；A62-B67；A62-B68；A62-B69；A62-B70；A62-B71；A62-B72；A62-B73；A62-B74；A62-B75；A62-B76；A62-B77；A62-B78；A62-B79；A62-B80；A62-B81；A62-B82；A62-B83；A62-B84；A62-B85；A62-B86；A62-B87；A62-B88；A62-B89；A62-B90；A62-B91；A62-B92；A62-B93；A62-B94；A62-B95；A62-B96；A62-B97；A62-B98；A62-B99；A62-B100；A62-B101；A62-B102；A62-B103；A62-B104；A62-B105；A62-B106；A62-B107；A62-B108；A62-B109；A62-B110；A62-B111；A62-B112；A62-B113；
A63-B1；A63-B2；A63-B3；A63-B4；A63-B5；A63-B6；A63-B7；A63-B8；A63-B9；A63-B10；A63-B11；A63-B12；A63-B13；A63-B14；A63-B15；A63-B16；A63-B17；A63-B18；A63-B19；A63-B20；A63-B21；A63-B22；A63-B23；A63-B24；A63-B25；A63-B26；A63-B27；A63-B28；A63-B29；A63-B30；A63-B31；A63-B32；A63-B33；A63-B34；A63-B35；A63-B36；A63-B37；A63-B38；A63-B39；A63-B40；A63-B41；A63-B42；A63-B43；A63-B44；A63-B45；A63-B46；A63-B47；A63-B48；A63-B49；A63-B50；A63-B51；A63-B52；A63-B53；A63-B54；A63-B55；A63-B56；A63-B57；A63-B58；A63-B59；A63-B60；A63-B61；A63-B62；A63-B63；A63-B64；A63-B65；A63-B66；A63-B67；A63-B68；A63-B69；A63-B70；A63-B71；A63-B72；A63-B73；A63-B74；A63-B75；A63-B76；A63-B77；A63-B78；A63-B79；A63-B80；A63-B81；A63-B82；A63-B83；A63-B84；A63-B85；A63-B86；A63-B87；A63-B88；A63-B89；A63-B90；A63-B91；A63-B92；A63-B93；A63-B94；A63-B95；A63-B96；A63-B97；A63-B98；A63-B99；A63-B100；A63-B101；A63-B102；A63-B103；A63-B104；A63-B105；A63-B106；A63-B107；A63-B108；A63-B109；A63-B110；A63-B111；A63-B112；A63-B113；
A64-B1；A64-B2；A64-B3；A64-B4；A64-B5；A64-B6；A64-B7；A64-B8；A64-B9；A64-B10；A64-B11；A64-B12；A64-B13；A64-B14；A64-B15；A64-B16；A64-B17；A64-B18；A64-B19；A64-B20；A64-B21；A64-B22；A64-B23；A64-B24；A64-B25；A64-B26；A64-B27；A64-B28；A64-B29；A64-B30；A64-B31；A64-B32；A64-B33；A64-B34；A64-B35；A64-B36；A64-B37；A64-B38；A64-B39；A64-B40；A64-B41；A64-B42；A64-B43；A64-B44；A64-B45；A64-B46；A64-B47；A64-B48；A64-B49；A64-B50；A64-B51；A64-B52；A64-B53；A64-B54；A64-B55；A64-B56；A64-B57；A64-B58；A64-B59；A64-B60；A64-B61；A64-B62；A64-B63；A64-B64；A64-B65；A64-B66；A64-B67；A64-B68；A64-B69；A64-B70；A64-B71；A64-B72；A64-B73；A64-B74；A64-B75；A64-B76；A64-B77；A64-B78；A64-B79；A64-B80；A64-B81；A64-B82；A64-B83；A64-B84；A64-B85；A64-B86；A64-B87；A64-B88；A64-B89；A64-B90；A64-B91；A64-B92；A64-B93；A64-B94；A64-B95；A64-B96；A64-B97；A64-B98；A64-B99；A64-B100；A64-B101；A64-B102；A64-B103；A64-B104；A64-B105；A64-B106；A64-B107；A64-B108；A64-B109；A64-B110；A64-B111；A64-B112；A64-B113；
A65-B1；A65-B2；A65-B3；A65-B4；A65-B5；A65-B6；A65-B7；A65-B8；A65-B9；A65-B10；A65-B11；A65-B12；A65-B13；A65-B14；A65-B15；A65-B16；A65-B17；A65-B18；A65-B19；A65-B20；A65-B21；A65-B22；A65-B23；A65-B24；A65-B25；A65-B26；A65-B27；A65-B28；A65-B29；A65-B30；A65-B31；A65-B32；A65-B33；A65-B34；A65-B35；A65-B36；A65-B37；A65-B38；A65-B39；A65-B40；A65-B41；A65-B42；A65-B43；A65-B44；A65-B45；A65-B46；A65-B47；A65-B48；A65-B49；A65-B50；A65-B51；A65-B52；A65-B53；A65-B54；A65-B55；A65-B56；A65-B57；A65-B58；A65-B59；A65-B60；A65-B61；A65-B62；A65-B63；A65-B64；A65-B65；A65-B66；A65-B67；A65-B68；A65-B69；A65-B70；A65-B71；A65-B72；A65-B73；A65-B74；A65-B75；A65-B76；A65-B77；A65-B78；A65-B79；A65-B80；A65-B81；A65-B82；A65-B83；A65-B84；A65-B85；A65-B86；A65-B87；A65-B88；A65-B89；A65-B90；A65-B91；A65-B92；A65-B93；A65-B94；A65-B95；A65-B96；A65-B97；A65-B98；A65-B99；A65-B100；A65-B101；A65-B102；A65-B103；A65-B104；A65-B105；A65-B106；A65-B107；A65-B108；A65-B109；A65-B110；A65-B111；A65-B112；A65-B113；
A66-B1；A66-B2；A66-B3；A66-B4；A66-B5；A66-B6；A66-B7；A66-B8；A66-B9；A66-B10；A66-B11；A66-B12；A66-B13；A66-B14；A66-B15；A66-B16；A66-B17；A66-B18；A66-B19；A66-B20；A66-B21；A66-B22；A66-B23；A66-B24；A66-B25；A66-B26；A66-B27；A66-B28；A66-B29；A66-B30；A66-B31；A66-B32；A66-B33；A66-B34；A66-B35；A66-B36；A66-B37；A66-B38；A66-B39；A66-B40；A66-B41；A66-B42；A66-B43；A66-B44；A66-B45；A66-B46；A66-B47；A66-B48；A66-B49；A66-B50；A66-B51；A66-B52；A66-B53；A66-B54；A66-B55；A66-B56；A66-B57；A66-B58；A66-B59；A66-B60；A66-B61；A66-B62；A66-B63；A66-B64；A66-B65；A66-B66；A66-B67；A66-B68；A66-B69；A66-B70；A66-B71；A66-B72；A66-B73；A66-B74；A66-B75；A66-B76；A66-B77；A66-B78；A66-B79；A66-B80；A66-B81；A66-B82；A66-B83；A66-B84；A66-B85；A66-B86；A66-B87；A66-B88；A66-B89；A66-B90；A66-B91；A66-B92；A66-B93；A66-B94；A66-B95；A66-B96；A66-B97；A66-B98；A66-B99；A66-B100；A66-B101；A66-B102；A66-B103；A66-B104；A66-B105；A66-B106；A66-B107；A66-B108；A66-B109；A66-B110；A66-B111；A66-B112；A66-B113；
A67-B1；A67-B2；A67-B3；A67-B4；A67-B5；A67-B6；A67-B7；A67-B8；A67-B9；A67-B10；A67-B11；A67-B12；A67-B13；A67-B14；A67-B15；A67-B16；A67-B17；A67-B18；A67-B19；A67-B20；A67-B21；A67-B22；A67-B23；A67-B24；A67-B25；A67-B26；A67-B27；A67-B28；A67-B29；A67-B30；A67-B31；A67-B32；A67-B33；A67-B34；A67-B35；A67-B36；A67-B37；A67-B38；A67-B39；A67-B40；A67-B41；A67-B42；A67-B43；A67-B44；A67-B45；A67-B46；A67-B47；A67-B48；A67-B49；A67-B50；A67-B51；A67-B52；A67-B53；A67-B54；A67-B55；A67-B56；A67-B57；A67-B58；A67-B59；A67-B60；A67-B61；A67-B62；A67-B63；A67-B64；A67-B65；A67-B66；A67-B67；A67-B68；A67-B69；A67-B70；A67-B71；A67-B72；A67-B73；A67-B74；A67-B75；A67-B76；A67-B77；A67-B78；A67-B79；A67-B80；A67-B81；A67-B82；A67-B83；A67-B84；A67-B85；A67-B86；A67-B87；A67-B88；A67-B89；A67-B90；A67-B91；A67-B92；A67-B93；A67-B94；A67-B95；A67-B96；A67-B97；A67-B98；A67-B99；A67-B100；A67-B101；A67-B102；A67-B103；A67-B104；A67-B105；A67-B106；A67-B107；A67-B108；A67-B109；A67-B110；A67-B111；A67-B112；A67-B113；
A68-B1；A68-B2；A68-B3；A68-B4；A68-B5；A68-B6；A68-B7；A68-B8；A68-B9；A68-B10；A68-B11；A68-B12；A68-B13；A68-B14；A68-B15；A68-B16；A68-B17；A68-B18；A68-B19；A68-B20；A68-B21；A68-B22；A68-B23；A68-B24；A68-B25；A68-B26；A68-B27；A68-B28；A68-B29；A68-B30；A68-B31；A68-B32；A68-B33；A68-B34；A68-B35；A68-B36；A68-B37；A68-B38；A68-B39；A68-B40；A68-B41；A68-B42；A68-B43；A68-B44；A68-B45；A68-B46；A68-B47；A68-B48；A68-B49；A68-B50；A68-B51；A68-B52；A68-B53；A68-B54；A68-B55；A68-B56；A68-B57；A68-B58；A68-B59；A68-B60；A68-B61；A68-B62；A68-B63；A68-B64；A68-B65；A68-B66；A68-B67；A68-B68；A68-B69；A68-B70；A68-B71；A68-B72；A68-B73；A68-B74；A68-B75；A68-B76；A68-B77；A68-B78；A68-B79；A68-B80；A68-B81；A68-B82；A68-B83；A68-B84；A68-B85；A68-B86；A68-B87；A68-B88；A68-B89；A68-B90；A68-B91；A68-B92；A68-B93；A68-B94；A68-B95；A68-B96；A68-B97；A68-B98；A68-B99；A68-B100；A68-B101；A68-B102；A68-B103；A68-B104；A68-B105；A68-B106；A68-B107；A68-B108；A68-B109；A68-B110；A68-B111；A68-B112；A68-B113；
A69-B1；A69-B2；A69-B3；A69-B4；A69-B5；A69-B6；A69-B7；A69-B8；A69-B9；A69-B10；A69-B11；A69-B12；A69-B13；A69-B14；A69-B15；A69-B16；A69-B17；A69-B18；A69-B19；A69-B20；A69-B21；A69-B22；A69-B23；A69-B24；A69-B25；A69-B26；A69-B27；A69-B28；A69-B29；A69-B30；A69-B31；A69-B32；A69-B33；A69-B34；A69-B35；A69-B36；A69-B37；A69-B38；A69-B39；A69-B40；A69-B41；A69-B42；A69-B43；A69-B44；A69-B45；A69-B46；A69-B47；A69-B48；A69-B49；A69-B50；A69-B51；A69-B52；A69-B53；A69-B54；A69-B55；A69-B56；A69-B57；A69-B58；A69-B59；A69-B60；A69-B61；A69-B62；A69-B63；A69-B64；A69-B65；A69-B66；A69-B67；A69-B68；A69-B69；A69-B70；A69-B71；A69-B72；A69-B73；A69-B74；A69-B75；A69-B76；A69-B77；A69-B78；A69-B79；A69-B80；A69-B81；A69-B82；A69-B83；A69-B84；A69-B85；A69-B86；A69-B87；A69-B88；A69-B89；A69-B90；A69-B91；A69-B92；A69-B93；A69-B94；A69-B95；A69-B96；A69-B97；A69-B98；A69-B99；A69-B100；A69-B101；A69-B102；A69-B103；A69-B104；A69-B105；A69-B106；A69-B107；A69-B108；A69-B109；A69-B110；A69-B111；A69-B112；A69-B113；
A70-B1；A70-B2；A70-B3；A70-B4；A70-B5；A70-B6；A70-B7；A70-B8；A70-B9；A70-B10；A70-B11；A70-B12；A70-B13；A70-B14；A70-B15；A70-B16；A70-B17；A70-B18；A70-B19；A70-B20；A70-B21；A70-B22；A70-B23；A70-B24；A70-B25；A70-B26；A70-B27；A70-B28；A70-B29；A70-B30；A70-B31；A70-B32；A70-B33；A70-B34；A70-B35；A70-B36；A70-B37；A70-B38；A70-B39；A70-B40；A70-B41；A70-B42；A70-B43；A70-B44；A70-B45；A70-B46；A70-B47；A70-B48；A70-B49；A70-B50；A70-B51；A70-B52；A70-B53；A70-B54；A70-B55；A70-B56；A70-B57；A70-B58；A70-B59；A70-B60；A70-B61；A70-B62；A70-B63；A70-B64；A70-B65；A70-B66；A70-B67；A70-B68；A70-B69；A70-B70；A70-B71；A70-B72；A70-B73；A70-B74；A70-B75；A70-B76；A70-B77；A70-B78；A70-B79；A70-B80；A70-B81；A70-B82；A70-B83；A70-B84；A70-B85；A70-B86；A70-B87；A70-B88；A70-B89；A70-B90；A70-B91；A70-B92；A70-B93；A70-B94；A70-B95；A70-B96；A70-B97；A70-B98；A70-B99；A70-B100；A70-B101；A70-B102；A70-B103；A70-B104；A70-B105；A70-B106；A70-B107；A70-B108；A70-B109；A70-B110；A70-B111；A70-B112；A70-B113；
A71-B1；A71-B2；A71-B3；A71-B4；A71-B5；A71-B6；A71-B7；A71-B8；A71-B9；A71-B10；A71-B11；A71-B12；A71-B13；A71-B14；A71-B15；A71-B16；A71-B17；A71-B18；A71-B19；A71-B20；A71-B21；A71-B22；A71-B23；A71-B24；A71-B25；A71-B26；A71-B27；A71-B28；A71-B29；A71-B30；A71-B31；A71-B32；A71-B33；A71-B34；A71-B35；A71-B36；A71-B37；A71-B38；A71-B39；A71-B40；A71-B41；A71-B42；A71-B43；A71-B44；A71-B45；A71-B46；A71-B47；A71-B48；A71-B49；A71-B50；A71-B51；A71-B52；A71-B53；A71-B54；A71-B55；A71-B56；A71-B57；A71-B58；A71-B59；A71-B60；A71-B61；A71-B62；A71-B63；A71-B64；A71-B65；A71-B66；A71-B67；A71-B68；A71-B69；A71-B70；A71-B71；A71-B72；A71-B73；A71-B74；A71-B75；A71-B76；A71-B77；A71-B78；A71-B79；A71-B80；A71-B81；A71-B82；A71-B83；A71-B84；A71-B85；A71-B86；A71-B87；A71-B88；A71-B89；A71-B90；A71-B91；A71-B92；A71-B93；A71-B94；A71-B95；A71-B96；A71-B97；A71-B98；A71-B99；A71-B100；A71-B101；A71-B102；A71-B103；A71-B104；A71-B105；A71-B106；A71-B107；A71-B108；A71-B109；A71-B110；A71-B111；A71-B112；A71-B113；
A72-B1；A72-B2；A72-B3；A72-B4；A72-B5；A72-B6；A72-B7；A72-B8；A72-B9；A72-B10；A72-B11；A72-B12；A72-B13；A72-B14；A72-B15；A72-B16；A72-B17；A72-B18；A72-B19；A72-B20；A72-B21；A72-B22；A72-B23；A72-B24；A72-B25；A72-B26；A72-B27；A72-B28；A72-B29；A72-B30；A72-B31；A72-B32；A72-B33；A72-B34；A72-B35；A72-B36；A72-B37；A72-B38；A72-B39；A72-B40；A72-B41；A72-B42；A72-B43；A72-B44；A72-B45；A72-B46；A72-B47；A72-B48；A72-B49；A72-B50；A72-B51；A72-B52；A72-B53；A72-B54；A72-B55；A72-B56；A72-B57；A72-B58；A72-B59；A72-B60；A72-B61；A72-B62；A72-B63；A72-B64；A72-B65；A72-B66；A72-B67；A72-B68；A72-B69；A72-B70；A72-B71；A72-B72；A72-B73；A72-B74；A72-B75；A72-B76；A72-B77；A72-B78；A72-B79；A72-B80；A72-B81；A72-B82；A72-B83；A72-B84；A72-B85；A72-B86；A72-B87；A72-B88；A72-B89；A72-B90；A72-B91；A72-B92；A72-B93；A72-B94；A72-B95；A72-B96；A72-B97；A72-B98；A72-B99；A72-B100；A72-B101；A72-B102；A72-B103；A72-B104；A72-B105；A72-B106；A72-B107；A72-B108；A72-B109；A72-B110；A72-B111；A72-B112；A72-B113；
A73-B1；A73-B2；A73-B3；A73-B4；A73-B5；A73-B6；A73-B7；A73-B8；A73-B9；A73-B10；A73-B11；A73-B12；A73-B13；A73-B14；A73-B15；A73-B16；A73-B17；A73-B18；A73-B19；A73-B20；A73-B21；A73-B22；A73-B23；A73-B24；A73-B25；A73-B26；A73-B27；A73-B28；A73-B29；A73-B30；A73-B31；A73-B32；A73-B33；A73-B34；A73-B35；A73-B36；A73-B37；A73-B38；A73-B39；A73-B40；A73-B41；A73-B42；A73-B43；A73-B44；A73-B45；A73-B46；A73-B47；A73-B48；A73-B49；A73-B50；A73-B51；A73-B52；A73-B53；A73-B54；A73-B55；A73-B56；A73-B57；A73-B58；A73-B59；A73-B60；A73-B61；A73-B62；A73-B63；A73-B64；A73-B65；A73-B66；A73-B67；A73-B68；A73-B69；A73-B70；A73-B71；A73-B72；A73-B73；A73-B74；A73-B75；A73-B76；A73-B77；A73-B78；A73-B79；A73-B80；A73-B81；A73-B82；A73-B83；A73-B84；A73-B85；A73-B86；A73-B87；A73-B88；A73-B89；A73-B90；A73-B91；A73-B92；A73-B93；A73-B94；A73-B95；A73-B96；A73-B97；A73-B98；A73-B99；A73-B100；A73-B101；A73-B102；A73-B103；A73-B104；A73-B105；A73-B106；A73-B107；A73-B108；A73-B109；A73-B110；A73-B111；A73-B112；A73-B113；
A74-B1；A74-B2；A74-B3；A74-B4；A74-B5；A74-B6；A74-B7；A74-B8；A74-B9；A74-B10；A74-B11；A74-B12；A74-B13；A74-B14；A74-B15；A74-B16；A74-B17；A74-B18；A74-B19；A74-B20；A74-B21；A74-B22；A74-B23；A74-B24；A74-B25；A74-B26；A74-B27；A74-B28；A74-B29；A74-B30；A74-B31；A74-B32；A74-B33；A74-B34；A74-B35；A74-B36；A74-B37；A74-B38；A74-B39；A74-B40；A74-B41；A74-B42；A74-B43；A74-B44；A74-B45；A74-B46；A74-B47；A74-B48；A74-B49；A74-B50；A74-B51；A74-B52；A74-B53；A74-B54；A74-B55；A74-B56；A74-B57；A74-B58；A74-B59；A74-B60；A74-B61；A74-B62；A74-B63；A74-B64；A74-B65；A74-B66；A74-B67；A74-B68；A74-B69；A74-B70；A74-B71；A74-B72；A74-B73；A74-B74；A74-B75；A74-B76；A74-B77；A74-B78；A74-B79；A74-B80；A74-B81；A74-B82；A74-B83；A74-B84；A74-B85；A74-B86；A74-B87；A74-B88；A74-B89；A74-B90；A74-B91；A74-B92；A74-B93；A74-B94；A74-B95；A74-B96；A74-B97；A74-B98；A74-B99；A74-B100；A74-B101；A74-B102；A74-B103；A74-B104；A74-B105；A74-B106；A74-B107；A74-B108；A74-B109；A74-B110；A74-B111；A74-B112；A74-B113；
A75-B1；A75-B2；A75-B3；A75-B4；A75-B5；A75-B6；A75-B7；A75-B8；A75-B9；A75-B10；A75-B11；A75-B12；A75-B13；A75-B14；A75-B15；A75-B16；A75-B17；A75-B18；A75-B19；A75-B20；A75-B21；A75-B22；A75-B23；A75-B24；A75-B25；A75-B26；A75-B27；A75-B28；A75-B29；A75-B30；A75-B31；A75-B32；A75-B33；A75-B34；A75-B35；A75-B36；A75-B37；A75-B38；A75-B39；A75-B40；A75-B41；A75-B42；A75-B43；A75-B44；A75-B45；A75-B46；A75-B47；A75-B48；A75-B49；A75-B50；A75-B51；A75-B52；A75-B53；A75-B54；A75-B55；A75-B56；A75-B57；A75-B58；A75-B59；A75-B60；A75-B61；A75-B62；A75-B63；A75-B64；A75-B65；A75-B66；A75-B67；A75-B68；A75-B69；A75-B70；A75-B71；A75-B72；A75-B73；A75-B74；A75-B75；A75-B76；A75-B77；A75-B78；A75-B79；A75-B80；A75-B81；A75-B82；A75-B83；A75-B84；A75-B85；A75-B86；A75-B87；A75-B88；A75-B89；A75-B90；A75-B91；A75-B92；A75-B93；A75-B94；A75-B95；A75-B96；A75-B97；A75-B98；A75-B99；A75-B100；A75-B101；A75-B102；A75-B103；A75-B104；A75-B105；A75-B106；A75-B107；A75-B108；A75-B109；A75-B110；A75-B111；A75-B112；A75-B113；
A76-B1；A76-B2；A76-B3；A76-B4；A76-B5；A76-B6；A76-B7；A76-B8；A76-B9；A76-B10；A76-B11；A76-B12；A76-B13；A76-B14；A76-B15；A76-B16；A76-B17；A76-B18；A76-B19；A76-B20；A76-B21；A76-B22；A76-B23；A76-B24；A76-B25；A76-B26；A76-B27；A76-B28；A76-B29；A76-B30；A76-B31；A76-B32；A76-B33；A76-B34；A76-B35；A76-B36；A76-B37；A76-B38；A76-B39；A76-B40；A76-B41；A76-B42；A76-B43；A76-B44；A76-B45；A76-B46；A76-B47；A76-B48；A76-B49；A76-B50；A76-B51；A76-B52；A76-B53；A76-B54；A76-B55；A76-B56；A76-B57；A76-B58；A76-B59；A76-B60；A76-B61；A76-B62；A76-B63；A76-B64；A76-B65；A76-B66；A76-B67；A76-B68；A76-B69；A76-B70；A76-B71；A76-B72；A76-B73；A76-B74；A76-B75；A76-B76；A76-B77；A76-B78；A76-B79；A76-B80；A76-B81；A76-B82；A76-B83；A76-B84；A76-B85；A76-B86；A76-B87；A76-B88；A76-B89；A76-B90；A76-B91；A76-B92；A76-B93；A76-B94；A76-B95；A76-B96；A76-B97；A76-B98；A76-B99；A76-B100；A76-B101；A76-B102；A76-B103；A76-B104；A76-B105；A76-B106；A76-B107；A76-B108；A76-B109；A76-B110；A76-B111；A76-B112；A76-B113；
A77-B1；A77-B2；A77-B3；A77-B4；A77-B5；A77-B6；A77-B7；A77-B8；A77-B9；A77-B10；A77-B11；A77-B12；A77-B13；A77-B14；A77-B15；A77-B16；A77-B17；A77-B18；A77-B19；A77-B20；A77-B21；A77-B22；A77-B23；A77-B24；A77-B25；A77-B26；A77-B27；A77-B28；A77-B29；A77-B30；A77-B31；A77-B32；A77-B33；A77-B34；A77-B35；A77-B36；A77-B37；A77-B38；A77-B39；A77-B40；A77-B41；A77-B42；A77-B43；A77-B44；A77-B45；A77-B46；A77-B47；A77-B48；A77-B49；A77-B50；A77-B51；A77-B52；A77-B53；A77-B54；A77-B55；A77-B56；A77-B57；A77-B58；A77-B59；A77-B60；A77-B61；A77-B62；A77-B63；A77-B64；A77-B65；A77-B66；A77-B67；A77-B68；A77-B69；A77-B70；A77-B71；A77-B72；A77-B73；A77-B74；A77-B75；A77-B76；A77-B77；A77-B78；A77-B79；A77-B80；A77-B81；A77-B82；A77-B83；A77-B84；A77-B85；A77-B86；A77-B87；A77-B88；A77-B89；A77-B90；A77-B91；A77-B92；A77-B93；A77-B94；A77-B95；A77-B96；A77-B97；A77-B98；A77-B99；A77-B100；A77-B101；A77-B102；A77-B103；A77-B104；A77-B105；A77-B106；A77-B107；A77-B108；A77-B109；A77-B110；A77-B111；A77-B112；A77-B113；
Subclass IA: amino acid B of the dipeptide prodrug element is N-alkylated glycine
In some embodiments, amino acid B of the dipeptide prodrug element is an N-alkylated glycine. Non-limiting examples of dipeptide prodrug elements having N-alkylated glycines as amino acid B are shown in the following table.
| Dipeptide prodrug element # | Amino acid "A" | Amino acid "B" |
| 1 | Aib | Gly(N-C1-C8Alkyl radical) |
| 2 | d-Ala | Gly(N-C1-C8Alkyl radical) |
| 3 | d-Lys | Gly(N-C1-C8Alkyl radical) |
| 4 | d-Cys | Gly(N-C1-C8Alkyl radical) |
| 5 | Aib | Gly (N-methyl) |
| 6 | d-Ala | Gly (N-methyl) |
| 7 | d-Lys | Gly (N-methyl) |
| 8 | d-Cys | Gly (N-methyl) |
| 9 | Aib | Gly (N-hexyl) |
| 10 | d-Ala | Gly (N-hexyl) |
| 11 | d-Lys | Gly (N-hexyl) |
| 12 | d-Cys | Gly (N-hexyl) |
Subclass IB: amino acid B of the dipeptide prodrug element is unsubstituted or monosubstituted at the beta-position
In some embodiments, amino acid B of the dipeptide prodrug element is unsubstituted or mono-substituted at the β position and has a relatively non-bulky side chain. Non-limiting examples of dipeptide prodrug elements having an amino acid B that is unsubstituted or mono-substituted at the beta position and a relatively non-bulky side chain are shown in the following table.
| Dipeptide prodrug element # | Amino acid "A" | Amino acid "B" |
| 13 | Aib | Ala(N-C1-C8Alkyl radical) |
| 14 | d-Ala | Ala(N-C1-C8Alkyl radical) |
| 15 | d-Lys | Ala(N-C1-C8Alkyl radical) |
| 16 | d-Cys | Ala(N-C1-C8Alkyl radical) |
| 17 | Aib | Leu(N-C1-C8Alkyl radical) |
| 18 | d-Ala | Leu(N-C1-C8Alkyl radical) |
| 19 | d-Lys | Leu(N-C1-C8Alkyl radical) |
| 20 | d-Cys | Leu(N-C1-C8Alkyl radical) |
| 21 | Aib | Met(N-C1-C8Alkyl radical) |
| 22 | d-Ala | Met(N-C1-C8Alkyl radical) |
| 23 | d-Lys | Met(N-C1-C8Alkyl radical) |
| 24 | d-Cys | Met(N-C1-C8Alkyl radical) |
| 25 | Aib | Asn(N-C1-C8Alkyl radical) |
| 26 | d-Ala | Asn(N-C1-C8Alkyl radical) |
| 27 | d-Lys | Asn(N-C1-C8Alkyl radical) |
| 28 | d-Cys | Asn(N-C1-C8Alkyl radical) |
| 29 | Aib | Glu(N-C1-C8Alkyl radical) |
| 30 | d-Ala | Glu(N-C1-C8Alkyl radical) |
| 31 | d-Lys | Glu(N-C1-C8Alkyl radical) |
| 32 | d-Cys | Glu(N-C1-C8Alkyl radical) |
| 33 | Aib | Asp(N-C1-C8Alkyl radical) |
| 34 | d-Ala | Asp(N-C1-C8Alkyl radical) |
| 35 | d-Lys | Asp(N-C1-C8Alkyl radical) |
| 36 | d-Cys | Asp(N-C1-C8Alkyl radical) |
| 37 | Aib | Gln(N-C1-C8Alkyl radical) |
| 38 | d-Ala | Gln(N-C1-C8Alkyl radical) |
| 39 | d-Lys | Gln(N-C1-C8Alkyl radical) |
| 40 | d-Cys | Gln(N-C1-C8Alkyl radical) |
| 41 | Aib | His(N-C1-C8Alkyl radical) |
| 42 | d-Ala | His(N-C1-C8Alkyl radical) |
| 43 | d-Lys | His(N-C1-C8Alkyl radical) |
| 44 | d-Cys | His(N-C1-C8Alkyl radical) |
| 45 | Aib | Lys(N-C1-C8Alkyl radical) |
| 46 | d-Ala | Lys(N-C1-C8Alkyl radical) |
| 47 | d-Lys | Lys(N-C1-C8Alkyl radical) |
| 48 | d-Cys | Lys(N-C1-C8Alkyl radical) |
| 49 | Aib | Arg(N-C1-C8Alkyl radical) |
| 50 | d-Ala | Arg(N-C1-C8Alkyl radical) |
| 51 | d-Lys | Arg(N-C1-C8Alkyl radical) |
| 52 | d-Cys | Arg(N-C1-C8Alkyl radical) |
| 53 | Aib | Ser(N-C1-C8Alkyl radical) |
| 54 | d-Ala | Ser(N-C1-C8Alkyl radical) |
| 55 | d-Lys | Ser(N-C1-C8Alkyl radical) |
| 56 | d-Cys | Ser(N-C1-C8Alkyl radical) |
| 57 | Aib | Cys(N-C1-C8Alkyl radical) |
| 58 | d-Ala | Cys(N-C1-C8Alkyl radical) |
| 59 | d-Lys | Cys(N-C1-C8Alkyl radical) |
| 60 | d-Cys | Cys(N-C1-C8Alkyl radical) |
| 61 | Aib | Pro |
| 62 | d-Ala | Pro |
| 63 | d-Lys | Pro |
| 64 | d-Cys | Pro |
| 65 | Aib | Ala (N-methyl) |
| 66 | d-Ala | Ala (N-methyl) |
| 67 | d-Lys | Ala (N-methyl) |
| 68 | d-Cys | Ala (N-methyl) |
| 69 | Aib | Leu (N-methyl) |
| 70 | d-Ala | Leu (N-methyl) |
| 71 | d-Lys | Leu (N-methyl) |
| 72 | d-Cys | Leu (N-methyl) |
| 73 | Aib | Met (N-methyl) |
| 74 | d-Ala | Met (N-methyl) |
| 75 | d-Lys | Met (N-methyl) |
| 76 | d-Cys | Met (N-methyl) |
| 77 | Aib | Asn (N-methyl) |
| 78 | d-Ala | Asn (N-methyl) |
| 79 | d-Lys | Asn (N-methyl) |
| 80 | d-Cys | Asn (N-methyl) |
| 81 | Aib | Glu (N-methyl) |
| 82 | d-Ala | Glu (N-methyl) |
| 83 | d-Lys | Glu (N-methyl) |
| 84 | d-Cys | Glu (N-methyl) |
| 85 | Aib | Asp (N-methyl) |
| 86 | d-Ala | Asp (N-methyl) |
| 87 | d-Lys | Asp (N-methyl) |
| 88 | d-Cys | Asp (N-methyl) |
| 89 | Aib | Gln (N-methyl) |
| 90 | d-Ala | Gln (N-methyl) |
| 91 | d-Lys | Gln (N-methyl) |
| 92 | d-Cys | Gln (N-methyl) |
| 93 | Aib | His (N-methyl) |
| 94 | d-Ala | His (N-methyl) |
| 95 | d-Lys | His (N-methyl) |
| 96 | d-Cys | His (N-methyl) |
| 97 | Aib | Lys (N-methyl) |
| 98 | d-Ala | Lys (N-methyl) |
| 99 | d-Lys | Lys (N-methyl) |
| 100 | d-Cys | Lys (N-methyl) |
| 101 | Aib | Arg (N-methyl) |
| 102 | d-Ala | Arg (N-methyl) |
| 103 | d-Lys | Arg (N-methyl) |
| 104 | d-Cys | Arg (N-methyl) |
| 105 | Aib | Ser (N-methyl) |
| 106 | d-Ala | Ser (N-methyl) |
| 107 | d-Lys | Ser (N-methyl) |
| 108 | d-Cys | Ser (N-methyl) |
| 109 | Aib | Cys (N-methyl) |
| 110 | d-Ala | Cys (N-methyl) |
| 111 | d-Lys | Cys (N-methyl) |
| 112 | d-Cys | Cys (N-methyl) |
| 113 | Aib | Ala (N-hexyl) |
| 114 | d-Ala | Ala (N-hexyl) |
| 115 | d-Lys | Ala (N-hexyl) |
| 116 | d-Cys | Ala (N-hexyl) |
| 117 | Aib | Leu (N-hexyl) |
| 118 | d-Ala | Leu (N-hexyl) |
| 119 | d-Lys | Leu (N-hexyl) |
| 120 | d-Cys | Leu (N-hexyl) |
| 121 | Aib | Met (N-hexyl) |
| 122 | d-Ala | Met (N-hexyl) |
| 123 | d-Lys | Met (N-hexyl) |
| 124 | d-Cys | Met (N-hexyl) |
| 125 | Aib | Asn (N-hexyl) |
| 126 | d-Ala | Asn (N-hexyl) |
| 127 | d-Lys | Asn (N-hexyl) |
| 128 | d-Cys | Asn (N-hexyl) |
| 129 | Aib | Glu (N-hexyl) |
| 130 | d-Ala | Glu (N-hexyl) |
| 131 | d-Lys | Glu (N-hexyl) |
| 132 | d-Cys | Glu (N-hexyl) |
| 133 | Aib | Asp (N-hexyl) |
| 134 | d-Ala | Asp (N-hexyl) |
| 135 | d-Lys | Asp (N-hexyl) |
| 136 | d-Cys | Asp (N-hexyl) |
| 137 | Aib | Gln (N-hexyl) |
| 138 | d-Ala | Gln (N-hexyl) |
| 139 | d-Lys | Gln (N-hexyl) |
| 140 | d-Cys | Gln (N-hexyl) |
| 141 | Aib | His (N-hexyl) |
| 142 | d-Ala | His (N-hexyl) |
| 143 | d-Lys | His (N-hexyl) |
| 144 | d-Cys | His (N-hexyl) |
| 145 | Aib | Lys (N-hexyl) |
| 146 | d-Ala | Lys (N-hexyl) |
| 147 | d-Lys | Lys (N-hexyl) |
| 148 | d-Cys | Lys (N-hexyl) |
| 149 | Aib | Arg (N-hexyl) |
| 150 | d-Ala | Arg (N-hexyl) |
| 151 | d-Lys | Arg (N-hexyl) |
| 152 | d-Cys | Arg (N-hexyl) |
| 153 | Aib | Ser (N-hexyl) |
| 154 | d-Ala | Ser (N-hexyl) |
| 155 | d-Lys | Ser (N-hexyl) |
| 156 | d-Cys | Ser (N-hexyl) |
| 157 | Aib | Cys (N-hexyl) |
| 158 | d-Ala | Cys (N-hexyl) |
| 159 | d-Lys | Cys (N-hexyl) |
| 160 | d-Cys | Cys (N-hexyl) |
In some embodiments, amino acid B of the dipeptide prodrug element is mono-substituted in the beta position and has a relatively bulky side chain, as shown in the table below.
| Dipeptide prodrug element # | Amino acid "A" | Amino acid "B" |
| 161 | Aib | Phe(N-C1-C8Alkyl radical) |
| 162 | d-Ala | Phe(N-C1-C8Alkyl radical) |
| 163 | d-Lys | Phe(N-C1-C8Alkyl radical) |
| 164 | d-Cys | Phe(N-C1-C8Alkyl radical) |
| 165 | Aib | Tyr(N-C1-C8Alkyl radical) |
| 166 | d-Ala | Tyr(N-C1-C8Alkyl radical) |
| 167 | d-Lys | Tyr(N-C1-C8Alkyl radical) |
| 168 | d-Cys | Tyr(N-C1-C8Alkyl radical) |
| 169 | Aib | Trp(N-C1-C8Alkyl radical) |
| 170 | d-Ala | Trp(N-C1-C8Alkyl radical) |
| 171 | d-Lys | Trp(N-C1-C8Alkyl radical) |
| 172 | d-Cys | Trp(N-C1-C8Alkyl radical) |
| 173 | Aib | Phe (N-methyl) |
| 174 | d-Ala | Phe (N-methyl) |
| 175 | d-Lys | Phe (N-methyl) |
| 176 | d-Cys | Phe (N-methyl) |
| 177 | Aib | Tyr (N-methyl) |
| 178 | d-Ala | Tyr (N-methyl) |
| 179 | d-Lys | Tyr (N-methyl) |
| 180 | d-Cys | Tyr (N-methyl) |
| 181 | Aib | Trp (N-methyl) |
| 182 | d-Ala | Trp (N-methyl) |
| 183 | d-Lys | Trp (N-methyl) |
| 184 | d-Cys | Trp (N-methyl) |
| 185 | Aib | Phe (N-hexyl) |
| 186 | d-Ala | Phe (N-hexyl)) |
| 187 | d-Lys | Phe (N-hexyl) |
| 188 | d-Cys | Phe (N-hexyl) |
| 189 | Aib | Tyr (N-hexyl) |
| 190 | d-Ala | Tyr (N-hexyl) |
| 191 | d-Lys | Tyr (N-hexyl) |
| 192 | d-Cys | Tyr (N-hexyl) |
| 193 | Aib | Trp (N-hexyl) |
| 194 | d-Ala | Trp (N-hexyl) |
| 195 | d-Lys | Trp (N-hexyl) |
| 196 | d-Cys | Trp (N-hexyl) |
Subclass IC: amino acid B of a dipeptide prodrug element disubstituted in the beta position
In some embodiments, amino acid B of the dipeptide prodrug element is disubstituted in the beta position. Non-limiting examples of dipeptide prodrug elements having amino acid B disubstituted in the beta position are shown in the following table.
| Dipeptide prodrug element # | Amino acid "A" | Amino acid "B" |
| 197 | Aib | Ile(N-C1-C8Alkyl radical) |
| 198 | d-Ala | Ile(N-C1-C8Alkyl radical) |
| 199 | d-Lys | Ile(N-C1-C8Alkyl radical) |
| 200 | d-Cys | Ile(N-C1-C8Alkyl)) |
| 201 | Aib | Val(N-C1-C8Alkyl radical) |
| 202 | d-Ala | Val(N-C1-C8Alkyl radical) |
| 203 | d-Lys | Val(N-C1-C8Alkyl radical) |
| 204 | d-Cs | Val(N-C1-C8Alkyl radical) |
| 205 | Aib | Thr(N-C1-C8Alkyl radical) |
| 206 | d-Ala | Thr(N-C1-C8Alkyl radical) |
| 207 | d-Lys | Thr(N-C1-C8Alkyl radical) |
| 208 | d-Cys | Thr(N-C1-C8Alkyl radical) |
| 209 | Aib | Ile (N-methyl) |
| 210 | d-Ala | Ile (N-methyl) |
| 211 | d-Lys | Ile (N-methyl) |
| 212 | d-Cys | Ile (N-methyl) |
| 213 | Aib | Val (N-methyl) |
| 214 | d-Ala | Val (N-methyl) |
| 215 | d-Lys | Val (N-methyl) |
| 216 | d-Cys | Val (N-methyl) |
| 217 | Aib | Thr (N-methyl) |
| 218 | d-Ala | Thr (N-methyl) |
| 219 | d-Lys | Thr (N-methyl) |
| 220 | d-Cys | Thr (N-methyl) |
| 221 | Aib | Ile (N-hexyl) |
| 222 | d-Ala | Ile (N-hexyl) |
| 223 | d-Lys | Ile (N-hexyl) |
| 224 | d-Cys | Ile (N-hexyl) |
| 225 | Aib | Val (N-hexyl) |
| 226 | d-Ala | Val (N-hexyl) |
| 227 | d-Lys | Val (N-hexyl) |
| 228 | d-Cys | Val (N-hexyl) |
| 229 | Aib | Thr (N-hexyl) |
| 230 | d-Ala | Thr (N-hexyl) |
| 231 | d-Lys | Thr (N-hexyl) |
| 232 | d-Cys | Thr (N-hexyl) |
Prodrugs
The dipeptide prodrug element is conjugated to any of the following glucagon related peptides (e.g., to the alpha amine of the N-terminal amino acid, an aliphatic amino group on the amino acid side chain of Q (e.g., lysine side chain), an aromatic amino group on the amino acid side chain of Q (e.g., aminophenylalanine, aminonaphthylalanine, aminotrryptophan, aminophenylglycine, aminohomophenylalanine), through any position that interferes with the activity of the glucagon related peptide, exemplary positions include positions 12, 16, 17, 18, 20, 28, or 29 of native glucagon (SEQ ID NO: 701) when a-B is attached to an aliphatic amino group on the amino acid side chain of Q, exemplary positions include positions 10, 13, 22, or 25 of native glucagon (SEQ ID NO: 701) when a-B is attached to an aromatic amino group on the amino acid side chain of Q, in some embodiments, the dipeptide prodrug element of the invention is conjugated to any of the following: SEQ ID NO: 1-564, 566-. For example, the dipeptide prodrug element can be conjugated to seq id NO: 742-768.
In some exemplary embodiments, Aib-Gly (N-hexyl), dLys-Gly (N-hexyl), dCys-Gly (N-hexyl), dAla-Gly (N-hexyl), Aib-Gly (N-methyl), dLys-Gly (N-methyl), dCys-Gly (N-methyl), dAla-Gly (N-hexyl), Aib-Phe (N-methyl), dLys-Phe (N-methyl), dCys-Phe (N-methyl), or dAla-Phe (N-methyl) is conjugated to SEQ ID NO: 742-745, 748-770, as set forth in SEQ ID NO: 769 and 794 and are shown in the table below.
Application method
Glucagon superfamily peptides
In general, prodrugs comprising glucagon superfamily peptides or glucagon related peptides (e.g., class 1, 2, 3, 4, or 5 peptides) can be used for any purpose for which glucagon superfamily peptides and glucagon related peptides have been used (see, e.g., the details above). For example, the disclosed prodrug analogs of bioactive peptides are considered suitable for any of the uses previously described for their corresponding parent bioactive peptides. Thus, the glucagon related peptide prodrug analogs described herein are useful for treating hypoglycemia, hyperglycemia, diabetes, or other metabolic diseases caused by high/low glucagon blood levels or high/low blood glucose levels. According to some embodiments, the patient to be treated with a prodrug disclosed herein is a domesticated animal, while in another embodiment, the patient to be treated is a human.
In some embodiments, the prodrugs are used to reduce or suppress appetite, reduce food intake, induce weight loss, or help maintain weight. Such methods of reducing appetite or promoting weight loss are expected to be useful for reducing weight, preventing weight gain, or treating various causes of obesity (including drug induced obesity) and reducing complications associated with obesity including vascular disease (coronary artery disease, stroke, peripheral vascular disease, ischemia reperfusion, etc.), hypertension, type II diabetes onset, hyperlipidemia, and musculoskeletal disease.
In other embodiments, the prodrug is used in combination with a nutrient for parenteral administration to a non-diabetic patient (e.g., a patient receiving parenteral nutrition or total parenteral nutrition) in a hospital setting. Non-limiting examples include surgical patients, coma patients, digestive tract disease patients or gastrointestinal nonfunctional patients (e.g., due to surgical resection, obstruction, or impaired absorptive capacity, Crohn's disease, ulcerative colitis, gastrointestinal obstruction, gastrointestinal fistulas, acute pancreatitis, intestinal ischemia, major gastrointestinal surgery, certain congenital gastrointestinal abnormalities, chronic diarrhea, or short bowel syndrome resulting from surgery), stroke patients, and patients who experience a healing process that often receives parenteral administration of carbohydrates, as well as various combinations of lipids, electrolytes, minerals, vitamins, and amino acids. The glucagon superfamily peptide prodrugs and the parenteral nutrition composition can be administered at the same time, at different times, before or after each other, provided that the glucagon superfamily peptide prodrugs exert the desired biological effect while the parenteral nutrition composition is being digested. For example, parenteral nutrition can be administered 1, 2, or 3 times daily, while glucagon superfamily peptide prodrugs are administered once every other day, three times a week, twice a week, once every two weeks, once every three weeks, or once a month.
Metabolic syndrome, also known as metabolic syndrome X, insulin resistance syndrome or Reaven's syndrome, is a condition that affects more than 5 million americans. Metabolic syndrome is typically characterized by the concentration of at least three or more of the following risk factors: (1) abdominal obesity (excess of adipose tissue in or around the abdomen), (2) atherogenic dyslipidemias (dyslipidemias, including high triglycerides, low HDL cholesterol and high LDL cholesterol that promote plaque accumulation in the arterial wall), (3) elevated blood pressure, (4) insulin resistance or glucose intolerance, (5) prothrombotic states (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), and (6) proinflammatory states (e.g., elevated C-reactive protein in the blood). Other risk factors may include aging, hormonal imbalances, and genetic predisposition.
Metabolic syndrome is associated with an increased risk of coronary heart disease and other conditions associated with vascular plaque accumulation, such as stroke and peripheral vascular disease, known as atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome can progress from their early insulin resistance state to full-blown type II diabetes with an increased risk of ASCVD. Without intending to be bound by any particular theory, the relationship between insulin resistance, metabolic syndrome, and vascular disease may include one or more concurrent pathogenic mechanisms, including impaired insulin-stimulated vasodilation, decreased NO availability associated with insulin resistance due to increased oxidative stress, and abnormalities in adipocyte-derived hormones (e.g., adiponectin) (Lteif and Mather, Can.J. Cardiol.20 (suppl B): 66B-76B (2004)).
According to the 2001 National Cholesterol expression Program additive treatment panel (ATP III) (2001 National Cholesterol education Program Adult treatment group), the same individual with any three of the following characteristics met the criteria for metabolic syndrome: (a) abdominal obesity (waist circumference of more than 102cm for men and more than 88cm for women); (b) serum triglyceride (150 m)g/dl or more); (c) HDL cholesterol (below 40mg/dl for men and below 50mg/dl for women); (d) blood pressure (above 130/85); and (e) fasting plasma glucose (above 110 mg/dl). According to the World Health Organization (WHO), individuals with high insulin levels (elevated fasting glucose or elevated postprandial glucose only) meet the criteria for metabolic syndrome with at least two of the following criteria: (a) abdominal obesity (waist/hip ratio greater than 0.9, body mass index at least 30kg/m2Or waist circumference of more than 37 inches); (b) cholesterol group with triglyceride level of at least 150mg/dl or HDL cholesterol of less than 35 mg/dl; (c) above blood pressure 140/90 or in the treatment of hypertension). (Mathur, Ruchi, "Metabolic Syndrome," Shiel master, Jr., William C., Medicine et. com, 11 months 5/2009).
For purposes herein, an individual is considered to have metabolic syndrome if the individual meets one or both of the criteria set by the adult treatment group or WHO of the 2001 national cholesterol education program.
Without being bound by any particular theory, the glucagon peptides described herein may be used to treat metabolic syndrome. Accordingly, the present invention provides a method of preventing or treating metabolic syndrome or reducing 1, 2, 3 or more risk factors thereof in a subject, the method comprising administering a glucagon peptide as described herein to the subject in an amount effective to prevent or treat metabolic syndrome or risk factors thereof.
Non-alcoholic fatty liver disease (NAFLD) refers to a wide range of liver diseases ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (NASH) to cirrhosis (irreversible advanced liver scarring). In liver cells (hepatocytes), NAFLD has common fat accumulation (fat infiltration) at all stages. Simple fatty liver is an abnormal accumulation of certain types of fats and triglycerides in hepatocytes without inflammation or scarring. In NASH, fat accumulation is associated with different degrees of inflammation (hepatitis) and scarring (fibrosis) of the liver. The inflammatory cells can destroy the hepatocytes (hepatocyte necrosis). In the terms "steatohepatitis" and "liponecrosis", fat (steato) refers to fatty infiltration, hepatitis refers to liver inflammation, and necrosis refers to damaged liver cells. NASH can eventually lead to scarring of the liver (fibrosis) followed by irreversible advanced scarring (cirrhosis). Cirrhosis caused by NASH is the last and most severe stage in the range of NAFLD. (Mendler, Michel, "Fatty Liver: NonalsoholicFatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatics (NASH)," Master Schoenfield, Leslie J., MedicinneNet. com., 8.29.2005).
Alcoholic or alcohol-induced liver disease includes 3 pathologically distinct liver diseases associated with or caused by excessive alcohol consumption: fatty liver (steatosis), chronic or acute hepatitis, and cirrhosis. Alcoholic hepatitis can range from mild hepatitis (where laboratory test abnormal results only indicate disease) to severe liver dysfunction with complications such as jaundice (yellowing of the skin due to bilirubin retention), hepatic encephalopathy (neurological dysfunction due to liver failure), ascites (accumulation of abdominal fluid), bleeding esophageal varices (varicose veins), blood clotting abnormalities and coma. Histologically, alcoholic hepatitis has the characteristic appearance of ballooning of hepatocytes, inflammation with neutrophils and sometimes malorosomes (abnormal aggregation of cellular intermediate filament proteins). Cirrhosis is characterized anatomically by widely distributed nodules in the liver with fibrosis. (Worman, Howard J., "Alcoholic LiverDisase", Columbia University Medical Center wet).
Without being bound by any particular theory, the class 2 and class 3 glucagon related peptides described herein may be used to treat alcoholic liver disease, NAFLD, or any stage thereof, including, for example, steatosis, steatohepatitis, hepatitis, liver inflammation, NASH, cirrhosis, or complications thereof. Accordingly, the present invention provides a method of preventing or treating alcoholic liver disease, NAFLD or any stage thereof in a subject, the method comprising administering to the subject a class 2 or class 3 glucagon peptide as described herein in an amount effective to prevent or treat alcoholic liver disease, NAFLD or a stage thereof. Such treatment methods include reducing/decreasing 1, 2, 3 or more of: liver fat content, incidence or progression of cirrhosis, incidence of hepatocellular carcinoma, signs of inflammation such as abnormal liver enzyme levels (e.g., aspartate aminotransferase AST and/or alanine aminotransferase ALT or LDH), elevated serum ferritin, elevated serum bilirubin, and/or signs of fibrosis, such as elevated TGF- β levels. In a preferred embodiment, class 2 or class 3 glucagon peptides are used to treat patients who develop beyond simple fatty liver (steatosis) and show signs of inflammation or hepatitis. Such methods may result in, for example, reduced AST and/or ALT levels.
GLP-1 and exendin-4 have been shown to have some neuroprotective effects. The invention also provides the use of glucagon superfamily peptides in the treatment of neurodegenerative diseases, including but not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, other demyelination-related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS-related dementia or other dementias, central nervous system cancer, traumatic brain injury, spinal cord injury, stroke or cerebral ischemia, cerebrovascular inflammation, epilepsy, Huntington's disease, Tourette's Syndrome, guillette's Syndrome, Guillain-barre Syndrome, hepatolenticular degeneration (Wilson's disease), Pick's disease, neuroinflammatory disorders, viral, fungal or bacterial encephalitis, encephalomyelitis or meningitis or other central nervous system infections, Prion diseases, cerebellar ataxia, cerebellar degeneration, spinocerebellar degeneration syndrome, friedreich's ataxia (friedreich ataxia), ataxia telangiectasia, spinal muscular dystrophy (spinodyskyotophy), progressive supranuclear palsy, dystonia, myospasticity, tremor, retinitis pigmentosa, striatal nigral degeneration, mitochondrial encephalomyopathy, neuronal ceroid lipofuscinosis, hepatic encephalopathy, renal encephalopathy, metabolic encephalopathy, toxin-induced encephalopathy, and radiation-induced brain injury.
Accordingly, the present invention provides a method of preventing or treating a neurodegenerative disease or reducing 1, 2, 3 or more risk factors thereof in a subject, the method comprising administering a glucagon peptide as described herein to the subject in an amount effective to prevent or treat the neurodegenerative disease or the risk factors thereof.
The methods of treatment of the present invention comprise the step of administering a prodrug disclosed herein to a patient using any standard route of administration, including parenterally, e.g., intravenously, intraperitoneally, subcutaneously or intramuscularly, intrathecally, transdermally, rectally, orally, intranasally, or by inhalation. In some embodiments, the composition is administered subcutaneously or intramuscularly, optionally into a depot or as part of a sustained release composition.
Compositions and combinations
The prodrugs of the invention may be administered alone or in combination with a second agent (e.g., an antidiabetic or antiobesity agent). In some aspects, the prodrug is administered in combination with a second prodrug or a glucagon superfamily member (including, e.g., glucagon related peptides). In certain embodiments, the prodrug is administered in combination with an antidiabetic agent, including but not limited to insulin; sulfonylureas, such as tolbutamide (Orinase), acetohexamide (demelor), tolazamide (Tolinase), chlorpropamide (trypticase), glipizide (glicotrol), glyburide (dabinetol), dabrymide (dabeta), glyburide (Micronase), dalianon (Glynase), glimepiride (alamide), or gliclazide (damacron)); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones, such as rosiglitazone (vindia), pioglitazone (Actos) or troglitazone (e.g. forest (Rezulin)) or other PPAR γ inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (acarbose/xylobay); exenatide (Byetta) or pramlintide; dipeptidyl peptidase-4 (DPP-4) inhibitors, such as vildagliptin or sitagliptin; SGLT (sodium dependent glucose transporter 1) inhibitors; or an FBPase (fructose 1, 6-bisphosphatase) inhibitor.
Antiobesity agents known in the art or under investigation include, but are not limited to, appetite suppressants including phenethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), amfepramoneBenzometrizineBenzphetamineSibutramineRimonabantOther cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (profac); qnexa (topiramate and phentermine), Excalia (bupropion and zonisamide), or Contrave (bupropion and naltrexone); or a lipase inhibitor similar to cenicrobile (orlistat) or cetilistat (also known as ATL-962) or GT 389-255.
Prodrugs of the invention may also be administered to patients suffering from catabolic wasting. It is estimated that more than half of cancer patients experience catabolic wasting characterized by involuntary progressive weight loss, weakness and low body fat and muscle levels. This syndrome is also common in AIDS patients and can also be present in bacterial and parasitic diseases, rheumatoid arthritis and chronic diseases of the intestine, liver, lung and heart. Catabolic wasting is often associated with anorexia and can be manifested as a result of an aging condition or physical trauma. Catabolic wasting is a symptom that reduces quality of life, worsens the underlying condition, and is the leading cause of death.
Pharmaceutical compositions comprising the prodrugs disclosed herein can be formulated and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also includes pharmaceutical compositions comprising one or more of the prodrugs disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the prodrug at a concentration of 1mg/ml in a phosphate buffer system at a pH of about 4.0 to about 7.0. The pharmaceutical compositions may comprise the prodrug as the sole pharmaceutically active ingredient, or the prodrug may be combined with one or more other active agents. According to some embodiments, there is provided a composition comprising a prodrug of the invention. Alternatively, compositions comprising the prodrugs and anti-obesity peptides can be provided for use in inducing weight loss or preventing weight gain. Suitable anti-obesity peptides include anti-obesity peptides disclosed in U.S. patent 5,691,309, 6,436,435 or U.S. patent application 20050176643.
According to some embodiments, pharmaceutical compositions are provided comprising any of the novel prodrugs disclosed herein, preferably sterile, and preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. Such compositions may contain a prodrug derivative of a biologically active peptide as disclosed herein, wherein the resulting active peptide is present at a concentration of at least 0.5mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or more. Such compositions may contain class 1, 2 or 3 bioactive peptide prodrug derivatives as disclosed herein, wherein the resulting active peptide is present at a concentration of at least A, wherein A is 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or higher. In other embodiments, such compositions may contain active peptides of class 1, 2 or 3 at a concentration of up to B, wherein B is 30mg/ml, 25mg/ml, 24mg/ml, 23, mg/ml, 22mg/ml, 21mg/ml, 20mg/ml, 19mg/ml, 18mg/ml, 17mg/ml, 16mg/ml, 15mg/ml, 14mg/ml, 13mg/ml, 12mg/ml, 11mg/ml 10mg/ml, 9mg/ml, 8mg/ml, 7mg/ml, 6mg/ml, 5mg/ml, 4mg/ml, 3mg/ml, 2mg/ml, 1mg/ml or 0.1 mg/ml. In some embodiments, the composition may contain glucagon related peptide class 1, 2 or 3 at a concentration ranging from A-B mg/ml, e.g., 0.001-30.0 mg/ml. In some embodiments, the pharmaceutical compositions comprise an aqueous solution that has been sterilized and optionally stored in various containers. According to some embodiments, the compounds of the present invention may be used to prepare a pre-prepared solution that is immediately ready for injection. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be further packaged as part of a kit that includes a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at ambient room temperature or at freezing temperatures.
All of the methods of treatment, pharmaceutical compositions, kits and other similar embodiments described herein contemplate that the prodrug compounds include all pharmaceutically acceptable salts thereof.
In some embodiments, kits are provided having means for administering the prodrug composition to a patient. The kit may further include various containers, such as vials, tubes, bottles, and the like. Preferably the kit may also include instructions for use. According to some embodiments, the device of the kit is an aerosol dispensing device, wherein the composition is packaged within the aerosol device. In another embodiment, the kit comprises a syringe and a needle, and in some embodiments, the prodrug composition is packaged within the syringe.
Pharmaceutical formulations of class 1, 2 and 3 glucagon related peptides
According to some embodiments, there is provided a pharmaceutical composition, wherein said composition comprises a glucagon peptide of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise any pharmaceutically acceptable ingredient including, for example, acidulants, additives, adsorbents, aerosol propellants, air release agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antimicrobials, binders, buffering agents, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizing agents, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, flavoring agents, flavorants, flow promoters, gelling agents, granulating agents, wetting agents, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, lozenge bases, pigments, plasticizers, polishing agents, preservatives, masking agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, Surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxic agents, viscosity increasing agents, water absorbing agents, water-soluble co-solvents, water-softening agents or wetting agents.
In some embodiments, the pharmaceutical composition comprises any one or a combination of the following components: gum arabic, acesulfame potassium, acetyl tributyl citrate, acetyl triethyl citrate, agar, albumin, alcohol, anhydrous alcohol, denatured alcohol, diluted alcohol, eleostearic acid, alginic acid, aliphatic polyesters, alumina, aluminum hydroxide, aluminum stearate, pullulan, alpha-amylose, ascorbic acid, ascorbyl palmitate, aspartame, bacteriostatic water for injection, bentonite slurry, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butyl paraben sodium, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, anhydrous calcium hydrogen phosphate, dehydrated calcium hydrogen phosphate, tribasic calcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate hemihydrate, Canola oil, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, beta-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate Cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetyltrimethylammonium bromide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbon (CFC), chlorophenoxyethanol, chloroxylenol, starch syrup dry powder, anhydrous, citric acid monohydrate, citric acid cocoa butter, a colorant, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, crosslinked carboxymethylcellulose sodium, crospovidone, cyclamic acid, cyclodextrin, glucose binder, dextrin, glucose, anhydrous glucose, diazolidinyl urea, dibutyl phthalate, sebacate, Diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-beta-cyclodextrin, compounds of the cyclodextrin type (for example) Dimethyl ether, dimethyl phthalate, dipotassium edetate, disodium hydrogen phosphate, calcium docusate, potassium docusate, sodium docusate, lauryl gallate, dodecyltrimethylammonium bromide, disodium calcium edetate, edetic acid (ethic acid), meglumine, ethanol, ethyl cellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethyl p-hydroxybenzoate, potassium ethyl p-hydroxybenzoate, sodium ethyl p-hydroxybenzoate, ethyl vanillin, fructose, liquid fructose, milled fructose, pyrogen-free fructose, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, a glycerol ester mixture of saturated vegetable fatty acids, glycerol, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, self-emulsifying glyceryl monostearate, glyceryl palmitostearate (glyceryl palmityl palmitstearate), Glycine, glycols, glycofurol, guar gum, Heptafluoropropane (HFC), cetyltrimethylammonium bromide, high fructose syrup, human serum albumin, Hydrocarbons (HC), dilute hydrochloric acid, hydrogenated vegetable oil type II, hydroxyethylcellulose, 2-hydroxyethyl-beta-cyclodextrin, hydroxypropyl Methylcellulose, low-substituted hydroxypropylcellulose, 2-hydroxypropyl-beta-cyclodextrin, hydroxypropylmethylcellulose phthalate, imidurea, indigo carmine, ion exchanger, iron oxide, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin alcohol, anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesium carbonate, normal magnesium carbonate, anhydrous magnesium carbonate, basic magnesium carbonate, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, malt, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium-chain triglyceride, meglumine, menthol, methylcellulose, methyl methacrylate, sodium alginate, sodium gluconate, sodium alginate, methyl oleate, methyl paraben, potassium methylparaben, sodium methylparaben, microcrystalline cellulose and sodium carboxymethylcellulose, mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum and lanolin alcohols, pharmaceutical glaze, phenol, liquefied phenol, phenoxyethanol, phenoxypropanol, phenylethanol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin, potassium polacrilin, poloxamer, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylate, polyethylene-polyoxypropylene-block polymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitol fatty acid ester, Polyoxyethylene stearate, polyvinyl alcohol, polyvinylpyrrolidone, potassium alginate, potassium benzoate, potassium hydrogen carbonate, potassium hydrogen sulfite, potassium chloride, potassium citrate, anhydrous potassium citrate, potassium hydrogen phosphate, potassium metabisulfite, potassium dihydrogen phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol alginate, propyl gallate, propyl p-hydroxybenzoate, potassium propyl p-hydroxybenzoate, sodium propyl p-hydroxybenzoate, protamine sulfate, rapeseed oil, ringer's solution, saccharin ammonium saccharin, calcium saccharin, sodium saccharin, red sage Flower oil, saponite, serum albumin, sesame oil, colloidal silica, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride, sodium cyclamate, sodium edetate, sodium lauryl sulfate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, anhydrous sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty esters), sorbitol, 70% sorbitol solutions, soybean oil, spermaceti, starch, corn starch, potato starch, pregelatinized, sterilized corn starch, stearic acid, pure stearic acid, stearyl alcohol, sucrose, sugar, compressible sugar, textured crushed sugar (conditioner's sugar), Sugar spheres, invert sugar, sucrose-invert sugar polymer (Sugartab), sunset FCF, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), cocoa butter, thimerosal, titanium dioxide, alpha tocopherol, vitamin E acetate, alpha vitamin E succinate, beta tocopherol, delta tocopherol, gamma tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl-beta-cyclodextrin, trimethyltetradecylammonium bromide, tris buffer, trisodium edetate, vanillin, hydrogenated vegetable oil type I, water, soft water, hard water, carbonless water, pyrogen-free water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for rinsing, wax, anionic emulsifying wax, carnauba wax, cationic wax, hexadecyl ester wax, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), cocoa butter, thimerosal, thiuram, trizan, thiuram, thim, thiuram, thi, Microcrystalline wax, nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, lanolin, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate or any excipient in the following literature: handbook of Pharmaceutical Excipients, 3 rd edition, a.h. kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980), which is incorporated by reference in its entirety, discloses various compositions for formulating pharmaceutically acceptable combinations The various components of (a) and known techniques for their preparation. Except insofar as any conventional substance is incompatible with the pharmaceutical composition, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.
The pharmaceutical formulations disclosed herein may be designed as short-acting, immediate-release, long-acting, or sustained-release formulations as described below. The pharmaceutical formulations may also be formulated for immediate, controlled or sustained release. The compositions of the invention may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form, thereby providing long term storage and/or delivery effects. The disclosed pharmaceutical formulations can be administered on any schedule, including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, biweekly, every three weeks, monthly, or every two months.
In some embodiments, the aforementioned components may be present in the pharmaceutical composition at any concentration, e.g., at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the aforementioned components may be present in the pharmaceutical composition at any concentration, e.g., up to B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In other embodiments, the aforementioned components may be present in the pharmaceutical composition in any concentration range, for example, from about a to about B. In some embodiments, a is 0.0001% and B is 90%.
The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 11, depending on the dosage form and route of administration. In certain embodiments, the pharmaceutical composition may comprise a buffering agent to achieve a physiologically compatible pH. The buffer may include any compound capable of buffering at a desired pH, such as phosphate buffer (e.g., PBS), triethanolamine, Tris, bicine, TAPS, troxin, HEPES, TES, MOPS, PIPES, cacodylate, MES, and the like. In certain embodiments, the buffer has a concentration of at least 0.5mM, at least 1mM, at least 5mM, at least 10mM, at least 20mM, at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 120mM, at least 150mM, or at least 200 mM. In some embodiments, the buffer has a concentration of no more than 300mM (e.g., at most 200mM, at most 100mM, at most 90mM, at most 80mM, at most 70mM, at most 60mM, at most 50mM, at most 40mM, at most 30mM, at most 20mM, at most 10mM, at most 5mM, at most 1 mM).
The prodrug compounds disclosed herein can be prepared by standard synthetic methods, recombinant DNA techniques, or any other method for preparing peptides and fusion proteins. Although certain unnatural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. In addition to standard peptide chemistry, where applicable, the compounds of the invention including non-peptide moieties can be synthesized by standard organic chemistry.