CN101035568A - Combination therapy using transferrin fusion proteins comprising GLP-1 - Google Patents

Abstract

The present invention provides combination therapy comprising transferrin fusion protein and DPP-IV inhibitors and/or neutral endopeptidase (NEP) inhibitors. The transferrin fusion protein comprises therapeutic polypeptides or peptides useful in the treatment of diseases such as diabetes.

Description

Use comprises the combined therapy of the transferrin fusion proteins of GLP-1

Related application

The application requires to enjoy the priority of the U.S. Provisional Patent Application of submitting on August 3rd, 2,004 60/598,031, and it is incorporated herein by reference fully at this.

The application relates to the PCT/US03/26818 partial continuous application of submission on August 28th, 2003, and PCT/US03/26818 is the partial continuous application of the U.S. Patent application 10/378,094 of submission on March 4th, 2003, and these two parts of applications are incorporated herein by reference fully at this.

Technical field

The present invention relates to comprise the transferrin fusion proteins of insulinotropic peptides (insulinotropic peptide), the half-life prolongs in the body of its effective therapeutic activity.The invention still further relates to the combined therapy that uses DPP-IV inhibitor and/or neutral endopeptidase inhibitor (NEP) and insulinotropic peptides.

Background technology

Protease

Proteolytic enzyme plays an important role these physiological process such as cell proliferation, differentiation, and signal processing by regulating protein conversion and processing in the regulation and control physiological process.Proteolytic enzyme is controlled the important proteic level of structural protein, enzyme and adjusting by proteolytic degradation.No matter hydrolase of proteolysis out of control is to raise or reduction, all relevant with various disease states, comprises inflammation, cancer, arteriosclerosis and degenerative disease.

International molecular biology community of biochemical society (IUBMB) recommends to use term " peptidase " to refer to peptide bond hydrolysis enzyme subclass (subclass E.C 3.4.).Widely the term protease of Shi Yonging has identical implication with peptidase.Peptidase comprises two groups of enzymes: endopeptidase and exopeptidase, and the peptide bond in its crack protein matter, and sequentially respectively from N-end or the terminal aminoacid of removing of C-.Term protease and endopeptidase have identical meanings.Proteolytic enzyme is according to its catalyst mechanism classification.IUBMB has approved four kinds of mechanism types: serine protease, cysteine proteinase, aspartic protease and metalloproteases.

Serine protease is extended familys of proteolytic enzyme, contains serine residue on the active enzymatic site of protein cleavage.They are prevalent in virus, antibacterial and the eukaryote.Serine protease has substrate specificity widely, can be subdivided into subfamily again based on these specificitys.More than 20 serine protease subfamily arranged, be divided into 6 branches (SA, SB, SC, SE, SF and SG).

Prolyl oligopeptidase is to be divided in the ramose a kind of serine protease of SC.It contains the peptide of proline in the carboxyl side hydrolysis of proline residue.It may be relevant with degraded with the maturation of peptide hormone and neuropeptide (people such as Wilk, 1983 Life Sci.33,2149-2157).The example of prolyl oligopeptidase comprises DPP IV (DPP-IV), dipeptidyl peptidase II (DPP-II), fibroblast activation protein and prolyl oligopeptidase.These enzymes have distinct specificity.

Proline is present in a large amount of peptide hormones.It has determined some architectural characteristic of these peptides, and for example the conformation of these peptides and stability prevent to be degraded by nonspecific protease.There are a large amount of peptidases of attacking the proline peptide bond.These peptidases not only fracture with X-Pro or Pro-X key are relevant, and participate in the degraded of corresponding alanyl key, but have the activity of decline.Because it is relevant with the biologic activity of regulating natural peptide substrates that some have an active peptidase of high special to the sequence that contains proline, these peptidases become the attractive object of study in medicinal chemistry field.For example, DPP-IV and level by regulating glucagon-like-peptide-1 (GLP-1) with treat diabetes and connect.In multiple disease, for example rheumatoid arthritis, multiple sclerosis, Graves disease (Grave ' s disease) and HashimotoShi thyroiditis, sarcoidosis (sarcoidosis) and cancer, DPP-IV is active to raise.In AIDS, Down's syndrome (Down ' s syndrome), anorexia/bulimia (anorexia/bulimia), pregnancy and hypogammag lobulinemia, the DPP-IV activity also raises.

The dipeptidyl peptidase that comprises DPP-IV

Two peptidyl amino peptidase activity are that catalysis is from peptide, polypeptide or and the terminal peptidase activity of removing dipeptides of proteinic N-.Usually, two peptidyl amino peptidases can be from cracking dipeptides XY on peptide, polypeptide and the proteinic unsubstituted N-terminal amino group, and wherein X and Y represent any amino acid residue.The example of dipeptidyl peptidase (DPPs) comprises dipeptidyl peptidase I (DPP-I), dipeptidyl peptidase II (DPP-II), dipeptidyl peptidase III (DPP-III) and dipeptidyl peptidase (DPP-IV).

DPP-I also is called cathepsin C, is a kind of lysosome cysteine proteinase of expressing in the great majority tissue.DPP-I is relevant with the processing of granzyme, and granzyme is the neutral serine at the cellulotoxic lymphocyte granule special secondary school gate expression that is activated.DPP-II is a kind of serine protease that is present in the lysosome.Identical with DPP-IV, the DPP-II cracking contains the peptide bond of proline.In fact, DPP-II has the substrate specificity similar to DPP-IV, but only works under acid pH.Dipeptidyl peptidase III (DPP-III) is a kind of metalloproteases.

DPP-IV is a kind of serine protease, comprises serine protease motif GWSYG, and has substrate specificity widely.It discharges aminoacid from amino terminal order range of hydrolysed peptides.Yet when the amino acid residue before the arrival proline, hydrolysis stops.As a result, the peptide with key X-Pro-Y-(X and Y are the aminoacid of choosing wantonly) is cleaved, obtains X-Pro and Y-.DPP-IV also can break at the dipeptides that has alanine on the penult position, though its efficient is lower than the dipeptides (people such as Yaron, 1993 Crit.Rev.Biochem.Mol.Biol.28:31-81) with proline.This enzyme can also other sequence of cracking, but efficient is still lower.

Verified, when discharging dipeptides from the N-of biologically active peptide is terminal, DPP-IV has high specific, and described bioactive peptide has proline or alanine on the penult position of the N-of peptide substrates end.The a large amount of possible peptide substrates of DPP-IV is identified.The DPP-IV substrate comprises peptide hormone and cytokine.The example of some peptide hormone is endorphine-2 (endomorphin-2), GLP-1, GLP-2, gastric inhibitory polypeptide (GIP), neuropeptide tyrosine, growth hormone releasing hormone (GHRH) and P material, and the example of some cytokine is RANTES, GCP-2, SDF-1 α, SDF-2 β, MDC, MCP-1, MCP-2 and MCP-3.DPP-II has the substrate specificity much at one with DPP-IV.

DPP-IV and diabetes

The diabetes of insulin dependency (IDDM or type i diabetes) are by treating for patient's administration of insulin at present.Noninsulindependent diabetes (NIDDM or type ii diabetes) is by diet, uses sulfonylureas (sulphonylureas) and stimulate insulin secretion or use biguanide and increase glucose uptake and treat.Individual need insulinize with resistance.Standard care requires to need intravenous injection every day insulin, and this can treat acute symptom, but the prolongation of treatment phase can cause angiopathy and nervous lesion.Modern method is very expensive, as transplanting, and the operation interference that need have risk.Therefore, need a kind of efficiently, treating diabetes replacement scheme cheaply of exploitation.

In the last few years, people were to interested day by day as the target spot of blood sugar lowering level with DPP-IV.Use inhibitor to block DPP-IV enzyme in the blood samples of patients or the activity of DPP-IV sample enzyme can cause reducing to degraded endogenic or the external insulinotropic peptides of using, these insulinotropic peptides such as GIP, GLP-1 or its analog.GIP and GLP-1 are the hormones of stimulating pancreas glucose induction formula ground excreting insulin, are the substrates of DPP-IV.Especially, because DPP-IV removes the aminoterminal His-Ala dipeptides of GLP-1, generate GLP-1-(9-36)-amide, GLP-1-(9-36)-amide can not cause that the glucose of islets of langerhans relies on formula ground excreting insulin, and the inhibition of the activity in vivo of this DPP-IV or DPP-IV sample enzyme will suppress the enzymatic activity of not expecting under the pathological condition effectively in mammalian organism.

PCT/DE97/00820 discloses the inhibitor of DPP-IV or DPP-IV sample enzymatic activity, alanyl pyrrolidinium (alanyl pyrrolidide) and isoleucyl-thiazoline salt (isoleucyl thiazolidide).DD 296075 discloses pyrrolidinium (pyrrolidide) and isoleucyl-thiazoline hydrochlorate.U.S. Pat 6,548,481 disclose the inhibitor that is similarly formed by aminoacid and thiazoline salt or pyrrolidinium group with dipeptide compound, and salt.Though these chemical compounds active functional inhibitor that is DPP-IV, but in some patient or particular type disease, it is problematic using these inhibitor, this is because these enzymes are responsible for activating or the so large-scale biologically active peptide of deactivation, that is to say that the DPP-IV inhibitor does not have specificity to expectation target spot GIP and GLP-1.

By the modification protection therapeutic peptide

Prevent that therapeutic protein and peptide such as GIP or GLP-1 from being that protein and peptide itself are modified by a kind of alternative of proteolytic enzyme cleaves, is exposed to proteolytic enzyme to hinder it.Verified, protein modification can improve stability, circulation time and the biologic activity of therapeutical peptide.The common method of some modified amino acids and peptide is disclosed in Chemistry and Biochemistry ofAmino Acid, Peptide and Proteins--A Survey of Recent Developments (Weinstein, B. edit, Marcel Dekker, Inc. publish, New York 1983), it is hereby incorporated by.In addition, the survey article of Francis (1992 Focus on Growth Factors 3:4-10, (Mediscript, London)) discloses protein modification and fusion rotein, and it is hereby incorporated by.

Along with the development of DNA recombinant technique and automatic technology, can easily prepare in a large number through modified polypeptides now, these peptides can be short, moderate-lengths or long.Can use automatic peptide synthesizer, solid-phase resin technology or recombinant technique, synthetic a large amount of little polypeptide hormones through modifying.For example, the substrate of a large amount of modifications of dipeptidyl peptidase can prepare with automatic peptide synthesizer, for example, and the substrate of DPP-IV such as GLP-1, GIP, neuropeptide tyrosine and Kallidin I.

Summary of the invention

The invention provides transferrin fusion proteins, it comprises the therapeutic peptide of protease cracking sensitivity or protein.The present invention also provides transferrin fusion proteins, and it comprises protease cracking sensitivity, the therapeutic peptide or the protein that have resistance or have partial resistance.Described protease can be DPP-IV or neutral endopeptidase (NEP).Yet, the invention provides and comprise the transferrin fusion proteins and the second combination of agents thing, described second reagent for example but be not limited to the inhibitor of DPP-IV and/or the inhibitor of NEP.In addition, said composition can be the pharmaceutical composition that is used for the treatment of various diseases.

The invention provides and use combined therapy, when being included in the treatment various diseases, use transferrin fusion proteins and at least a second reagent.Transferrin fusion proteins can be used simultaneously with one or more second reagent.Alternately, before using second reagent or afterwards, order is used transferrin fusion proteins.Preferably, second reagent is the inhibitor of DPP-IV or the inhibitor of NEP.

Modify GLP-1 peptide moiety of the present invention and comprise one or more sudden changes, make them partly or completely resist the cracking of protease, for example the cracking of DPP-IV.The GLP-1 peptide can be GLP-1 (7-37) (SEQ ID NO:32) or GLP-1 (7-36) (amino acid/11-30 of SEQ ID NO:2).For example, modify these peptides by A8 being sported G and/or K34A.

Brief description

Fig. 1 shows the Restriction Enzyme collection of illustrative plates of pREX0094.

Fig. 2 shows the Restriction Enzyme collection of illustrative plates of plasmid pREX0198.

Fig. 3 shows the Restriction Enzyme collection of illustrative plates of pSAC35.

Fig. 4 shows the Restriction Enzyme collection of illustrative plates of plasmid pREX0240.

Fig. 5 shows the Restriction Enzyme collection of illustrative plates of pREX0052.

Fig. 6 shows the Restriction Enzyme collection of illustrative plates of pREX0367.

Fig. 7 shows the Restriction Enzyme collection of illustrative plates of pREX0368.

Fig. 8 shows the time-histories of GLP-1 and H-GLP-1 and DPP-IV incubation.The residue content of this pictorialization active full-length peptide detects by the ELISA that is specific to active GLP-1.

Detailed Description Of The Invention

1. general description

The present invention part is a kind of more effectively based on the needs exploitation, the replacement scheme for the treatment of diabetes cheaply. Insulinotropic peptides such as GLP-1, is very promising non-insulin-dependent 2 type diabetes and relevant metabolic disorder such as prediabetes (pre-diabetes), metabolic syndrome and the fat therapeutic agent of being used for the treatment of. Other useful insulinotropic peptides (insulinotropic peptide) comprises exciting peptide (exendin) 3 of Heloderma suspectum and the exciting peptide 4 of Heloderma suspectum. Yet the body of these insulinotropic peptides is short interior plasma half-life, mainly is owing to being removed from serum rapidly and being degraded by proteolysis. Also carried out extensive work and suppressed to be responsible for the enzyme DPP-IV of degraded GLP-1, perhaps modified GLP-1 and slow down its degradation speed and still keep its BA. Although carry out these a large amount of effort, also do not prepare lasting active GLP-1. Therefore, need to modify GLP-1, the exciting peptide 3 of Heloderma suspectum, the exciting peptide 4 of Heloderma suspectum and other insulinotropic peptides, with the longer activity in vivo duration of generation, and still keep its hypotoxicity and treatment advantage.

2. definition

As used in this, term " is derived " and is meant and uses or do not use enzyme, the modification of one or more amino acid residues of peptide being carried out by chemical means, for example, by alkylation, acidylate, formation ester or form amide.

As used in this, term " derives from " and is meant from particular source and obtains a kind of molecule, for example obtains a kind of molecule from parent's molecule.

As used in this, term " two peptidyl amino peptidase activity (dipeptidyl aminopeptidaseactivity) " is meant from the peptidase activity of the terminal cracking dipeptides of peptide, polypeptide or proteinic N-.Usually, two peptidyl amino peptidases can be from cracking dipeptides XY on peptide, polypeptide or the proteinic unsubstituted N-terminal amino group, wherein X and Y representative is selected from any amino acid residue of the group of being made up of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, but is at least Ala, Arg, Asp and/or Gly.Preferably, Y is Pro or Ala.All X can be different or identical with Y.The example of two peptidyl amino peptidases includes, but are not limited to DPP-I, DPP-II, DPP-III and DPP-IV.

As used in this, term " glucagon-like-peptide-1 (GLP-1) " and " GLP-1 derivant " are meant intestinal hormones, and it is usually at hyperglycemia process moderate stimulation insulin secretion, and glucagon suppression is secreted, stimulate the insulin biosynthesis, and slow down gastric emptying and acid secretion.As U.S. Pat 5,574, disclosed in 008, some GLP-1 and GLP-1 derivant promote the cellular uptake glucose, but do not stimulate insulin expression, and this patent is hereby incorporated by.

As used in this, term " insulinotropic peptides " is meant and has the active peptide of short islets of langerhans.Insulinotropic peptides stimulates or causes stimulating the synthetic or expression of hormone insulin.This peptide comprises propeptide, analog, fragment, has the active peptide of short islets of langerhans as glucagon-like peptide 1, the exciting peptide 3 of Heloderma suspectum and the exciting peptide 4 of Heloderma suspectum and other.

As used in this, " pharmaceutically acceptable " be meant tolerate on the physiology and be applied the material and the compositions that can not produce hypersensitive or similar untoward reaction such as regurgitation, giddy etc. to man-hour usually.Usually, as used in this, term " pharmaceutically acceptable " is meant to the approval of the administrative organization of federation or state government or is set forth in American Pharmacopeia or other approved usually particularly pharmacopeia of people of animal that is used for.

As used in this, term " pharmaceutical composition " is meant the compositions that comprises medicine and add pharmaceutically acceptable carrier or diluent when needing.Select pharmaceutically acceptable carrier and additive, make the side effect of medical compounds be minimized and the performance of this chemical compound can not be eliminated or be suppressed to the degree of failing to respond to any medical treatment that makes.

As used in this, " physiology effective dose " is the amount that the patient produces slowing down of expectation or curative effect of passing.This amount is specific to various medicines and final approved dosage level thereof.

As used in this, " treatment effective dose " be meant when the patient that is applied to needs, be enough to realize to treat through the therapeutical peptide modified or the amount of peptide.Constitute changing of " treatment effective dose " through the therapeutical peptide of modification or the amount of peptide, it depends on used therapeutic protein, the order of severity of situation or disease, patient's age of being treated and body weight can be determined according to himself knowledge and this paper disclosure routinely by those of ordinary skills.

As used in this, " therapeutic protein " is meant protein, polypeptide, the antibody with one or more therapeutic activities and/or biologic activity, or its fragments of peptides or variant.The included therapeutic protein of the present invention includes, but are not limited to protein, polypeptide, peptide, antibody and biological product (biologics).Term peptide, protein and polypeptide are exchanged use at this.Alternately, term " therapeutic protein " is meant the endogenic or naturally occurring correlative of therapeutic protein.Have the polypeptide of " therapeutic activity " or peptide or " therapeutic activity " protein and be meant such polypeptide, peptide or protein, it has one or more known biologic activity and/or therapeutic activities, this activity and therapeutic protein such as one or more other human cytokines qualitative correlations disclosed herein or known in the art.As a non-limitative example, " therapeutic protein " is such protein, polypeptide or peptide, and it can be used for treatment, prevents or improve disease, situation or unusual.This disease, situation or be present in unusually among people or the non-human animal, for example veterinary purpose.

As used in this, term " processing " or " treatment " are meant that any of chemical compound of the present invention uses, and comprise: (1) wards off disease and takes place on one's body animal, but this animal tends to ill pathology or the symptom of also not suffering from or not showing this disease; (2) suppress the intravital disease of animal, this animal is just suffering from or is showing ill pathology or symptom (that is, further developing of pathology and/or symptom being stopped); Perhaps (3) improve the intravital disease of animal, and this animal is just suffering from or showing ill pathology or symptom (that is, pathology and/or symptom being reversed).

As used in this, term " biologic activity " is meant the ability of regulating biological function." biologic activity " comprises functional activity and structure-activity.

As used in this, term " slows down " and is meant and alleviates or relax disease or unusual symptom and the ability that realizes curing.For example, ease the pain and the preparation of not curing this situation or disease is a kind of preparation that slows down.

As used in this, term " prevention " is meant to have protective effect, for example works to resist or stop something, particularly disease or situation.

As used in this, " be purified " protein or the nucleic acid that protein or nucleic acid are meant that separation obtains from cell component." being purified " protein or nucleic acid has been purified in the non-existent purity level of occurring in nature.

As used in this, term " pure basically (substantially pure) " protein or nucleic acid are meant a kind of protein or nucleic acid, and it does not have all other cell component.

As used in this, term " curative " be meant have healing, recovery or salvage.For example, " therapeutic preparation " or " therapeutic composition " has the healing effect.

3. the specific embodiment

Dipeptidyl peptidase

Dipeptidyl peptidase is a hydrolytic enzyme, and it removes dipeptides from peptide, polypeptide or proteinic unsubstituted N-terminal amino group group.The example of dipeptidyl peptidase (DPP) includes, but are not limited to DPP-I, DPP-II, DPP-III, DPP-IV, lures albumen (attractin) and fibroblast activation protein matter (FAP).This family or have similar functions and the different new enzyme of structure is just found gradually.

Dipeptidyl peptidase I (DPP-I) also is called cathepsin C, is a kind of lysosome cysteine proteinase that belongs to papain family.DPP-I can remove dipeptides from the free amine group of various peptides and protein substrate is terminal successively, thereby works with the pattern of exopeptidase (particularly dipeptidyl peptidase).If cut key is positioned at the either side of proline residue, perhaps the N-terminal residue is lysine or arginine, and cracking can not take place effectively.

DPP-II be a kind ofly be present in the lysosome, the serine protease of Unknown Function.Identical with DPP-IV, its main cracking contains the peptide bond of proline.In fact, DPP-II has the similar substrate specificity with DPP-IV, but it only works at acid pH.Mammiferous DPP-II and DPP-IV can distinguish with inhibitor puromycin and bacitracin (bacitracin); Puromycin only suppresses DPP-II, and bacitracin only suppress DPP-IV (1988 J.Biol.Chem.263,6613-6618).Dipeptidyl peptidase III (DPP-III) is a kind of metalloproteases.DPP-V discharges X-Ala, His-Ser and the Ser-Tyr dipeptides of N-end.

DPP-VII also is called the rest cell prolidase, is a kind of dipeptidase of proline-specific.Based on the sequence of people DPP-VII and rat DPP-II relatively (78% homogeneity) (people such as Araki, 2001 J.Biochem.129 279-288), it is generally acknowledged that DPP-VII and DPP-II are identical protease.

DPP-VIII is people's back proline (postproline) two peptidyl amino peptidases, with DPP-IV and FAP homology (Abbott, people such as CA., 2000 European Journal of Biochemistry 267,6140).Similar with DPP-IV, DPP-VIII is ubiquitous.882 amino acid whose protein of the DPP-VIII cDNA of total length coding, its and DPP-IV have about 27% homogeneity, have about 51% similarity with FAP, but do not have membrane spaning domain, and the glycosylation that the glycosylation of N-connection is connected with O-does not take place yet.Reorganization DPP-VIII hydrolysis DPP-IV substrate A la-Pro, the Arg-Pro and the Gly-Pro that are purified.Therefore, reorganization DPP-VIII and DPP-IV and FAP have back proline dipeptides base amino peptidase activity.The DPP-VIII enzymatic activity has and the corresponding to neutral pH optimum activity of non-lysosome.Show that at the similarity on tissue expression pattern and the substrate latent effect and the DPP-IV of DPP-VIII on T cell activation and immunologic function is similar between DPP-VIII and the DPP-IV.

(2002 Gene 299 185-93) report that they have identified and characterized new DPP-IV sample molecule to people such as Olsen C., and this molecule is called dipeptidyl peptidase sample 3-protein d PP-IX.Similar with DPP-IV, DPP-IX comprises serine protease motif GWSYG (SEQ ID NO:110).In DPP-IX, there is this motif and constitutes catalysis three combination (triad) aminoacid Ser, Asp among the DPP-IV and the conservative order of His residue and at interval, make DPP-IX DPP-IV gene family due to.

Luring albumen (DPPT-L) is the soluble sugar albumen of a kind of 175kDa, and it is in the news can hydrolysis Gly-Pro.Lure albumen to contain kelch repetitive structure territory, do not have significant sequence homogeneity with DPP-IV or any other peptidase.Fibroblast activation protein (FAP) is the conjugated protein enzyme of cell surface at the prolyl oligopeptidase gene family of tissue reconstruction position expression.

Prolyl endopeptidase (PEP) also is called proline oligopeptidase (PO), is at first found by Walter and colleague thereof, is enzyme people such as (, Science173,827-829 (1971)) Walter of a kind of oxytocin of degrading in the people uterus.This enzymatic breaking contains the peptide bond of the carboxyl side of the proline in the peptide of X-Pro-Y sequence, wherein X is the substituted aminoacid of a kind of peptide or N-end, and Y is a kind of peptide, aminoacid, amide or alcohol (people such as Yoshimoto, J.Biol.Chem.253,3708-3716 (1979)).This enzyme has high degree of specificity (Lin ﹠amp to the conformation conversion (trans-conformation) of the peptide bond of the imino group side of proline; Brandts, Biochemistry 22,4480-4485 (1983)).

Prolyl oligopeptidase hydrolysis angiotensin I and Angiotensin II, this causes that angiotensin (1-7) discharges.Angiotensin (1-7) has hemangiectasis activity, and regulates vassopressin release, influences the memory process, and this is by confirming to the specific PEP-inhibitor of rat injection.This injection can reverse the amnesia that is caused by scopolamine.The example that may have physiological function of this enzyme of proof is not only in this experiment, and it causes a kind of hypothesis, be the inhibitor of PEP can influence the memory process and can dementia (people such as Yoshimoto, 1987 J.Pharmacobio-Dyn.10,730-735).

Dipeptidyl peptidase (DPP-IV) and substrate

DPP-IV is a kind of by the molecule of wide expression, and its degraded with multiple peptide and hormone is relevant.Purified various types of DPP-IV, and disclosed its enzymatic property.For example, from rat liver (people such as Hopsu-Havu V.K., 1966 Histochem., 7:197-201), Ren sus domestica (people such as BarthA., 1974 Biol.Med.Chem., 32:157-174), (Svensson B.1978Eur.J.Biochem. for pig small intestine, 90:489-498), pig liver (people 1981Biochim.Biophys.Acta such as Fukasawa K.M., 657:179-189), people's submaxillary gland (people such as Oya H., 1972Biochim.Biophys.Acta, 258:591-599), sheep kidney (people such as Yoshimoto T., 1977Biochim.Biophys.Acta, 485:391-401; People such as Yoshimoto T., 1978 J.Biol.Chem., 253:3708-3716) or microorganism (Fukusawa K.M.1981 Biochem.Biophys., 210:230-237; Yoshimoto is J.Biochem. T.1982,91:1899-1906 (1982)) obtain DPP-IV middle the separation.

In human immune system, DPP-IV is identical with t cell surface antigen CD26, and CD26 is expressed by the lymphocyte that is activated (T lymphocyte, bone-marrow-derived lymphocyte and natural killer cell).CD26/DPP-IV is an II type glycoprotein, has intrinsic DPP IV activity, and can be in conjunction with I type ADA Adenosine deaminase (ADA-1).Express on its composing type ground in epithelial cell, but in the T lymphocyte, it is only expressed under strict cell regulate and control, and its expression is raised when cell activation.Verified, the ectodomain of CD26/DPP-IV has DPP IV activity (people such as Hegen, 1990 J.Immunol 144:2908-2914; People such as Ulmer, 1990 Scand.J.Immunol.31:429-435), as and if altogether stimulating activity partly depends on activity people such as (, 1993Proc.Natl.Acad.Sci.USA 90:4586-4590) Tanaka of this enzyme.DPP-IV is relevant with the cytokine function regulation and control, may play an important role in HIV infects.

U.S. Pat 6,265,551 disclose the DPP-IV/CD26 of a kind of circulation, soluble form, and it separates from human serum.The DPP-IV/CD26 of this serum form has similar enzymatic property and antigenic characteristic to the DPP-IV/CD26 of ubiquitous form membrane; But it is completely different aspect a plurality of biochemistrys.Especially, the molecular weight of the DPP-IV/CD26 of circulation serum form is 175kDa, is different from the molecular weight of the 105kDa of form membrane, and it is not in conjunction with ADA-1.Yet, the DPP IV activity of the DPP-IV/CD26 display functionality of circulation form, and keep the ability that stimulates T lymphocyte reaction recall antigen (recall antigen) altogether.

The proteolytic activity of DPP-IV is positioned on about 200 amino acid whose fragments of this PROTEIN C-end.Catalytic residue (Ser-629, Asp-708, His-740) is to be different from unique sequence arrangement of traditional serine protease such as chymase and subtilisin.The dipeptidyl peptidase enzyme activity change large number of biological reactive protein of proline-specific and the biologic activity of polypeptide, wherein, these protein and polypeptide comprise GLP-1, neurotransmitter P material, human growth hormone releasing factor, erythropoietin, interleukin-22 or the like.Possible DPP-IV substrate is set forth in table 1, table 2 and the table 3.Regulating these peptides influences the DPP-IV cracking and can be used for treating clinical disease, includes, but are not limited to diabetes, inflammation, angiopathy, autoimmune disease, multiple sclerosis, arthrosis and transforms relevant disease with optimum with malignant cell

Table 1: have human cell factor, somatomedin, neuropeptide and the vasoactive peptide of penultimate proline, they are the substrates of inferring of DPP-IV.

Polypeptide	SEQ ID NO：	The N-end sequence
			Il-1 β	1	Ala-Pro-Val-Arg-Ser-
Interleukin-2	2	Ala-Pro-Thr-Ser-Ser-
			Interleukin-5	3	Ile-Pro-Thr-Glu-Ile-
Interleukin-6	4	Val-Pro-Pro-Gly-Glu-
			IL-10 INTERLEUKIN-10	5	Ser-Pro-Gly-Gln-Gly-
Interleukin-13 (reorganization)	6	Ser-Pro-Gly-Pro-Val-
			Complement C4a	7	Lys-Pro-Arg-Leu-Leu-

Granulocyte chemoattractant protein II	8	Gly-Pro-Val-Ser-Ala-
			Granulocyte macrophage colony stimulating factor	9	Ala-Pro-Ala-Arg-Ser-
Granulocyte-colony stimulating factor	10	Thr-Pro-Leu-Gly-Pro-
			Erythropoietin	11	Ala-Pro-Pro-Arg-Leu-
The gastrin releasing peptide growth hormone	12	Phe-Pro-Thr-Ile-Pro-
			Interferon-induced peptide 10 (γ IP10)	13	Val-Pro-Leu-Ser-Arg-
Interferon regulatory factor 1 (IRF-1)	14	Met-Pro-Ile-Thr-Arg
			Interferon regulatory factor 2 (IRF-2)	15	Met-Pro-Val-Glu-Arg
Insulin-like growth factor-i	16	Gly-Pro-Glu-Thr-Leu-
			Melanoma growth-stimulating activity	17	Ala-Pro-Leu-Ala-Thr-
Migration inhibitory factor	18	Met-Pro-Met-Phe-Ile-
			Monocyte chemoattractant protein I	19	Glu-Pro-Asp-Ala-Ile-
Neuropeptide tyrosine	20	Tyr-Pro-Ser-Lys-Pro-
			Pancreatic polypeptide	21	Ala-Pro-Leu-Glu-Pro-
Peptide YY	22	Try-Pro-Ile-Lys-Pro-
			Prolactin antagonist	23	Leu-Pro-Ile-Cys-Pro-
RANTES	24	Ser-Pro-Tyr-Ser-Ser-
			The P material	25	Arg-Pro-Lys-Pro-Gln-
Thrombopoietin	26	Ser-Pro-Ala-Pro-Pro-
			1 type transforming protein (N-myc)	27	Met-Pro-Gly-Met-Ile-
2 type transforming proteins (N-myc)	28	Met-Pro-Ser-Cys-Ser-
			Tumor necrosis factor	29	Leu-Pro-Gly-Val-Leu-
VEGF	30	Ala-Pro-Met-Ala-Glu-

Table 2: have the human peptide and the protein of penultimate alanine, they are the substrates of inferring of DPP-IV.

ADA Adenosine deaminase
	Annexin
Mammary gland alkalescence conservative protein (breast basic conserved protein)
	Cofilin
Natural killer cell enhancer b
	The precursor of alpha-interferon
Interleukin-11, α and 1, the precursor of β and interleukin-11 3
	The precursor of macrophage inflammatory protein-2-α and 2-β
The precursor of melanophorin
	The precursor of oxytocin-neurophysin 1
Growth hormone releasing hormone
	Amyloid beta (1-28)
Anxiety peptide (anxiety peptide)
	The pro-opiomelanocortin connection peptides

The present invention can utilize through the DPP substrate of modifying, and comprises one or more additional aminoacid at the N-of substrate end, avoids the active effect of DPP with the protection substrate.Be used for being disclosed in table 3 according to the preferred substrate of modification of the present invention.

The cracked substrate of table 3:DPP-IV (CD26)

The DPP-IV substrate	SEQ ID NO：	Sequence
			GIP	31	YAEGTFISDY SIAMDKIHQQ DFVNWLLAQK GKKNDWKHNI TQ
GLP-1	32 (amino acid/11s-29)	HAEGTFTSDV SSYLEGQAAK EFIAWLVKG
			GLP-2	33	HADGSFSDEM NTILDNLAAR DFINWLIQTK ITD
Growth hormone releasing hormone	34	YADAIFTNSY RKVLGQLSAR KLLQDIMSRQ QGESNQERGA RARL

The DPP-IV substrate	SEQ ID NO：	Sequence
			Glucagon (slowly deactivation is different from GIP and GLP)	35	HSQGTFTSDY SKYLDSRRAQ DFVQWLMNT
Peptide histidine-methionine	36	HADGVFTSDF SKLLGQLSAK KYLESLM
			IGF-1	37	G PETLCGAELV DALQFVCGDR GFYFNKPTGY GSSSRRAPQT GIVDECCFRS CDLRRLEMYC APLKPAKSAR SVRAQRHTDM PKAQKEVHLK NASRGSAGNK TY
Kallidin I	38	RPPGFSPFR
			The P material	39	RPKPQQFFGL M
CLIP	40	RPVKVYPNGA EDESAEAFPL EF
			Neuropeptide tyrosine	41	YPSKPDNPGE DAPAEDMARY YSALRHYINL ITRQRY
Peptide YY (being activated) by DPP-IV	42	YPIKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY
			Prolactin antagonist	43	LPICPGGAA RCQVTLRDLF DRAVVLSHYI HNLSSEMFSE FDKRYTHGRG FITKAINSCH TSSLATPEDK EQAQQMNQKD FLSLIVSILR SWNEPLYHLV TEVRGMQEAP EAILSKAVEI EEQTKRLLEG MELIVSQVHP ETKENEIYPV WSGLPSLQMA DEESRLSAYY NLLHCLRRDS HKIDNYLKLL KCRIIHNNNC
Human chorionic gonadotropin (HCG)	44	(alpha subunit) APDVQDCPEC TLQEDPFFSQ PGAPILQCMG CCFSRAYPTP LRSKKTMLVQ KNVTSESTCC VAKSYNRVTV MGGFKVEDHT ACHCSTCYYH KS
			Human chorionic gonadotropin (HCG)	45	(β subunit) SKEPLRPRCR PINATLAVEK EGCPVCITVN TTICAGYCPT MTRVLQGVLP ALPQVVCNYR NVRFESIRLP GCPRGVNPVV SYAVALSCQC ALCRRSTTDC GGPKDHPLTC DDPRFQDSSS SKAPPPSLPS PSRLPKPSDT PILPQ
Intestinal presses down peptide (enterostatin)	46	APGPR
			Gastrin releasing peptide	47	VPLPAGGGTV LTKMYPRGNH WAVGHLM
IL-2	48	APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT
			IL-1b	49	APVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ DMEQQVVFSM SFVQGEESND KIPVALGLKE KNLYLSCVLK DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFES AQFPNWYIST SQAENMPVFL GGTKGGQDIT DFTMQFVSS
Endorphine-2	50	YPFF
			The tyr-melanostatin	51	YPLG

The DPP-IV substrate	SEQ ID NO：	Sequence
			Aprotinin (aprotinin)	52	RPDFCLEPPY TGPCKARIIR YFYNAKAGLC QTFVYGGCRA KRNNFKSAED CMRTCGGA
RANTES	53	SPYSSDTTPC CFAYIARPLP RAHIKEYFYT SGKCSNPAVV FVTRKNRQVC ANPEKKWVRE YINSLEMS
			Trypsinogen	54	NPLLILTFV AAALAAPFDD DDKIVIVGGYNC EENSVPYQVS LNSGYHFCGG SLINEQWVVS AGHCYKSRIQ VRLGEHNIEV LEGNEQFINA AKIIRHPQYD RKTLNNDIML IKLSSRAVIN ARVSTISLPT APPATGTKCL ISGWGNTASS GADYPDELQC LDAPVLSQAK CEASYPGKIT SNMFCVGFLE GGKDSCQGDS GGPVVCNGQL QGVVSWGDGC AQKNKPGVYT KVYNYVKWIK NTIAANS
α 1-microglobulin	55	G PVPTPPDNIQ VQENFNISRI YGKWYNLAIG STCPWLKKIM DRMTVSTLVL GEGATEAEIS MTSTRWRKGV CEETSGAYEK TDTDGKFLYH KSKWNITMES YVVHTNYDEY AIFLTKKFSR HHGPTITAKL YGRAPQLRET LLQDFRVVAQ GVGIPEDSIF TMADRGECVP GEQEPEPILI PRV
			Interferon inducible protein matter 10 (IP10)	56	VPLSRTVRCT CISISNQPVN PRSLEKLEII PASQFCPRVE IIATMKKKGE KRCLNPESKA IKNLLKAVSK ERSKRSP
Eotaxin	57	GPASVPTTCC FNLANRKIPL QRLESYRRIT SGKCPQKAVI FLTKLAKDIC ADPKKKYVQD SMKYLDQKSP TPKP
			Mononuclear cell chemical attractants albumen 1 (MCP-1)	58	QPDAINAPVT CCYNFTNRKI SVQRLASYRR ITSSKCPKEA VIFKTIVAKE ICADPKQKWV QDSMDHLDKQ TQTP
Mononuclear cell chemical attractants albumen 2 (MCP-2)	59	QPDSVSIPIT CCFNVINRKI PIQRLESYTR ITNIQCPKEA VIFKTKRGKE VCADPKERWV RDSMKHLDQI FQNLKP
			Mononuclear cell chemical attractants albumen 3 (MCP-3)	60	QPVGINTSTT CCYRFINKKI PKQRLESYRR TTSSHCPREA VIFKTKLDKE ICADPTQKWV QDFMKHLDKK TQTPKL
Granulocyte chemoattractant protein-2	61	GPV SAVLTELRCT CLRVTLRVNP KTIGKLQVFP AGPQCSKVEVV ASLKNGKQVC LDPEAPFLKK VIQKILDSGN KKN
			SDF-1a	62	KPVSLSYRCP CRFFESHVAR ANVKHLKILN TPNCALQIVA RLKNNNRQVC IDPKLKWIQE YLEKALNK
SDF-1b	63	KPVSLSYRCP CRFFESHVAR ANVKHLKILN TPNCALQIVA RLKNNNRQVC IDPKLKWIQE YLEKALNKRF KM
			Macrophage derived chemotactic factor	64	GPYGANMEDS VCCRDYVRYR LPLRVVKHFY WTSDSCPRPG VVLLTFRDKE ICADPRVPWV KMILNKLSQ
B-cheese deltorphin delta (casomorphin)	65	YPFVEPI
			Former colipase (procolipase)	66	APG PRGIIINLEN GELCMNSAQC KSNCCQHSSA LGLARCTSMA SENSECSVKT LYGIYYKCPC ERGLTCEGDK TIVGSITNTN FGICHDAGRS KQ

The DPP-IV substrate	SEQ ID NO：	Sequence
			Vasoactive intestinal peptide (VIP)	67	HSDAVFTDNYTRLRKQMAVKKYLNSILN
Hypophysis adenylyl cyclase activating peptide 38 (PACAP38)	68	HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK
			Secrete sour regulin (oxyntomodulin)	69	HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Growth hormone (1-43)	70	FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYS
			Secretin (secretin)	71	HSDGTFTSELSRLREGARLQRLLQGLV
The natriuretic peptide in brain source		SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH

The substrate that is used to modify comprises aminoterminal X-Pro-Y, X-Ala-Y, X-Ser-Y or X-Gly-Y.Preferably, the substrate that is used to modify is GLP-1.

The modified polypeptide of avoiding the DPP active function

The invention provides through modified polypeptides, for example the process modified polypeptides substrate of DPP comprises one or more additional aminoacid at the N-of substrate end, avoids the active effect of DPP to protect this peptide substrate.In one embodiment, compare, have an additional aminoacid at its N-end through modified polypeptides with wild type peptide.In another embodiment, have 5 additional aminoacid through modified polypeptides at its N-end.Alternately, has 1-5 additional aminoacid through modified polypeptides at its N-end.Any N-end that is added to peptide substrate of 20 seed amino acids perhaps adds non-natural aminoacid.

Can expect, avoid the cracked protection of DPP, after using, can all can from according to modification of the present invention, benefit in body circulation or the cleaved any pharmaceutical polypeptide in any position in vivo with peptide bond owing to the invention provides.

According to this aspect of the present invention, might remove at least about 30%, preferably at least about 50%, more preferably at least about 70% even more preferably at least about 90% with most preferably at least about 99% dipeptidyl peptidase enzymatic activity.Adopt method of the present invention, also may remove two peptidyl amino peptidase activity fully.

Equally, may reduce at least about 30%, preferably at least about 50%, more preferably at least about 70% even more preferably at least about 90% with most preferably at least about the dipeptidyl peptidase sensitivity of 99% substrate.Adopt method of the present invention, also may remove two peptidyl amino peptidase sensitivity fully.

Although process modified polypeptides of the present invention or peptide substrates are partially or substantially avoided the active effect of DPP, but through the modified polypeptides substrate still keep at least about 10%, preferably at least about 30%, more preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90% with most preferably at least about 99% functional activity and effect.In some cases, the process modified polypeptides or the peptide substrates of the decline of functional activity and effect are useful.For example, when merging, form that serum stability improves and during the fusion rotein of body-internal-circulation half-life prolongation, the process modified polypeptides or the peptide substrates of functional activity or effect decline are useful through modified polypeptides or peptide and other polypeptide such as transferrin.

Under other situation, with process modified polypeptides or peptide are not compared the effect rising of process modified polypeptides or peptide.

With not relatively by the homopolypeptide mutually of modified forms, of the present inventionly avoid the cracked effect of dipeptidyl peptidase basically through the modified polypeptides molecule.Relatively more different with the analysis of not passing through modified polypeptides to the evaluation meeting that the protective effect of this essence is carried out through modified polypeptides because of being used for.Yet,, in this is analyzed, the dipeptidyl peptidase enzymatic lysis is had detectable resistance level through modified polypeptides in order to show the protective effect of essence.This analysis includes, but are not limited to that (2002Endocrinology 142 people such as Doyle, 4462-4468), people such as O ' Harte (1999 Diabetes 48,758-765) and people such as Siegel (1999 Regulatory Peptide 79,93-102) in those disclosed method.

Compare with non-stabilized peptide substrate, more stable when DPP of the present invention stablizes peptide substrate and has DPP in vivo.The non-stabilized polypeptide identical with sequence compared, and the therapeutical peptide substrate that DPP is stable has the longer active half-life usually.By will not comparing, determine the stability of peptidase through the half-life of modified polypeptides substrate in serum or blood and the half-life of corresponding therapeutic peptide in serum or blood through modification.By collection serum or blood sample behind the peptide of the peptide of using the process modification and process modification, and the activity of definite this peptide, determine the half-life.Except determining activity, also measure the length of peptide substrate by HPLC or mass spectral analysis.

The present invention also provides through modified polypeptides or peptide, and it has according to reformed amino terminal of the present invention avoiding the cracked effect of DPP, and has functional activity or the inside of effect and/or the amino acid change of C-end that does not influence polypeptide.These have less amino acid change through modified polypeptides, though these amino acid changes also comprise nonconservative replacement, and the aminoacid replacement of Bao Chiing normally.

Process modified polypeptides of the present invention or peptide also have the functional activity of change.For example, the process modified polypeptides or the peptide of functional activity rising are useful.Alternately, can active process modified polypeptides or the peptide that descends of function of use.Therefore, process modified polypeptides of the present invention or peptide also contain the amino acid change that does not influence functional activity or effect.For example, the amino terminal of the GLP-1 analog that functional activity is changed is modified, and makes it avoid the cracked effect of DPP.

The example that conserved amino acid replaces is the replacement of carrying out on the same group mutually, for example at basic amino acid group (for example arginine, lysine, histidine), acidic amino acid group (for example glutamic acid and aspartic acid), polar amino acid group (for example glutamine and agedoite), hydrophobic amino acid group (for example leucine, isoleucine, valine), ArAA group (for example phenylalanine, tryptophan, tyrosine) and p1 amino acid group (for example glycine, alanine, serine, threonine, methionine).

Non-conservation replaces and comprises that the aminoacid in the group is by the aminoacid replacement of another group.For example, non-conservation replaces and to comprise with polar amino acid and replace hydrophobic amino acid.The generality that nucleotide replaces is described referring to people (1991) such as for example Ford, Prot.Exp.Pur.2:95-107.

The invention provides obvious variant through the aminoacid sequence of modified polypeptides and peptide, the for example variant of the recombinant sources of the non-natural of the allele variant/sequence variants of the polypeptide of naturally occurring mature form or peptide, polypeptide, peptide existence, and the directly congener in congener and kind of polypeptide or peptide.Use the known technology in nucleic acid recombinant technique and protein biochemistry field, can prepare this variant at an easy rate.Use molecular engineering and sequence information, can easily identify/prepare this variant.In addition, according to itself and of the present invention through modified polypeptides or the sequence of peptide and/or the homology of structure, be easy to this variant and other peptide are distinguished.

Preferably, the peptide through modification of the present invention is to comprise one or more additional amino acid whose GLP-1 and analog thereof at the N-end.

In some cases, DPP for example DPP-IV can activating peptide, rather than by this peptide of cracking deactivation.In this case, the modification to peptide can reduce, delays or stop the peptide activation basically.

Coding is through the nucleic acid of modified polypeptides

The invention provides the nucleic acid molecules of coding, these polypeptide and peptide moiety ground or avoid the cracked effect of DPP basically and have functional activity and effect through modified polypeptides and peptide.In one embodiment, nucleic acid molecule encoding provided by the present invention with not comparing through modified polypeptides of its wild type, has at least one additional aminoacid through modified polypeptides and peptide at its N-end through modified polypeptides and peptide.In another embodiment, nucleic acid molecule encoding has 5 additional amino acid whose process modified polypeptides and peptide at its N-end.Alternately, nucleic acid molecule encoding has 1-5 additional amino acid whose process modified polypeptides and peptide at its N-end.Preferably, the nucleic acid molecules of the GLP-1 that modifies of coding comprises and is coded in its N-end and has one or more additional amino acid whose sequences.

Nucleic acid molecules of the present invention comprises DNA (deoxyribonucleic acid) (DNAs), strand and DNA (deoxyribonucleic acid) two strands.Yet, can also be ribonucleic acid (RNAs), and the RNA:DNA duplex molecule of heterozygosis.The nucleic acid molecules that is comprised also comprises genomic DNA, cDNA, mRNA, and antisense molecule.Nucleic acid molecules of the present invention also comprises natural or synthetic RNA, DNA or the cDNA of coding through modified polypeptides, or its complementary strand.

In order to make up through modified polypeptides, it is partially or substantially avoided the active effect of DPP and has functional activity and/or effect with not comparing through modified polypeptides of wild type, the nucleic acid of the not process modified polypeptides of encoding wild type can be used as starting point, and is modified the process modified polypeptides of the expectation of encoding.Known have many methods to be used to add sequence or the nucleic acid encoding sequence that is used to suddenly change, and determine by these functions through the sequence encoded polypeptide of modification.

The present invention also provides the nucleic acid of coded polypeptide and peptide, and this polypeptide has the amino terminal of the process modification of avoiding the DPP splitting action and do not influence the functional activity of polypeptide basically or inside and the amino acid change C-end of effect.These have less amino acid change through modified polypeptides, though these amino acid changes also can be non-conservative replacements, and their normally conserved amino acid replacements.The nucleotide that is used to realize the technology of site-specific mutagenesis is substituted in this area and knows.Preferably, nucleic acid coding has one or more additional amino acid whose GLP-1 analog at the N-end.

As known in the art, article two, " similarity " between polynucleotide or the polypeptide is by the nucleotide of polynucleotide or polypeptide or the sequence of aminoacid sequence and conservative nucleotide or aminoacid replacement thing and second polynucleotide or polypeptide are relatively come to determine.As known in the art, " homogeneity " is meant the sequence degree of correlation between two polypeptide or two polynucleotide sequences, and the matching degree that is tested and appraised between two chains of this sequence is determined.Homogeneity and similarity all are easy to calculating, and (A.M. edits for ComputationalMolecular Biology, Lesk, Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D.W. edits, Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A.M. and Griffin, H.G. edits, Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J. edits, M StocktonPress, New York, 1991).

In the method that has homogeneity between two polynucleotide of big quantitative determination or the peptide sequence and similarity simultaneously, term " homogeneity " and " similarity " also are known (the Sequence Analysisin Molecular Biology of technical staff, von Heinje, G., Academic Press, 1987; Sequence AnalysisPrimer, Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991; And Carillo, H., and Lipman, D., SIAM J.Applied Math., 48:1073 (1988).Being generally used for measuring the homogeneity between the two sequences or the method for similarity comprises, but be not limited to Computers at Guide to Huge, Martin J.Bishop edits, Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., those disclosed method among the SIAM J.Applied Math.48:1073 (1988).

The method for optimizing of homogeneity is determined in design, to produce maximum match between the two sequences that is detected.The method of determining homogeneity and similarity is written as computer program.Determine that the homogeneity between the two sequences and the preferred computer program technic of similarity comprise, but be not limited to GCG program package (people such as Devereux, 387 (1984)), BLASTP, BLASTN, FASTA (people such as Atschul, J.Molec.Biol.215:403 (1990)) Nucleic acid Research 12 (1):.Above the size of mentioned similarity or homogeneity be confirmed as same degree between the two sequences, expression article one sequence is derived from the second sequence.Article two, the same degree between the nucleotide sequence can be determined by the computer program that this area is known, for example the GAP that provides in GCG program software bag (Needleman and Wunsch (1970) Journal of Molecular Biology 48:443-453).In order to determine the same degree between two nucleotide sequences of the present invention, use GAP, have following setting: the GAP that 5.0 GAP produces (creation) point penalty and 0.3 prolongs (extension) point penalty.

Codon optimized

The feasible nucleotide sequence that can change polypeptide of the degeneracy of genetic code, and still generate the modified polypeptide that comprises the aminoacid sequence identical with the first DNA sequence encoded polypeptide.This operation is called " codon optimized " (be described in U.S. Pat 5,547, in 871, it is incorporated herein by reference in full at this), and the method for this reformed DNA sequence of design is provided.The design of codon optimized gene should be considered various factors, the original DNA operation that comprises probability, the synthesis path that the frequency of codon utilization in the organism, immediate neighbor frequency, rna stability, secondary structure form and be intended to this gene is carried out.Especially, when adopting yeast expression system, adopt obtainable method to come to change the codon of coding specific fusion proteins with those the easiest codons of being discerned by yeast.

The degeneracy of genetic code makes identical aminoacid sequence to encode and translate with multitude of different ways.For example, leucine, serine and arginine are respectively by six kinds of different codon codings, and valine, proline, threonine, alanine and glycine are respectively by four kinds of different codon codings.But in eukaryotic cell and prokaryotic cell, the frequency of utilization of this synonymous codon is different between genome.For example, the preference pattern of synonymous codon is very similar between mammal, and at evolutionary distance organism far away for example in yeast (for example saccharomyces cerevisiae), antibacterial (for example escherichia coli) and the insecticide (for example D.Melanogaster), then show visibly different genome password and utilize frequency mode (Grantham, R. wait the people, Nucl.Acid Res., 8,49-62 (1980); Grantham, people such as R., Nucl.Acid Res., 9,43-74 (1981); Maroyama, people such as T., Nucl.Acid Res., 14,151-197 (1986); Aota, S. etc., Nucl.Acid Res., 16,315-402 (1988); Wada, people such as K., Nucl.Acid Res., 19 Supp., 1981-1985 (1991); Kurland, C.G., FEBSLetters, 285,165-169 (1991)).As if these difference of codon selection mode by regulating the whole expression that peptide elongation ratio influences each gene.(Kurland, C.G., FEBS Letters, 285,165-169 (1991); Pedersen, S., EMBO J., 3,2895-2898 (1984); Sorensen, M.A., J.Mol.Biol., 207,365-377 (1989); Randall, people such as L.L., Eur.J.Biochem., 107,375-379 (1980); Curran, J.F. and Yarus, M., J.Mol.Biol., 209,65-77 (1989); Varenne, people such as S., J.Mol.Biol., 180,549-576 (1984), Varenne, people such as S., J.Mol, Biol., 180,549-576 (1984); Garel, J.-P., J.Theor.Biol., 43,211-225 (1974); Ikemura, T., J.Mol.Biol., 146,1-21 (1981); Ikemura, T., J.Mol.Biol., 151,389-409 (1981)).

The preferred codon of synthetic gene utilizes frequency to reflect and derives from the codon utilization of the genomic karyogene of (perhaps closely related as far as possible) cell/organism accurately, this cell/organism will be used to the cell/organism of Recombinant Protein Expression, particularly Saccharomyces.As discussed above, in a preferred embodiment, before or after modification described herein, codon optimized to be used for yeast expression through modified polypeptides process.

Carrier

Be used for ceneme of the present invention and comprise following element usually, with 5 ' be operably connected to 3 ' direction: transcripting promoter, secretory signal sequence, coding are through the DNA sequence of modified polypeptides, and transcription terminator.As discussed above, any arrangement of process modified polypeptides and peptide can be used in the carrier of the present invention.Selection to suitable promoter, signal sequence and terminator will be determined by selected host cell, be obvious for a person skilled in the art, will more specifically discuss hereinafter.

The suitable yeast carrier that is used for the present invention is described in U.S. Pat 6,291, in 212, comprise YRp7 (people such as Struhl, Proc.Natl.Acad.Sci.USA 76:1035-1039,1978), YEpl3 (people such as Broach, Gene 8:121-133,1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108,1978), pPPC0005, pSeCHSA, pScNHSA, pC4 and derivant thereof.Useful yeast plasmid carrier also comprises pRS403-406, pRS413-416 and derives from Stratagene CloningSystems, La Jolla, and CA 92037, the pichia of USA (Pichia) carrier.Plasmid pRS403, pRS404, pRS405 and pRS406 are yeast integrative plasmids (YIps) but and have included yeast selected marker HIS3, TUPI, LEU2 and URA3 in.Plasmid pRS413-41.6 is yeast centromeric plasmid (YCps).

This carrier generally includes selectable labelling, can be to have one of gene of dominant phenotype in a large number arbitrarily, thus, can carry out phenotype analytical and select transformant.But preferred selected marker is that those remedy the auxotrophic labelling of host cell, produce antibiotic resistance or make cell can utilize the sign of specific carbon source, comprise LEU2 (people such as Broach, ditto), URA3 (people such as Botstein, Gene8:17,1979), HIS3 people such as (, the same) Struhl or POT1 (Kawasaki and Bell, EP171,142).Other suitable selected marker comprises the CAT gene, and it gives the yeast cells chlorampenicol resistant.The preferred promoter that is used for yeast comprises promoter (people such as Hitzeman, JBiol.Chem.225:12073-12080,1980 from Yeast sugar zymolysis gene; Alber and Kawasaki, J.Mol.Appl.Genet.1:419-434,1982; Kawasaki, U.S. Pat 4,599,311) or the promoter (people such as Young of alcohol dehydrogenase gene, be stated from Genetic Engineering ofMicroorganisms for Chemicals, people such as Hollaender (editor), 355, Plenum, New York, 1982; Ammerer, Meth.Enzymol.101:192-201,1983).From this point, particularly preferred promoter is TPI1 promoter (Kawasaki, U.S. Pat 4,599,311) and ADH2-4^C(referring to U.S. Pat 6,291,212) promoter (people such as Russell, Nature 304:652-654,1983).Ceneme may also comprise transcription terminator.Preferred transcription terminator is TPI1 terminator (Alber and Kawasaki, the same).More preferably, promoter is a disclosed PRB1 promoter among the EP 431880, and terminator is a disclosed ADH1 terminator among the EP 60057, and these two parts of patents are incorporated herein by reference fully at this.

Except yeast, process modified polypeptides of the present invention and peptide can be expressed in filamentous fungi, for example, and in each strain of Eurotium.The example of useful promoter comprises that those derive from the promoter of aspergillus nidulans (Aspergillus nidulans) glycolysis gene, for example ADH3 promoter (people such as McKnight, EMBO J.4:2093-2099,1985) and tpiA promoter.The example of suitable terminator is ADH3 terminator people such as (, the same) McKnight.Utilize the ceneme of these elements to be cloned into to be inserted in the carrier in the chromosomal DNA of Eurotium for example.

Be used to realize that mammalian expression vector of the present invention will comprise the promoter that can guide process modified polypeptides and peptide to transcribe.Preferred promoter comprises viral promotors and cell promoter.Preferred viral promotors comprises major late promoter (Kaufman and Sharp, Mol.Cell.Biol.2:1304-13199,1982) from adenovirus 2 and SV40 promoter people such as (, Mol.Cell.Biol.1:854-864,1981) Subramani.Preferred cell promoter comprises that mice metallothionein-1 promoter people such as (, Science 222:809-814,1983) Palmiter and mice V κ are (referring to U.S. Pat 6,291,212) promoter (people such as Grant, Nuc.Acids Res.15:5496,1987).Particularly preferred promoter is mice VH (referring to U.S. Pat 6,291, a 212) promoter.These expression vectors can also contain a cover RNA shearing site of the DNA sequence upstream that is positioned at promoter downstream and coding process modified polypeptides or peptide.Preferred RNA shearing site can obtain from adenovirus and/or immunoglobulin gene.

In expression vector, also contain the polyadenylation signal that is positioned at interested coded sequence downstream.Polyadenylation signal comprise from the early stage of SV40 or late period polyadenylation signal (Kaufman and Sharp, ditto), polyadenylation signal (people such as DeNoto, Nucl.Acid Res.9:3719-3730,1981) fromadenovirus 5 E1B districts and human growth hormone gene terminator.Particularly preferred polyadenylation signal is VH (referring to U.S. Pat 6,291, a 212) gene terminator.Expression vector can comprise noncoding virus leader sequence, and the tripartite leader[of adenovirus 2 for example is between promoter and RNA shearing site.Preferred carrier can also comprise enhancer sequence, for example SV40 enhancer and mice μ (referring to U.S. Pat 6,291,212) enhancer (Gillies, Cell 33:717-728,1983).Expression vector can also comprise the sequence of encoding adenovirus VA RNA.

As mentioned below, expression vector also is used for expressed fusion protein, and this fusion rotein comprises and is fused to for example of the present invention through modified polypeptides or peptide on the transferrin of second peptide species or peptide, is used to prolong the half-life through modified polypeptides or peptide.In addition, will be fused to labelling (tag) and/or the cracking site that is used to express and discharge process modified polypeptides or peptide through modified polypeptides or peptide.

Transform

The technology that is used to transform fungus is known in this area, be described in for example people (Proc.Natl.Acad.Sci.USA 75:1929-1933 such as Beggs (ditto), Hinnen, 1978), people such as Yelton, (Proc.Natl.Acad.Sci.USA 81:1740-1747,1984) and among the Russell (Nature301:167-169,1983).The genotype of host cell has genetic defect usually, and this defective is by existing the selected marker on the expression vector to remedy.Select within specific host and the selectable ability that is marked at those of ordinary skills.

Can be by the transfection of the mediation of calcium phosphate for example, the DNA sequence that will comprise the clone through modified polypeptides and peptide of the present invention imports in the mammalian cell of being cultivated (people such as Wigler, Cell14:725,1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456,1973).Can also adopt cloned dna sequence is imported other technology in the mammalian cell, for example electroporation (people such as Neumann, EMBOJ.1:841-845,1982) or fat transfection.In order to identify the cell of the DNA that has integrated the clone, but usually selected marker and interested gene or cDNA are inserted in the cell.The marks packets of preferably can selecting that is used for cultivating mammalian cell is drawn together the gene of giving the resistance of medicine, and these medicines are neomycin, hygromycin and methotrexate for example.But selected marker can be the selected marker that can increase.Preferably the selected marker that can increase is the DHFR gene.Particularly preferred amplifiable marker is DHFRr (referring to U.S. Pat 6,291,212) cDNA (Simonsen and Levinson, Proc.Natl.Acad.Sci.USA 80:2495-2499,1983).(Stoneham comments in Mass.), and selects in the selectable those of ordinary skills' of being marked at the ability for MammalianCell Technology, Butterworth Publishers can to select to be marked at Thilly.

Host cell

The present invention also comprises the cell of being expressed process modified polypeptides of the present invention or peptide by being converted, and is preferably yeast cells.Except by transformed host cells itself, the present invention also comprises the culture of these cells in Nutrient medium, preferred monoclonal (clone's homogeneous) culture or derive from the culture of monoclonal culture.If polypeptide is secreted, then contain this polypeptide and cell in the culture fluid, if perhaps cell is filtered or centrifugally falls, then do not contain cell.

Being used to realize that host cell of the present invention comprises eukaryotic cell, is prokaryotic cell sometimes, can transform or transfection by enough foreign DNAs, and grows in culture fluid, for example the mammal of Pei Yanging, insecticide, fungus, plant and bacterial cell.The carrier that will comprise nucleotide sequence of the present invention imports in the host cell, makes this carrier be retained as the outer carrier of chromosome of a kind of chromosomal integration body or self replication.Because nucleotide sequence more may maintain in the cell with being stabilized, integration is considered to a kind of advantage usually.By homologous recombination or non-homogeneous reorganization with vector integration in host chromosome.

Gene and source thereof that being chosen in of host cell depended on coded polypeptide to a great extent.Host cell can be single celled microorganism, prokaryotic cell for example, perhaps non-single celled microorganism, for example eukaryotic cell.Prokaryotic cell or eukaryotic cell all are spendable.When being prokaryotic host cell, can use the cell such as escherichia coli or the bacillus subtilis that are used usually.

When using prokaryotic cell, use reproducible carrier in host cell as host cell.The preferred expression plasmid that uses, it provides initiation protein to synthesize required promoter, SD sequence (Shine-Dalgarno sequence) and start codon (for example ATG) in the carrier of upstream region of gene of the present invention, to impel expression of gene.The example of above-mentioned carrier comprises and derives from the colibacillary plasmid that is used usually such as pBR322, pBR325, pUC12, pUC13 etc.Yet applicable carrier is not limited to these examples and can also uses various known carriers.Can be used for using the commercial example that acquires carrier in the colibacillary expression system to comprise pGEX-4T (Amersham PharmaciaBiotech), pMAL-C2, pMAl-P2 (New England Biolabs), pET21/lacq (Invitrogen), pBAD/His (Invitrogen) etc.

The example of eukaryotic host cell comprises yeast cells etc.The example of the preferred craniate zooblast that uses comprises COS cell (from the cell of monkey) (1981 Cell, 23,175), dihydrofolate reductase deficient cells strain (1980 Proc.Natl.Acad.Sci. in Chinese hamster ovary cell and source thereof, USA., 77,4216) or the like, the example of the preferred yeast cells that uses comprises saccharomyces cerevisiae etc.Yet used cell is not limited to these examples.Preferably, yeast cells is used to express through modified polypeptides or peptide.

The fungal cell comprises yeast specie (pichia belongs to for Saccharomyces for example, Schizosaccharomyces), can be used as the host cell among the present invention.Comprise and be supposed to be used to realize the present invention, the example that comprises zymic fungus as the host who expresses process modified polypeptides of the present invention or peptide is that pichia yeast belongs to (kind that is classified as Hansenula (Hansenula) before comprising) in the present invention, Saccharomyces, Kluyveromyces, Eurotium, mycocandida, Candida, Torulopsis (Torulopsis), spore torulopsis (Torulaspora), Schizosaccharomyces, Citeromycesbaodingensis belongs to (Citeromyces), Pachysolen, zygosaccharomyces belongs to (Zygosaccharomyces), Debaryomyces (Debaryomyces), trichoderma (Trichoderma), cephalosporium, Humicola (Humicola), Mucor, Neurospora, Ye Shi Saccharomyces (Yarrowia), Metschunikowia, Rhodosporidium, basidiomycetes Leucosporidium (Leucosporidium), Botryoascus, lock Sporobolomyces (Sporidiobolus), intend endomyces (Endomycopsis) etc.The example of Saccharomycodes is saccharomyces cerevisiae, S.italicus and Lu Shi yeast (S.Rouxii).The example of Kluyveromyces is K..fragilis, K..lactis and K..marxianus.Suitable spore torulopsis kind is T.delbrueckii.The example of pichia Pseudomonas is P.angusta (being called H.polymorpha in the past), P.anomala (being called H.anomala in the past) and pichia pastoris (P.pastoris).

Be used to prepare the useful especially host cell through modified polypeptides or peptide of the present invention and be methanol auxotype (methanoltrophic) pichia pastoris (Pichia pastoris) people (1995) Protein Express.Purif 6:619-624 such as () Steinlein.Since the alcohol oxidase promoter of pichia pastoris separated and clone since, pichia pastoris be developed as the preparation foreign protein important host; Its conversion was reported in 1985 first.When lacking glucose, pichia pastoris can utilize methanol as carbon source.The alcohol oxidase (alcoholoxidase) that the pichia pastoris expression system can utilize methanol induction is promoter (AOX1), and this promoter control coding is used for the gene of the expression of alcohol oxidase, metabolic first step of alcohol oxidase catalysis methanol.This promoter is characterized, and is inserted in a series of pichia pastoris expression vectors.Because the protein that generates in pichia pastoris can correctly fold usually, and is secreted in the culture fluid, the fermentation of genetically engineered pichia pastoris provides a kind of and has substituted as the good of escherichia expression system.

Wine brewing yeast strain is another kind of preferred host.In a preferred embodiment, using yeast cells, perhaps more specifically is the Saccharomyces cerevisiae host cell that has genetic defect in the required gene of the glycosylation that connects of the agedoite at glycoprotein.Saccharomyces cerevisiae host cell with this defective prepares by the sudden change and the selection technology of routine, although many yeast strains that can obtain have been modified and prevented or reduce glycosylation or super mannose groupization.

In order to optimize the preparation of heterologous protein, preferred host strain has sudden change, and for example pep4 of saccharomyces cerevisiae sudden change (Jones, Genetics 85:23-33,1977) causes the active decline of proteolysis.Particularly advantageously use (people 1998Biochem.J.330 such as Copley the gene of coding aspartyl protease yeast aspartase 1 (YAP3) or coding yeast aspartase (yapsin) 2 gene (MKC7) or both, the host who has sudden change 1333-1340) makes the proteolytic activity that is oriented to alkaline residue be lowered or eliminate.The host strain that contains sudden change in other protease-encoding district is used to prepare a large amount of therapeutical peptide or peptides through modifying of the present invention especially.

The host cell that contains DNA construct of the present invention is suitably being grown in the grown cultures liquid.As used in this, term " suitable grown cultures liquid " is meant the culture fluid that contains cell growth desired nutritional.Cell growth desired nutritional comprises carbon source, nitrogenous source, essential amino acids, vitamin, mineral and somatomedin.Grown cultures liquid is generally used for screening the cell that contains DNA construct, by for example medicament selection or lack crucial nutrition, but malnutrition remedy by the selected marker on the DNA construct, perhaps use the DNA construct cotransfection.Yeast cells for example, is preferably grown in the culture fluid that chemical constituent is determined, contains non-amino acid whose nitrogenous source, inorganic salt, vitamin and essential amino acids additive in the culture fluid.The pH of culture fluid preferably maintains greater than 2 and less than 8 pH, is preferably pH 5.5-6.5.The method of keeping stable p H comprises the pH control that cushions and continue, and preferably realizes pH control by adding ammonia, ammonium hydroxide or sodium hydroxide.Preferred buffer reagent comprise citric acid, phosphoric acid, succinic acid and Bis-Tris (Sigma Chemical Co., St.Louis, Mo.).The yeast cells of the required gene of glycosylation that the disappearance agedoite connects is preferably grown in containing the culture fluid of permeating stablizer.Preferred permeating stablizer is the sorbitol that is added in the culture fluid, and its concentration is preferably 0.5M or 1.0M between 0.1M and 1.5M.

Generally in the commercially available culture fluid that contains serum or serum-free, grown by the mammalian cell cultivated.Selection is suitable for the culture fluid of specific cells system in those of ordinary skills' ability.Transfected mammalian cell growth a period of time is generally 1-2 days, begins to express interested DNA sequence.Carry out medicament selection then, but select to express the auxocyte of selected marker with stable manner.With the selected marker cells transfected that can increase, can progressively improve the cloned sequence that drug level selects copy number to increase for, thereby improve expression.

Baculovirus/insect cell expression system also can be used to prepare therapeutical peptide or the peptide through modifying of the present invention.BacPAK^TMBaculovirus expression expression system (BD Biosciences (Clontech)) is high level ground express recombinant protein in insect host cell.Target gene is inserted in the conversion carrier, this carrier with linearizing BacPAK6 viral DNA by cotransfection in insect host cell.The genomic key component of BacPAK6 DNA disappearance baculovirus.When DNA and carrier reorganization, this key element is resumed, and target gene is transferred in the genome of baculovirus.After the reorganization, the minority viral plaque is verified the reorganization phenotype by picking and purification.Then, the new isolating recombinant virus that increases, and be used for the infected insect cell culture and prepare a large amount of expectation protein.

Secretory signal sequence

Term " secretory signal sequence " or " signal sequence " or " secretion targeting sequencing " can exchange use mutually, are described in for example U.S. Pat 6,291,212 andU.S. Pat 5, in 547,871, these two parts of patents are incorporated herein by reference fully at this.Secretory signal sequence or signal sequence or secretion targeting sequencing coding secretion peptide.The secretion peptide is to guide mature polypeptide or protein to secrete the aminoacid sequence that comes out from cell.Usually, the secretion peptide is characterised in that to have the hydrophobic amino acid core, and typically but whether utterly () be present in new synthetic proteinic amino terminal.In secretion process, the secretion peptide is cleaved from the mature protein usually to get off.The secretion peptide may contain the processing site, makes through secretion during the path, and signal peptide cracking from the mature protein is got off.The processing site is coded in the signal peptide, perhaps for example is added in the signal peptide by in vitro mutagenesis.

The secretion peptide can be used to guide the secretion through modified polypeptides and peptide of the present invention.Can be used for secreting the bonded a kind of such secretion peptide of peptide with other is the 3rd domain of yeast barrier albumen (Barrier protein).Secretory signal sequence or signal sequence or secretion targeting sequencing are that the translation post-treatment step of a series of complexity is required, and these steps cause proteinic secretion.If there is complete signal sequence, entered the inner chamber of rough endoplasmic reticulum by expressed protein, be transported in the excretion vesicles by Golgi body then, be transported to the extracellular at last.Usually, signal sequence is closelyed follow in the start codon back, and coding is by excretory proteinic aminoterminal signal peptide.In most of the cases, the signal sequence specific protein enzymatic lysis that is known as signal peptidase falls.Preferred signal sequence improves by the processing of the expressed recombinant protein of virus, mammal or vilolitin carrier and effluxes efficient.Preferred signal sequence is mammal or people's a transferrin signal sequence.In some cases, natural substrate signal sequence can be used for expressing and secretes of the present invention through modified polypeptides or peptide.In order to ensure removing signal sequence effectively, in some cases, preferably between signal sequence and maturation protein, comprise short propetide (pro-peptide) sequence, wherein the C-end portion of propetide comprises the protease recognition site, for example yeast kex2p protease.Preferably, propeptide sequence is about 2-12 aminoacid, more preferably is about 4-8 aminoacid.The example of this propetide is Arg-Ser-Leu-Asp-Lys-Arg, Arg-Ser-Leu-Asp-Arg-Arg, Arg-Ser-Leu-Glu-Lys-Arg and Arg-Ser-Leu-Glu-Arg-Arg (being respectively SEQ ID NO:111-114).

Avoid the preparation of the process modified polypeptides substrate of DPP splitting action

Synthetic method, DNA recombinant technique or any method that other prepares peptide and fusion rotein by standard prepare process modified polypeptides of the present invention, and it partly or basically resists the DPP activity.

Solid-phase peptide synthetic method: Merrifield, J.Am.Chem.Soc, 888:2149,1963 are described in following list of references prevailingly; Barany and Merrifield, In the Peptides, E.Gross and J.Meinenhofer edit, Academic Press, New York, 3:285 (1980); S.B.H.Kent.Annu.Rev.Biochem., 57:957 (1988).Adopt the solid-phase peptide synthetic method, can by progressively with aminoacid addition to the peptide chain of covalently bound elongation gradually to the hard resin granule, the peptide that preparation has desired length and sequence.Automatically synthetic can being used in this method.

As mentioned above, also can use Protocols in Molecular Biology, use the nucleotide sequence of these polypeptide of coding, obtain of the present invention through modified polypeptides.These sequences can be RNA or DNA, and link to each other with control sequence and/or be inserted in the carrier.Then with the carrier transfection in host cell such as antibacterial.The preparation of this carrier and in the host, produce or molecular biology and the gene engineering expressed by routine realize.

In addition, can use easy synthetic DNA sequence, in commercially available expression system, prepare of the present invention through modified polypeptides by recombinant technique.

Obtain of the present inventionly through modified polypeptides by recombination method, this recombination method comprises: (a) cultivate host cell being of value under the condition of producing polypeptide; (b) reclaim this polypeptide.Use methods known in the art, in the nutrient medium that is fit to the production polypeptide, cultivate host cell.For example; by shake-flask culture, small-scale or large scale fermentation (comprising the fermentation of successive, batch-wise, fed-batch or solid phase) cultured cell in the fermentation tank of laboratory or industry, in suitable culture fluid and under permission expression of polypeptides and/or the isolating condition, carry out.The program that adopts this area to know, in comprising the suitable nutrient medium of carbon source and nitrogenous source and inorganic salt, cultivate (referring to for example, antibacterial and zymic list of references; Bennett, J.W. and LaSure, L. edits, More Gene Manipulations in Fungi, Academic Press, California, 1991).Suitable culture fluid can be from supplier's acquisition or according to the composition preparation of announcing (for example, U.S. typical case culture collection catalogue).If be secreted in the nutrient medium, can from culture fluid, directly reclaim polypeptide through modified polypeptides.If do not secreted through modified polypeptides, from product of cell lysis, reclaimed polypeptide.

As an example, comprise through the of the present invention of modified polypeptides or peptide fusion protein preparing by disclosed fermentation process in WO 0044772 that this patent is incorporated herein by reference fully at this through modified polypeptides or peptide.

The method of knowing with this area through modified polypeptides detects, and this method is specific to this polypeptide.These detection methods comprise the use of specific antibody, and the formation of enzyme product or the disappearance of zymolyte are in conjunction with special receptor, perhaps by detect the activation of special receptor in cell analysis.For example, determine the activity of process modified polypeptides with the enzyme analysis.By conventional program, include, but are not limited to centrifugal, filtration, extraction, spray drying, evaporation or precipitation, from nutrient medium, reclaim the process modified polypeptides that is generated.

Polypeptide of the present invention comes purification by the method that multiple this area is known, (for example include, but are not limited to chromatography, ion-exchange chromatography, affinity chromatograph, hydrophobic chromatography, chromatofocusing, size exclusion chromatography), (for example, preparation isoelectronic focusing, differential dissolubility are (for example for electrophoresis method, ammonium sulfate precipitation), SDS-PAGE or extraction is (referring to for example, Protein Purification, J.C.Janson and Lars Ryden edit, VCH Publishers, New York, 1989).

Fusion rotein and protein conjugate

The invention provides through modified polypeptides or peptide, it is by recombination method or covalently bound being attached on the heterologous molecule.Be connected on heterologous molecule such as the plasma protein, can make the activity of process modified polypeptides or peptide prolong a couple of days to several weeks.In some cases, only need to use once this therapeutical peptide or peptide in this period through modifying.Because reactive compound will be mainly in conjunction with macromole, and the be ingested probability of other physiological process of cell internal interference of this macromole is less, thereby can obtain bigger specificity.

In another embodiment, process modified polypeptides of the present invention or peptide are attached on the heterologous sequence by recombination form, form chimeric protein or fusion rotein.This chimeric protein or fusion rotein comprise process modified polypeptides or the peptide that is operably connected on the heterologous protein, through modified polypeptides or peptide moiety ground or avoid the cracked effect of DPP basically, and described heterologous protein has basically not and pass through modified polypeptides or the homologous aminoacid sequence of peptide." be operably connected " and be meant that process modified polypeptides or peptide and heterologous protein are at the frame endomixis.Heterologous protein is fused to N-end or the C-end through modified polypeptides or peptide.

In one embodiment, fusion rotein does not influence the activity through modified polypeptides itself of the present invention.For example, fusion rotein includes, but not limited to the fusion rotein of enzymatic, for example beta galactosidase fusions, the two heterozygosis GAL fusions of yeast, poly His fusions, MYC-labelling fusions, HI-labelling fusions and Ig fusions.This fusion rotein, particularly poly-His fusions, the purification of the process modified polypeptides of being convenient to recombinate.In another example, fusion rotein comprises of the present invention by the aminoacid sequence between modified peptides and the other parts (moiety), described aminoacid sequence provides recognition sequence, makes to discharge the peptide through modifying of the present invention behind chemistry or enzymatic lysis.In some host cell (for example, mammalian host cell), proteic expression and/or secretion can increase by using the allos signal sequence.In another embodiment, will be fused on the molecule that can prolong its serum stability or serum half-life through modified polypeptides or peptide, for example on the plasma protein.Preferably, will be fused on serum albumin, immunoglobulin or its part such as the Fc domain through modified polypeptides or albumen.More preferably, will be fused on transferrin, lactotransferrin, melanocyte transferrin or its heterocomplex through modified polypeptides or peptide.U.S. Patent application 10/231,494 and US10/378,094 and International Patent Application PCT/US03/26818 in the method for preparing this fusion rotein is provided, these patent applications are incorporated herein by reference fully at this.

Such as in these patent applications discussion, the transferrin that can modify and be connected through modified polypeptides or peptide.It can present degree of glycosylation and reduce.Optional through the transferrin polypeptide of modifying: single transferrin N domain, single transferrin C-structure territory, transferrin N and C-structure territory, two kinds of transferrin N domains and two kinds of transferrin C-structure territories from following one or more.

When the C-structure territory of Tf during as fusion rotein a part of, amino acid residue (the SEQ ID NO:3 of PCT/US03/26818 corresponding to N413 and N611, it is incorporated herein by reference fully at this) the glycosylation site that connects of two N-suddenlyd change, in Yeast system, to express, avoid taking place glycosylation or super mannose groupization, and the serum half-life that prolongs fusion rotein and/or human cytokines (prepares sialyl (asialo) Tf, or under some situation, single sialyl (monosialo)-Tf or two sialyl (disialo)-Tf).Except Tf aminoacid, can also the contiguous residue in the N-X-S/T glycosylation site be suddenlyd change, to prevent or to reduce glycosylation basically corresponding to N413 and N611.U.S. Pat 5,986,067 referring to people such as Funk.The glycosylation that the N domain that is reported that the Tf that expresses in pichia pastoris is connected by single hexose O-at the S32 place, site S32 also can be suddenlyd change or modify prevents this glycosylation.

Therefore, in one embodiment of the invention, transferrin fusion proteins comprises that through the transferrin molecule of modifying, wherein the degree of glycosylation of transferrin reduces, and includes, but are not limited to the Tf of sialyl, single sialyl and two sialyl forms.In another embodiment, the transferrin of transferrin fusion proteins partly comprises the transferrin mutant of reorganization, and it is suddenlyd change to prevent glycosylation.In another embodiment, the transferrin of transferrin fusion proteins partly comprises by complete glycosylated reorganization transferrin mutant.In another embodiment, the transferrin of transferrin fusion proteins partly comprises the human serum transferrin mutant of reorganization, it is suddenlyd change to prevent glycosylation, wherein one of Asn413 and Asn611 (the SEQ ID NO:3 of PCT/US03/26818, it is incorporated herein by reference fully at this) can not be carried out glycosylated aminoacid by being sported at least.In another embodiment, the transferrin of transferrin fusion proteins partly comprises the human serum transferrin mutant of reorganization, it is suddenlyd change prevents or reduces glycosylation basically, wherein can the contiguous residue in the N-X-S/T glycosylation site be suddenlyd change.And, reduce or prevent glycosylation by sudden change serine or threonine residues.In addition, knownly X is become proline can suppress glycosylation.

Can prepare chimeric protein or fusion rotein by the DNA recombinant technique of standard.For example, the dna fragmentation of coding different proteins links together in frame according to conventional method.In another embodiment, fusion gene is synthetic by routine techniques, comprises automatic dna synthesizer.Alternately, carry out the pcr amplification of genetic fragment with anchor primer, the feasible complementary jag that generates between two successive genetic fragments of this anchor primer, then annealing and amplification again generate chimeric gene order (referring to Ausubel 1992 Current Protocols in Molecular Biology).And many expression vectors are commercially available, the encoded a kind of fusion part of these carriers (fusion moiety) (for example, GST albumen).With coding through the nucleic acid clone of modified polypeptides or peptide in this expression vector, make that merging part is connected to through on modified polypeptides or the peptide in the mode in the frame.

In another embodiment, be coupled on the heterologous molecule by covalent bond, improve its stability and avoid the active effect of DPP through therapeutical peptide or the peptide of modifying.

As an example, through modified polypeptides or peptide by being coupled on the blood constituent the not essential linking group of described blood constituent through the covalent bond that forms between the reactive group of the peptide modified and the blood constituent.Blood constituent can be fixed or movably.The example of fixed blood constituent is immovable blood constituent, comprise tissue, membrane receptor, a matter albumen (interstitial protein), fibrin, collagen protein, platelet, endotheliocyte, epithelial cell and relevant film and membrane receptor, somatic cell, Skeletal Muscle Cell and smooth muscle cell, neuron composition, osteocyte and osteoclast thereof, and institute's organism tissue, particularly those tissues relevant with blood circulation and lymphsystem.Movably the example of blood constituent is the blood constituent that does not have the fixed position in the time period of any prolongation, described time expand section be no more than 5 minutes usually, more generally be no more than 1 minute.These blood constituents are not that film is relevant, and have the time that prolongs in blood, and exist with the Cmin of at least 0.1 μ g/ml.Movably blood constituent comprises serum albumin, transferrin, immunoglobulin such as IgM and IgG, α₁Protease inhibitor, Antithrombin III and α₂-antiplasmin.Movably the half-life of blood constituent is usually at least about 12 hours.

In blood constituent and therapeutical peptide or (invivo) or (ex vivo) formation of exsomatizing in vivo of the covalent bond between the peptide through modifying.For exsomatize forming covalent bond, will add blood, serum to through modified polypeptides or peptide or contain in the normal saline solution just like the blood constituent of human serum albumin or IgG, between through modified polypeptides or peptide and blood constituent, to form covalent bond.In addition,, and in normal saline solution, react with maleimide or similar reactive chemistry base group modification through modified polypeptides or peptide with blood constituent.In case therapeutical peptide or peptide and blood constituent reaction through modifying when forming through modified polypeptides or peptide conjugate, are administered to the patient with this conjugate.Alternately, can directly be administered to the patient through therapeutical peptide or the peptide of modifying, therapeutical peptide that feasible formation process is in vivo modified or the covalent bond between peptide and the blood constituent.In addition, can with recombiant protein for example albumin carry out identical reaction.

The various positions that the therapeutical peptide that nonspecific process is modified or the chemical reaction group of peptide can react with it in vivo comprise cell, particularly erythrocyte (erythrocyte) and platelet, and protein, as immunoglobulin, comprise IgG and IgM, serum albumin, ferritin, steroid binding protein, transferrin, thyroxine-binding protein, α-2-macroglobulin etc.

Through modified polypeptides or peptide can contain or can be by chemical modification to contain the reactive group in conjunction with mercaptan (thiol).In one embodiment of the invention, through modified polypeptides or peptide can with polyethylene glycol conjugation.Alternately, through modified polypeptides or peptide can with polyethyleneglycol modified glycolipid or with polyethyleneglycol modified fatty acid coupling.

An aspect can be coupled to modified polypeptides or peptide on fatty acid or the derivative of fatty acid, improves its stability.The example of fatty acid includes, but are not limited to, lauric acid, Palmic acid, oleic acid and stearic acid.The example of derivative of fatty acid comprises ethyl ester, propyl ester, cholesterol ester, coenzyme A ester, nitrobenzophenone ester, naphthyl ester, monoglyceride, diglyceride and triglyceride, aliphatic alcohol, aliphatic alcohol acetas etc.

Another aspect can be prepared into the affine complex of medicine (drugaffinity complex) (DAC with modified polypeptides or peptide engineering^TM) on.The affine complex of medicine has three parts: the ingredient of being responsible for biologic activity; Ingredient is attached to connector on the reactive chemistry group; And the reactive chemistry group that is positioned at the connector other end, be responsible for this construction is permanently attached on the specific objective protein in the body.For example, people such as Kim (2003, Diabetes 52 (3): 751) disclose the affine complex of a kind of GLP-1-albumin medicine.People such as Kim prove that the link coupled DAC:GLP-1 of albumin is attached on the GLP-1 receptor (GLP-1R), and activation forms cAMP in the allos fibroblast of expressing this receptor.This result shows, the natural GLP-1 of the link coupled DAC:GLP-1 simulation of albumin.People such as Kim provide a kind of activatory new method of GLP-1R signal that is used to prolong.

Design modified polypeptides or the affine complex of peptide medicine to be using by subcutaneous injection, and albumin-binding fast and optionally in vivo.Formed biological conjugate has therapeutic activity identical with endogenous polypeptide or peptide and similar effect, and has in animal body and more approach albuminous pharmacokinetics pattern.

Pharmaceutical composition

The invention provides pharmaceutical composition, it comprises through therapeutical peptide and the peptide of modifying, and this polypeptide and peptide moiety ground or avoid the cracked effect of DPP basically still still keep its functional activity and effect.This pharmaceutical composition can be oral, parenteral is used, and uses as approach such as (IV), intra-arterial (IA), intramuscular (IM), subcutaneous (SC), intraperitoneal, percutaneous in the blood vessel.Can suitably use by perfusion under the situation.In some cases, can carry out the oral cavity, intranasal, rectum, percutaneous, by aerosol administration, and can be transferred to vascular system through modified polypeptides.For example, fusions that process modified polypeptides of the present invention and transferrin partly constitute or conjugate can be transferred to vascular system through modified polypeptides, perhaps by striding across blood brain barrier in conjunction with transferrin receptor, described in International Patent Application PCT/US03/26778, this application is incorporated herein by reference fully at this.Though can carry out multiple injection if desired, carry out a shot usually.Can use therapeutical peptide or the peptide that process is modified by any instrument easily, comprise syringe, the trocar, catheter etc.Specific method of application will change according to the amount that will use, no matter be single pill (a singlebolus) or continuous administration or the like mode.Preferably, can carry out using in the blood vessel, wherein the position of Dao Ruing is not crucial for purposes of the invention, preferably at the position that has quick blood flow, is peripheral vein or central vein when for example intravenous is used.More preferably, can the subcutaneous administration pharmaceutical composition.When using when combining with slow release method or protectiveness matrix phase, other approach also is useful.Purpose is to make through the therapeutic peptide of modifying or polypeptide as being distributed effectively in blood, so as can with blood constituent or component of organization reaction.

Usually, the present invention includes pharmaceutical composition, it comprises therapeutical peptide or peptide and pharmaceutically acceptable diluent, antiseptic, solubilizing agent, emulsifying agent, adjuvant and/or the carrier through modifying of the present invention of effective dose.This compositions comprises the diluent of have various buffer components (for example, Tris-HCl, acetate, phosphate), pH and ionic strength; Additive such as detergent (detergent) and solubilizing agent (for example, polysorbate80), antioxidant (for example, ascorbic acid, sodium metabisulfite), antiseptic (for example, thimerosal, benzyl alcohol) and dilatant (for example, lactose, mannitol); Described material is brought in the preparation of granules thing of polymerizable compound such as PLA, poly glycolic acid etc., perhaps brought in the liposome.Can also use hyaluronic acid, this has the prolongation effect of the persistent period in circulation.This compositions may influence the interior rate of release of physical state, stability, body and the interior removing speed of body of current protein and derivant.Referring to for example, Remington ' sPharmaceutical Sciences, the 18th edition (1990, Mack Publishing Co., Easton, Pa.18042) 1435-1712 page or leaf is hereby incorporated by.

For example, can on the physiology, use therapeutical peptide or peptide in the acceptable solution through modifying, described solution for example, deionized water, phosphate-buffered saline (PBS), normal saline, aquiferous ethanol or other alcoholic solution, blood plasma, protein solution, mannitol, dextrose hydrate, alcohol, plant wet goods.Other additive comprises buffer agent, salt, the last acceptable stabilizing agent of physiology or the like, and wherein said solution is buffered in the pH scope of about 5-10 usually, and the concentration range of buffer agent is usually at about 50-250mM, and the concentration range of salt is usually at about 5-500mM.The example of physiological buffer especially for what inject, comprises Hank ' s liquid and Ringer ' s liquid.Percutaneous preparation can contain penetrating agent, as bile salts or fusidate (fusidates).

Pharmaceutical composition can be prepared into tablet or lozenge, Sublingual tablet (sublingual tablets), sachet (sachets), paquets, Perle, suppository, Emulsion (cream), ointment (ointment), skin gel agent, transcutaneous device, aerosol, drinkable and injectable injection.Compositions can also be prepared into liquid form, perhaps is prepared into exsiccant powder, for example is convenient to the lyophilized form that stores and transport.Also comprise implantable slow releasing preparation.

Oral dosage form

In one embodiment, the invention provides the pharmaceutical composition of oral administration solid dosage form, it comprises therapeutical peptide or peptide through modifying, described dosage form is at Remington ' sPharmaceutical Sciences (1990), the 18th edition, among the Mack Publishing Co.Easton Pa.18042 general description is arranged, it is hereby incorporated by.The solid dosage form comprises tablet, capsule, pill, lozenge (troche) or rhombus sheet (lozenge), flat colloid (cachet) or piller (pellet).In addition, packing liposome or albuminoid can be used for preparing compositions of the present invention (for example U.S. Pat 4,925, the proteinlike granule of report in 673).Can use liposome packing, this liposome can be with various polymer-derivedization (for example U.S. Pat 5,013,556).Being described in by G.S.Banker and C.T.Rhodes of possible solid dosage form for treatment usefulness edited, Marshall, and K. provides in the 10th chapter of Modern Pharmaceutics (1979), and it is hereby incorporated by.In a word, described preparation will comprise through therapeutical peptide or peptide and the inert fraction of modifying, and inert fraction makes the effect of avoiding gastric environment, and in intestinal the delivery of biologically active material.

If desired, to carrying out chemical modification, make that it is effective carrying out that the oral cavity presents through therapeutical peptide or the peptide of modifying.Usually, included chemical modification be with at least one part (one moiety) be attached to through the therapeutical peptide modification or peptide originally on one's body, wherein said part is feasible can be absorbed the blood flow from stomach or intestinal.Also expectation is the stability that comprehensively improves chemical compound, and is increased in the circulation time of body.The part that can be used as the covalent attachment carrier in the present invention also can be used for this purpose.The example of this part comprises: the copolymer of PEG, ethylene glycol and propylene glycol, carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone and polyproline.Referring to, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts edit, Wiley-Interscience, New York, N.Y., 367-83; People such as Newmark (1982), J.Appl.Biochem.4:185-9.Spendable other polymer is for poly--1,3-dioxolanes and poly--1,3,6-three butyl oxide links (tioxocane).As noted above, be used for medicinal usage and be preferably peg moiety.

Equally, can be fused on the another kind of polypeptide, improve its general stability or improve its oral cavity and present through the therapeutical peptide or the peptide reorganization of modifying.For example, will be fused on transferrin, melanocyte transferrin or the lactotransferrin through therapeutical peptide or the peptide of modifying.The method that is used to prepare fusion rotein is at U.S. Patent application US10/378, describes in 094, and this application is incorporated herein by reference fully at this.

For the dosage form that present in the oral cavity, also may use salt through the aliphatic amino acid of modifying, strengthen the absorption of therapeutic compound of the present invention as carrier as N-(8-[2-hydroxy benzoyl] amino) sodium caprylate (SNAC).Use the clinical efficacy of the heparin preparations of SNAC to be confirmed by the 2 phases clinical experiment that Emisphere Technologies implements.Referring to, U.S. Pat 5,792,451, " mouth cavity medicine is presented compositions and method " (Oral drug delivery composition andmethods), it is incorporated herein by reference fully at this.

Therapeutical peptide or peptide through modification of the present invention can be included in the preparation as the granule of the about 1mm of granular size or the ultrafine particle of bead form.The preparation of the material of using with capsule also can be the suppository or or even the tablet of powder type, mild compression.Therapeutic preparation can prepare by compressing.

Can also comprise coloring agent and flavoring agent.For example, therapeutical peptide or the peptide (as by liposome or microsphere packing) of preparation through modifying further is included in the edible product then, for example contains the chilled beverage of coloring agent and flavoring agent.

Can dilute pharmaceutical composition of the present invention or increase its volume with a kind of inert material.These diluent comprise carbohydrate, particularly the glucosan and the starch of mannitol, cc-lactose, Lactis Anhydrous, cellulose, sucrose, modification.Some inorganic salt also is used as implant, comprises calcium phosphate, magnesium carbonate and sodium chloride.Some commercially available diluent are Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

In the therapeutic preparation of solid dosage form, comprise disintegrating agent.Material as disintegrating agent includes, but are not limited to starch, comprises the commercial disintegrating agent based on starch, Explotab.Primojel, amberlite (Amberlite), sodium carboxymethyl cellulose, ultramylopectin, sodium alginate, gelatin, Pericarpium Citri junoris (orange peel), acid carboxymethyl cellulose, natural sponge (natural sponge) and bentonite (bentonite) can use.The disintegrating agent of another kind of form is insoluble cation exchange resin.The glue of powdered can be as disintegrating agent with as binding agent, pulverous glue that can comprise, for example agar, karaya (karaya) or tragacanth.Alginic acid and sodium salt thereof also can be used as disintegrating agent.

Useful binders forms hard tablet, and comprises the material from natural product, for example Radix Acaciae senegalis, tragacanth, starch and gelatin being fixed together through therapeutical peptide or the peptide of modifying.Other comprise methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinylpyrrolidone (PVP) and hydroxypropyl emthylcellulose (HPMC) can be used in the alcoholic solution, make therapeutic composition become graininess.

In drug combination preparation of the present invention, comprise the anti-friction liniment, to prevent the thickness that in the preparation processing process, becomes.Lubricant can be used as through therapeutical peptide of modifying or the sealing coat between peptide and the die wall, and these lubricants include, but are not limited to: stearic acid comprises its magnesium salt and calcium salt, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil and wax.Soluble lubricant also can use, sodium lauryl sulfate for example, lauryl magnesium sulfate, various molecular weight polyethylene glycol, Carbowax 4000 and 6000.

Add fluidizer and may improve therapeutical peptide or the mobile performance of peptide in process for preparation that process is modified, and help in compression process, to rearrange.Fluidizer comprises starch, Pulvis Talci, pyrogenic silica and hydration siallitization thing.

In order to help therapeutical peptide or the peptide through modification of the present invention to be dissolved in the water environment, add surfactant as wetting agent.Surfactant comprises anionic detergent, as sodium lauryl sulfate, cetyl sodium sulfosuccinate and sodium cetanesulfonate.Cationic detergent be can use, benzalkonium chloride (benzalkonium chloride) or benzethonium chloride (benzethonium chloride) comprised.Be included in that the possible nonionic detergent as surfactant is polidocanol (lauromacrogol) 400,many polyoxies 40 stearate, polyoxyethylene hydrogenatedOleum Ricini 10,50 and 60 in the preparation, glyceryl monostearate, polysorbate40,60,65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose.These surfactants are present in the preparation of albumen or derivant, and this albumen or derivant are used separately or constituted mixture in varing proportions.

Also comprise additive in the preparation, improve through the therapeutical peptide of modification and the picked-up of peptide.The additive that may have this specific character is for example fatty acid, oleic acid, linoleic acid plus linolenic acid.

The sustained release preparation is also expected.Can bring in the inert base through therapeutical peptide or the peptide modified of the present invention, for example in the natural gum, this substrate makes and can discharge by diffusion or leaching mechanism.Also slow matrix degradation can be brought in the preparation, described substrate is alginate, polysaccharide for example.The another kind of form of the sustained release of chemical compound of the present invention is by the method preparation based on Oros therapeuticsystem (Alza Corp.), be that medicine is encapsulated in the semipermeable membrane, this film makes water to enter, and owing to osmosis is released medicine from single aperture.Some sugar-coat also has the effect that postpones release.

Other bag also be can be used in the preparation by mode.These bags are comprised various sugar, and it can be used for bag by in the dish (coating pan).Also can give in the tablet of film bag quilt through therapeutical peptide or the peptide of modifying, the material of Shi Yonging is divided into two groups in this case.First group is the material of non-intestinal, comprises methylcellulose, ethyl cellulose, hydroxyethyl-cellulose, methyl hydroxyl-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methylcellulose, carboxy-methyl cellulose sodium, providone and Polyethylene Glycol.Constitute by the intestinal material second group phthalic acid ester normally.

Multiple mixtures of material also can be used for providing the film bag quilt of the best.Can the flat board bag by or fluid bed or carried out film bag quilt by compressed package.

Form is presented by pulmonary

In another embodiment, the present invention is provided for also that pulmonary presents comprises through the therapeutical peptide modified or the pharmaceutical composition of peptide.When sucking, pharmaceutical composition is presented to mammiferous pulmonary, and enters blood flow through the lung epithelial internal layer.

The invention provides and be designed for the purposes that a large amount of machinerys of therapeutic products are presented by pulmonary, include, but are not limited to nebulizer, metered dose inhaler and powder inhalator, all be familiar with to those skilled in the art.Some the special example that is suitable for implementing commercially available device of the present invention is Mallinckrodt, Inc., St.Louis, the Ultravent nebulizer that Mo. makes; MarquestMedical Products, Englewood, the Acorn II nebulizer that Colo. makes; Glaxo Inc., Research Triangle Park, the Ventolin metered dose inhaler that N.C. makes; Fisons Corp., Bedford, the Spinhaler powder inhalator that Mass makes.

All these devices need use and be fit to disperse through the therapeutical peptide of modification and the preparation of peptide.Typically, various preparations are specific to the type of equipment therefor, and except the diluent, adjuvant and/or the carrier that are used for the treatment of, comprise and use suitable propelling material.

The most advantageously be prepared into particle form through therapeutical peptide or the peptide of modifying, be used for being presented to most effectively the deep (distal lung) of lung, average particle size particle size most preferably is 0.5-5 μ m less than 10 μ m.

Pharmaceutically acceptable carrier comprises carbohydrate for example trehalose, mannitol, xylitol, sucrose, lactose and Sorbitol.Other composition that is used for preparation comprises DPPC, DOPE, DSPC and DOPC.Can use natural or synthetic surfactant.Use PEG (even except it is used for derived protein or analog).Also can use glucosan, for example encircle glucosan.Can use the relevant reinforcing agent of bile salts with other.Can use cellulose and cellulose derivative.Also can use aminoacid, as be used for buffer preparation.

In addition, also comprise use liposome, microcapsule or microsphere, the carrier of inclusion complex (inclusion complex) or other type.

The preparation that is fit to use with jet-propelled nebulizer or soniclizer generally includes this creative chemical compound, and this chemical compound is dissolved in the water with the concentration of the about 0.1-25mg biological activity protein of every ml soln.Described preparation also comprises buffer agent and monosaccharide (for example, being used for stable protein and adjusting osmotic pressure).The nebulizer preparation also contains surfactant, reduces or prevents because the spatial induction polymerization of protein that the atomizing of the solution when forming aerosol causes.

The preparation that uses with metered dose inhaler comprises usually and contains the powder that grinds that is suspended in this creationary chemical compound in the propellant by surfactant.Propellant can be any conventional material that can be used for this purpose, for example Chlorofluorocarbons, contain hydrochlorofluorocarazeotropic, hydrogeneous fluorine carbon or hydrocarbon, comprise Arcton 11, dichlorofluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane, perhaps their combination.Suitable surfactant comprises Sorbitol trioleate and soybean lecithin.Oleic acid is useful as surfactants also.

Be used for comprising the dried powder that grinds that contains this creationary chemical compound from the dispersive preparation of powder inhalator device, and comprise the filler of being convenient to powder dispersion amount from this device, for example lactose, Sorbitol, sucrose, mannitol, trehalose or xylitol, its content for example accounts for the 50-90% of described weight of formulation.

Nose is presented form

Comprise that also nose presents the pharmaceutical composition through modified polypeptides or peptide of the present invention.Nose is presented and is made after will being administered to nose through the therapeutical peptide modified or peptide, this albumen directly can be transferred in the blood flow, and need be at this product of pulmonary deposition.Be used for the preparation that nose presents and comprise that those have the preparation of glucosan or ring glucosan.Also disclose by crossing over presenting of other mucosa transportation.

Dosage

The dosage relevant with the method for treatment above-mentioned condition can be by the doctor in charge, consider to change pharmaceutically-active various factors, for example, patient's age, situation, body weight, sex and diet, the order of severity of any infection, administration time and other clinical factor are determined.Usually, the daily dose scheme is preferably per kilogram of body weight 0.1-150 microgram in per kilogram of body weight 0.01-1000 microgram should the scope of creativeness chemical compound.

With therapeutic protein treatment disease through modifying

The invention provides various transferrin fusion proteins, it can be used for treating multiple disease.For example, the pharmaceutical composition that comprises fused polypeptide of the present invention or peptide is used for the treatment of disease, for example still be not limited to insulin resistance (insulin resistance), hyperglycemia (hyperglycemia), hyperinsulinemia (hyperinsulinemia), perhaps the blood levels of free fatty or glycerol raises, hyperlipemia (hyperlipidemia), obesity (obesity), the X syndrome, the Developmental and Metabolic Disorder syndrome, inflammation, diabetic complication, impaired glucose dynamic equilibrium, impaired glucose tolerance, type ii diabetes, prediabetes (prediabetes), hypertriglyceridemia arteriosclerosis (hypertriglyceridemiaatherosclerosis), nerve problems, congestive heart failure, dyspepsia (dyspepsia) and irritable bowel trace integration disease (irritable bowel syndrome).Process modified polypeptides and peptide also can be used for inducing the angst resistance effect to CNS, are used to activate CNS or are used for aftertreatment.

Owing to of the present inventionly be fused on transferrin or the modified transferrin through the therapeutical peptide modified and peptide; perhaps partially or substantially be protected and be not subjected to the active effect of DPP, so this therapeutical peptide and peptide be not in vivo than more stable through therapeutical peptide and the peptide modified.Therefore, use a small amount of molecule and can realize effective treatment.In some cases, can reduce side effect than low dosage.

In one embodiment, therapeutical peptide and the peptide through modification of the present invention can be used as a kind of tranquillizer.Therefore, the invention provides a kind of use process of the present invention modified polypeptides or peptide and make mammalian subject quelling method, this patient has cause the central or peripheral nervous system activity to increase unusual.This method comprises to the patient uses therapeutical peptide or the peptide through modifying that is enough to this patient is produced calm or angst resistance effect amount.Can Intraventricular (intracerebroventriculary), oral cavity, subcutaneous, intramuscular or intravenous use therapeutical peptide or peptide through modifying.Such method can be used for treatment or improves nervous system situation, for example anxiety, movement disorder, attack (aggression), mental disorder, epilepsy (seizure), panic attack (panic attack), hysteria and dyssomnias (sleepdisorder).

And, the present invention includes the method for the activeness that strengthens mammalian subject, comprise to the patient and use therapeutical peptide or the peptide that is enough to the patient is produced the amount of activation through modifying.Described patient suffers from the situation of the activeness decline that can cause central or peripheral nervous system.Can be used for treatment or improve the attention deficiency syndrome, memory loss (memoryloss) of depression (depression), schizoaffective disorder, sleep apnea (sleep apnea), wholwe-hearted degree difference, forgetful and Gelineau's syndrome (narcolepsy) or the like being useful situation through the therapeutical peptide modified or peptide to wake the central nervous system up.

Equally, the insulin resistance of the operation of particular type, selectivity abdominal postoperative had the greatest impact to postoperative in first day, continued at least 5 days, and may need just to recover in 3 weeks normal.Therefore, the needs of patients of postoperative is used insulinotropic peptides a period of time through modifying of the present invention behind surgical injury.Therefore, therapeutical peptide or the peptide through modification of the present invention can be used for aftertreatment.Needs of patients was used the about 1-16 of insulinotropic peptides hour through modification of the present invention before operation, the patient is being implemented use in the operation process and use behind corrective surgery to be no more than one period of 5 days.

And, therapeutical peptide and the peptide through modifying of the present invention, for example insulinotropic peptides is used for the treatment of insulin resistance, is independent of it and is used for post-operative treatment.Insulin resistance may be because insulin combines minimizing with cell surface receptor, perhaps changes owing to endocellular metabolism.First type that is characterised in that insulin sensitivity decline can overcome by improving insulin concentration usually.Second type that is characterised in that the insulin response reduction can not overcome by a large amount of administration of insulin.Insulin resistance after the wound (trauma) can overcome by the amount of insulin that gives to be directly proportional with the insulin resistance degree, and it obviously is to reduce institute by insulin sensitivity to cause.

Preferably, the insulinotropic peptides that the invention provides through modifying makes hyperglycemia normalization, and this realizes by glucose dependence, mechanism insulin dependency and that do not rely on insulin.Equally, be used as the main preparation, particularly type ii diabetes of treatment diabetes through the insulinotropic peptides of modifying.The present invention is particularly suitable for treating the patient who suffers from diabetes, comprise type i diabetes and type ii diabetes, because the effect of described peptide depends on the concentration of glucose of blood, therefore, when using current Therapeutic Method, the risk of hypoglycemic side effect is greatly reduced.

Effectively make the dosage that passes through the insulinotropic peptides of modifying of patient's blood sugar level normalization will depend on multiple factor; comprising; but be not limited to; patient's sex, body weight and age, the order of severity that can not blood sugar regulation, potential cause that can not blood sugar regulation; no matter be that glucose or other carbohydrate source are used simultaneously; path of using and bioavailability, persistence in vivo, preparation and effect.

Preferably, therapeutic peptide such as the insulinotropic peptides through modification of the present invention is used for the treatment of the impaired tolerance of (impaired) glucose, glycosuria, hyperlipemia, metabolic acidosis, diabetes, diabetic neuropathy and nephropathy.More preferably, GLP-1 and the analog thereof that the peptide of modifying is the process modification that pass through that is used for the treatment of type ii diabetes.

Monitoring is through the therapeutical peptide of modification and the existence of peptide

Use is used for the active analytical method of measurement function, HPLC-MS or at the antibody of described polypeptide or peptide, can monitors therapeutical peptide and peptide through modifying.For example, in the blood of monitoring mammalian hosts through the active of the therapeutical peptide modified or peptide and/or whether through the existence of the therapeutical peptide modified or peptide.Gather the part or the sample of host's blood in different time points, can determine whether to be attached on the persistent blood constituent with the amount of enough generation therapeutic activities through the therapeutical peptide modified or peptide, determine subsequently in the blood through the therapeutical peptide modified or the level of peptide.If desired, also determine therapeutical peptide or peptide, as being attached on which kind of blood constituent through the insulinotropic peptides of modifying through modifying.

As an example, used and analyzed the insulinotropic peptides that the short active method of islets of langerhans can be monitored process modification of the present invention.The insulinotropic peptides of modifying that passes through of the present invention has short islets of langerhans activity, and its activity is identical with the short islets of langerhans activity of not passing through the insulinotropic peptides of modifying at least.To offer zooblast through the peptide of modifying, and perhaps this peptide be injected to animal, and the insulin of monitoring immune response activity respectively is discharged in the blood circulation of solution or animal, determine short islets of langerhans characteristic through the insulinotropic peptides of modifying.But use the radioimmunoassay of specific detection insulin, detect the existence of the insulin of immune response activity.Can detect any radioimmunoassay that IRI exists though can adopt, preferably use Albano, the improved form of people's such as J.D.M. (1972 Acta Endocrinol.70:487-509) analytical method, it is hereby incorporated by fully.

Determine through the therapeutical peptide of modification or the short islets of langerhans characteristic (Penhos, J.C wait people 1969 Diabetes 18:733-738, and it is hereby incorporated by) of peptide by the pancreas perfusion.Wherein implement, improve and analyze dabbling mode and preferably adopt Weir, people's such as G.C (J.Clin.Investigat.54:1403-1412 (1974)) method is hereby incorporated by.

Analyze through the therapeutical peptide of modification and the existence of peptide with the HPLC that combines mass spectral analysis (MS), this is well-known to those skilled in the art.Typically, use two mobile phases, as 0.1%TFA/ water and 0.1%TFA/ acetonitrile.Column temperature and gradient condition can change.

Be used to monitor use and be specific to through the therapeutical peptide modified and the antibody of peptide through the another kind of method of the existence of the therapeutical peptide modified and peptide.Use has specific monoclonal antibody or polyclonal antibody to specific therapeutical peptide or peptide through modifying, helps to solve any such problem.Can from the host, generate or the antibody of deriving, therapeutical peptide or peptide immunity that described host modifies with specific process, or, perhaps use synthetic immunogen immune corresponding to the antigenic determinant of described therapeutical peptide or peptide with the immunogenic fragments immunity of this therapeutical peptide or peptide.Preferred antibody is to having high specific and affinity through therapeutical peptide or the peptide of modifying.Such antibody can also carry out labelling with enzyme, fluorescent dye or radioactive label.

Can through therapeutical peptide and the peptide of modification with whether existing in the antibody detection blood flow.Can be by SDS-PAGE and western engram analysis blood and/or serum sample.This technology makes can analyzing blood or serum, determines whether be attached on the blood constituent through therapeutical peptide or the peptide modified.

Glucagon-like-peptide-1 (GLP-1)

Can generate stability and the enhanced recruit of biologic activity with the DNA recombinant technique.These molecules are to be fused to the biological activity protein on the stabilize proteins and the combination of peptide, and this stabilize proteins has natural the long half-lift, for example immunoglobulin Fc part, albumin and transferrin.The biologic activity of these fusion molecule retentive activity parts has than natural not Fused albumen or the longer pharmacokinetics of peptide homologue.Pharmacokinetics strengthens also can improve biologic activity, reduces the side effect of not expecting, and more conveniently provides to the patient.The example that many this fusion rotein are arranged is as interferon-albumin, interferon-Fc, BNP-albumin, GLP-1-albumin, GLP-1-transferrin.

Though fusion rotein is stable to degraded and degraded is had resistance that possible the protease mechanism of degrading activity part can cause this molecule by slow deactivation.Especially, many peptides such as GLP-1, dynorphin (dynorphin) (Berman YL, Juliano L, Devi LA J Biol Chem.1995 October 6; 270:23845-50), enkephalin (Gu ZF, Menozzi D, Okamoto A, Maton PN, Bunnett NW Exp Physiol.1993 January; 78:35-48), BNP, ANP, angiotensin, Kallidin I and PYY are very responsive to protease such as DPP IV, neutral endopeptidase.These protease cause the peptide quick inactivating in the circulation either alone or in combination.Peptide and large protein such as albumin, Fc and transferrin merge the remarkable resistance of giving protease.Yet this can not eliminate the deactivation that is caused by protease fully.Therefore, the combination of fusion rotein and protease inhibitor has PK and the PD higher than fusion rotein itself.

The invention provides transferrin fusion proteins, it comprises the therapeutic peptide that protease is had resistance.Preferably, of the present invention is that it partly or basically is protected and avoids the DPP active function through the insulinotropic peptides of modification through the therapeutic peptide of modifying.More preferably, be GLP-1 peptide and analog and the fragment that process is modified through the insulinotropic peptides of modifying.GLP-1 peptide that process is modified and analog thereof and fragment can be used for treating diabetes, particularly type ii diabetes.The N-end sequence of wild type GLP-1 is His-Ala-Glu; The N-end sequence that comprises the group that is selected from following composition through the GLP-1 polypeptide of modifying of the present invention: His-His-Ala-Glu (SEQ ID NO:115), Gly-His-Ala-Glu (SEQ ID NO:116), His-Gly-Glu, His-Ser-Glu, His-Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu (SEQ ID NO:82), His-His-Gly-Glu (SEQ IDNO:83), His-His-Ser-Glu (SEQ ID NO:84), Gly-His-Ala-Glu (SEQ ID NO:85), Gly-His-Gly-Glu (SEQ ID NO:86), Gly-His-Ser-Glu (SEQ ID NO:87), His-X-Ala-Glu, His-X-Gly-Glu and His-X-Ser-Glu, wherein X is any aminoacid.As described below, can carry out other and modify and reduce and prevent proteasome degradation, these molecules can be used in the method and composition of the present invention.In addition, any GLP-1 part or GLP-1 analog, derivant, or analogies can (as a kind of fusion rotein) be used for method and composition of the present invention.

With the N-end of aminoacid addition,, can prevent second amino acids of dipeptidyl peptidase enzymatic lysis GLP-1 owing to sterically hindered to GLP-1.Therefore, GLP-1 is with the reservation function activity.With any or non-natural aminoacid addition of 20 seed amino acids N-end to GLP-1.Histidine also is a kind of preferred amino acids.In some cases, use the aminoacid of uncharged or positive charge.Preferably, add less aminoacid, as glycine.The GLP-1 through modifying that will have additional amino acid then is fused on the transferrin, the preparation fusion rotein.In one embodiment, the GLP-1 peptide is modified, to contain at least one additional aminoacid at its amino terminal.In another embodiment, the GLP-1 peptide is modified, to contain at least 5 additional aminoacid at its amino terminal.Alternately, the GLP-1 peptide is modified, to contain 1-5 additional aminoacid at its amino terminal.

Glucagon-like-peptide-1 (GLP-1) is a kind of gut hormone of regulating and control insulin secretion, belongs to so-called entero-insular axis.Entero-insular axis is specified one group of hormone that discharges from gastrointestinal mucosa, and they are to the existence and the absorption response of nutritional labeling in the digestive tract, and the promotion insulin discharges in early days and enhancing discharges.Incretin (incretin) effect is the potentiation to insulin secretion, and this may be important for normal glucose tolerance.Therefore GLP-1 is insulinotropic hormone very important on a kind of physiology because be responsible for the incretin effect.

GLP-1 be Proglucagon (proglucagon) gene product (people such as Bell, Nature, 1983,304:368-371).It is synthetic in the intestinal endocrine cell with two kinds of main macromole forms, GLP-1 (7-36) amide and GLP-1 (7-37).This peptide was identified after the cDNAs of Proglucagon and gene are cloned in early days first the eighties in 20th century.

The original research that full-length peptide GLP-1 (1-37 and 1-36 amide) is carried out shows that bigger GLP-1 molecule lacks biologic activity.1987, three independently research group prove, remove the biologic activity that preceding 6 aminoacid can improve the GLP-1 molecule.

People such as Schmidt (1985 Diabetologia 28704-707) disclose the aminoacid sequence of GLP-1.People GLP-1 is a kind of peptide of 37 amino acid residues, derives from Proglucagon, and is synthetic in the L of ileum far-end, pancreas and brain cell.Proglucagon is processed into GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 and takes place in the L cell.The aminoacid sequence of GLP-1 (7-37) is SEQ ID NO:32 (X=Gly):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly。

In GLP-1 (7-36) amide, terminal Gly is by NH₂Substitute.

In the human patients of suffering from type ii diabetes (noninsulin dependent diabetes (NIDDM)), sometimes even in suffering from the patient of type i diabetes, GLP-1 sample molecule has anti-diabetic activity.Only when glucose level raises, produce actively with GLP-1 treatment, for example increase insulin secretion and biosynthesis, reduce glucagon secretion, delay gastric emptying, thus provide a kind of may be than insulin or the safer Therapeutic Method of sulfonylureas.Use suitable GLP-1 treatment, the intravital GLPP level of patient can be got back to normal level.Also have report to show, GLP-1 sample molecule can be protected and even recover pancreatic beta cell function among the type ii diabetes patient.

Any GLP-1 sequence can be modified by adding one or more aminoacid at its amino terminal, comprises GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36) and GLP-1 (7-37).GLP-1 has powerful effect to gastrointestinal tract.Measure perfusion GLP-1 with the physiology and can suppress the inductive and food-induced gastric acid secretion of pentagastrin (people such as Schjoldager, Dig.Dis.Sci.1989,35:703-708 effectively; People such as Wettergren, Dig Dis Sci 1993; 38:665-673).It also suppresses enzyme secretion (people such as Wettergren, the Dig DisSci 1993 of gastric emptying speed and pancreas; 38:665-673).With carbohydrate or when containing lipoprotein solution perfusion people's ileum, can produce similar inhibitory action (people such as Layer, Dig Dis Sci1995,40:1074-1082 to the secretion of harmonization of the stomach pancreas and motion; People such as Layer, Digestion 1993,54:385-38).Concomitantly, GLP-1 secretion is greatly stimulated, inferred GLP-1 may be responsible at least in part this so-called " ileum lock (ileal-brake) " act on (people such as Layer, Digestion 1993; 54:385-38).In fact, studies show that recently that the ileum lock effect of GLP-1 is more important than its effect to islets of langerhans on the physiology.Therefore, in dose response research, GLP-1 under the same slow rate of flooding of required with influencing islet secretion at least speed, influence gastric emptying speed (people such as Nauck, Gut 1995; 37 (supplements 2): A124).

As if GLP-1 influence food intake.Use food intake (people (editor) such as people .in Ditschuneit such as Schick, Obesity in Europe, John Libbey﹠amp that GLP-1 suppresses rat significantly in the ventricle; Company ltd, 1994; 363-367; People such as Turton, Nature 1996,379:69-72).This effect is high degree of specificity seemingly.Therefore, the terminal GLP-1 (1-36 that extends of N-^Amide) be non-activity, and the suitable GLP-1 antagonist of dosage, the exciting peptide 9-39 of Heloderma suspectum, effect (people such as Tang-Christensen, Am.J.Physiol., 1996,271 (4Pt 2): R848-56) of eliminating GLP-1 fully.In rat, sharply periphery is used GLP-1 and can not suppressed food intake (people such as Tang-Christensen, Am.J.Physiol., 1996,271 (4Pt 2): R848-56 tempestuously; People such as Turton, Nature 1996,379:69-72).Yet it also is possible can be used as also from the GLP-1 of intestinal L emiocytosis that a satiety signal works.

In diabetics, the short islets of langerhans effect of GLP-1 and GLP-1 to gastrointestinal effect be saved (people such as Willms, Diabetologia 1994; 37, supplement 1:A118), this helps to reduce food-induced glucose skew, and still the more important thing is also to influence food intake.Verified, continue intravenous with the speed of 4ng/kg/min and use GLP-1 one weekly assembly and improve control significantly the intravital glycerol mass formed by blood stasis of NIDDM patient (glycaemic), and do not have significant side effects (people such as Larsen, Diabetes 1996; 45, supplement 2:233A.).

The GLP-1 that process is modified partially or substantially is protected and avoids the active effect of DPP, and can be used for treating I type and type ii diabetes and obesity through the GLP-1 analog of modifying.

As used in this, term " GLP-1 molecule " is meant GLP-1, GLP-1 analog or GLP-1 derivant.

As used in this, term " GLP-1 analog " is defined as comparing with GLP-1 the molecule with one or more aminoacid replacement, disappearance, inversion or interpolation.Many GLP-1 analog are known in this area, comprise, for example, GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Val⁸-GLP-1 (7-37), Gln⁹-GLP1 (7-37), D-Gln⁹-GLP-1 (7-37), Thr¹⁶-Lys¹⁸-GLP-1 (7-37) and Lys¹⁸-GLP-1 (7-37) (SEQ ID NO:72) U.S. Pat 5,118,666 discloses the example of GLP-1 analog, for example GLP-1 (7-34) and GLP-1 (7-35).

Term " GLP-1 derivant " is defined as a kind of molecule, it has the aminoacid sequence of GLP-1 or GLP-1 analog, but its amino acid side chain gene, alpha-carbon atom, the terminal amino group group, or one or more in the terminal carboxylic acid group also have chemical modification.Chemical modification includes, but not limited to add chemical part, produces new key and removes chemical part.

As used in this, term " GLP-1 related compound " is meant any chemical compound of the definition that falls into GLP-1, GLP-1 analog or GLP-1 derivant.

WO 91/11457 discloses the analog of active GLP-1 peptide 7-34,7-35,7-36 and 7-37, and it partly is useful as GLP-1.

EP 0708179-A2 (Eli Lilly ﹠amp; Co.) disclose GLP-1 analog and derivant, it comprises the terminal imidazole group of N-and randomly is connected to unbranched C on the lysine residue of position 34₆-C₁₀Carboxyl groups.

EP 0699686-A2 (Eli Lilly ﹠amp; Co.) disclose the terminal truncated segment of some GLP-1N-, it is reported that they have biologic activity.

U.S. Pat 5,545,618 disclose basically by GLP-1 (7-34), GLP1 (7-35), GLP-1 (7-36) or GLP-1 (7-37), or its amide form, and the GLP-1 molecule of pharmaceutically acceptable salt composition, have at least a following modification that is selected from: (a) lysine on glycine, serine, cysteine, threonine, agedoite, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine or D-lysine the position of substitution 26 and/or the position 34; Or with the arginine on glycine, serine, cysteine, threonine, agedoite, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine or D-arginine the position of substitution 36 (SEQ ID NO:73); (b) tryptophan on the antioxidative aminoacid replacement position 31 (SEQ ID NO:74); (c) at least a replacement in the following replacement: the valine on tyrosine the position of substitution 16; Serine on lysine the position of substitution 18; Glutamic acid on aspartic acid the position of substitution 21; Glycine on serine the position of substitution 22; Glutamine on arginine the position of substitution 23; Alanine on arginine the position of substitution 24; And the lysine on glutamine the position of substitution 26 (SEQ ID NO:75); (d) at least a replacement in the following replacement: the alanine on glycine, serine or cysteine the position of substitution 8; Aspartic acid, glycine, serine, cysteine, threonine, agedoite, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine replace the glutamic acid of going up position 9; Glycine on serine, cysteine, threonine, agedoite, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine the position of substitution 10; And the aspartic acid on glutamic acid the position of substitution 15 (SEQID NO:76); (e) with glycine, serine, cysteine, threonine, agedoite, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine, or the histidine on histidine the position of substitution 7 of D-or N-acidylate or alkylated forms (SEQ ID NO:77); Wherein, replace (a) and (b), (d) and (e) in, it is the D type that substituted aminoacid can be chosen wantonly, the aminoacid of the position of substitution 7 selectively is N-acidylate or N-alkylated forms.

U.S. Pat 5,118,666 disclose a kind of active GLP-1 molecule of short islets of langerhans that has.This molecule is selected from the peptide with following aminoacid sequence: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (GLP-1,7-34, see SEQ ID NO:32) or His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (GLP-1,7-35 sees SEQ ID NO:32); And the derivant of described peptide, wherein said peptide is selected from: the pharmaceutically-acceptable acid addition of described peptide; The pharmaceutically acceptable carboxylate of described peptide; The pharmaceutically acceptable lower alkyl esters of described peptide; Pharmaceutically acceptable amide with the described peptide that is selected from amide, low alkyl group amide and lower dialkyl amide.

U.S. Pat 6,277,819 have instructed the mortality rate that reduces after myocardial infarction forms and the method for sickness rate, comprise to the patient and use GLP-1, GLP-1 analog and GLP-1 derivant.The GLP-1 analog with following structural formula represent (SEQ ID NO:^*): R₁-X₁-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X₂-Gly-Gln-Ala-Ala-Lys-X₃-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R₂(SEQ ID NO:78) and pharmaceutically acceptable salt thereof, wherein: R1 is selected from: L-histidine, D-histidine, deaminizating-histidine, 2-amino-histidine, beta-hydroxy-histidine, same histidine (homohistidine), α-methyl fluoride-histidine and Alpha-Methyl-histidine; X₁Be selected from: Ala, Gly, Val, Thr, Ile and Alpha-Methyl-Ala; X₂Be selected from: Glu, Gln, Ala, Thr, Ser and Gly; X₃Be selected from: Glu, Gln, Ala, Thr, Ser and Gly; R₂Be selected from: NH₂And Gly-OH; As long as the scope of the isoelectric point, IP of GLP-1 analog about 6.0 to about 9.0, and if R₁Be His, X₁Be Ala, X₂Be Glu, and X₃When being Glu, R₂Must be NH₂

People such as Ritzel (Journal of Endocrinology, 1998,159:93-102) a kind of GLP-1 analog, [Ser are disclosed⁸] GLP-1, wherein the alanine of second N-end substitutes with serine.This modification does not influence the short islets of langerhans activity of this peptide, but produces a kind of plasma stability analog higher than GLP-1.

U.S. Pat 6; 429; 197 are taught in Acute Stroke (stroke) or hemorrhage back is a kind of ideal Therapeutic Method with the GLP-1 treatment; preferably carrying out intravenous uses; this is because this processing provides a kind of means; be used to optimize insulin secretion, strengthen the brain anabolism, strengthen insulin effectiveness, and keep the normal or slight hypoglycemia of blood glucose, and do not have the serious hypoglycemia or the risk of other side effect by glucagon suppression.The invention provides a kind of in Acute Stroke or hemorrhage back with GLP-1 or its biologic activity analogue treatment ischemia or the method for pouring into brain again; optimize insulin secretion; strengthening insulin by the glucagon suppression antagonism renders a service; and keep the normal or slight hypoglycemia of blood glucose, and do not have serious hypoglycemic risk.

U.S. Pat 6; 277; 819 provide a kind of method that reduces mortality rate and sickness rate after the myocardial infarction, comprise so that the effective dose of blood glucose normalization uses the chemical compound of a kind of being selected from: GLP-1, GLP-1 analog, GLP-1 derivant and pharmaceutically acceptable salt thereof for the patient of needs.

U.S. Pat 6,191,102 disclose a kind of method that reduces the patient's who needs the reduction body weight body weight, by to be enough to cause that the dosage of weight loss uses one period that effectively causes weight loss of a kind of compositions to the patient, the described time was at least for 4 weeks, and said composition comprises glucagon-like-peptide-1 (GLP-1), glucagon-like peptide-1 analogs (GLP-1 analog), pancreas glucagon sample peptide-1-derivative (GLP-1 derivant) or its pharmaceutically acceptable salt.

After the subcutaneous administration, GLP-1 be have fully active (people such as Ritzel, Diabetologia 1995; 38:720-725), but mainly due to DPP IV sample enzymatic degradation by fast degraded (people such as Deacon, J Clin Endocrinol Metab 1995,80:952-957; People such as Deacon, J ClinEndocrinol Metab 1995,80:952-957; People such as Deacon, 1995, Diabetes 44:1126-1131).Therefore, unfortunately, GLP-1 and many its analog in human body, have short plasma half-life (people such as Orskov, Diabetes 1993; 42:658-661).Therefore, the purpose of this invention is to provide GLP-1 or its analog through modifying, they have the binding mode of prolongation with respect to GLP-1 (7-37).Derivant and the analog thereof that also has a purpose to provide GLP-1 of the present invention, their removing speed is lower than GLP-1 (7-37).And, an object of the present invention is to provide pharmaceutical composition, said composition comprises enhanced GLP-1 or the GLP-1 analog through modifying of stability.Therefore, the present invention includes the purposes that is used for the treatment of the disease relevant with GLP-1 through the GLP-1 that modifies or GLP-1 analog, these diseases for example still are not limited to above-mentioned those.

One aspect of the present invention relates to and comprising through the GLP-1 of modification and the preparation of drug combination of GLP-1 analog, can prepare described comprising through the GLP-1 of modification and the pharmaceutical composition of GLP-1 analog by any of having set up of compounding pharmaceutical method for compositions, for example, as Remington ' s Pharmaceutical Sciences, described in 1985.Said composition can be to be fit to systemic injection or dabbling form, and can prepare with suitable liquid-carrier equally, for example sterilized water or isotonic saline solution or glucose solution.Said composition is sterilized by conventional sterilizing methods well known in the art.Resulting liquid solution can wire up uses or filtration and lyophilizing under aseptic condition lyophilized preparation and aseptic aqueous solution combination before using.Described compositions contains the required pharmaceutically acceptable auxiliary substance of similar physiological situation, buffer reagent for example, tension adjustment reagent etc., for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride etc.

GLP-1 and GLP-1 analog through modification of the present invention also is suitable for nose, percutaneous, lung or rectal administration.The pharmaceutically acceptable carrier or the diluent that are used for compositions can be any conventional solid carriers.The example of described solid carrier is lactose, Gypsum Fibrosum powder (terra alba) sucrose, Pulvis Talci, gelatin, agar, pectin, Radix Acaciae senegalis, magnesium stearate and stearic acid.Similarly, carrier or diluent can comprise any slow-release material that this area is known, and for example glyceryl monostearate or glycerol distearate use separately or mix with wax (wax).

It is particularly advantageous providing compositions of the present invention with the extended release preparation form.Equally, compositions is mixed with and contains through the GLP-1 of modification or the microcapsule or the microgranule of GLP-1 analog, pack or disperse with suitable pharmaceutically acceptable biodegradable polymer, this polymer is polylactic acid, polyglycolic acid or lactic acid/ethanol copolymer for example.

Be used for nose and use, prepared product can contain and is dissolved or suspended in particularly GLP-1 or the GLP-1 analog through modifying in the aqueous carrier of liquid-carrier, is used for aerosol and uses.Described carrier contains additive, for example solubilizing agent, for example propylene glycol, surfactant, absorption enhancer, for example lecithin (phosphatidalcholine) or cyclodextrin, perhaps antiseptic such as p-Hydroxybenzoate.

Usually, process modified polypeptides of the present invention or peptide are dispersed in the unit dosage form with pharmaceutically acceptable carrier with per unit dosage.

And, the present invention includes the GLP-1 of process modification and the purposes that the GLP-1 analog is used to prepare medical product, this medical product can be used for treating the disease (metabolic disease) relevant with the blood sugar level rising, for example still is not limited to above-mentioned those diseases.Especially, the present invention includes the GLP-1 of process modification and the purposes that the GLP-1 analog is used for the treatment of diabetes, comprise type ii diabetes, obesity, serious burn (severe burn) and heart failure, comprise congestive heart failure and acute coronary syndrome.

The present invention also provides part or is protected basically and avoids the exciting peptide-3 of the Heloderma suspectum through modifying and the exciting peptide-4 of Heloderma suspectum of DPP active function.The exciting peptide-the 4th of exciting peptide-3 of Heloderma suspectum and Heloderma suspectum, insulinotropic peptides comprises 39 aminoacid (residue 2 is different with residue 3), has about 53% homology with GLP-1.Exciting peptide-3 sequence of Heloderma suspectum is HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:79), and the sequence of the exciting peptide-4 of Heloderma suspectum is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS (SEQ IDNO:80).The present invention comprises also that through exciting peptide-4 fragment of the Heloderma suspectum of modifying it comprises for example exciting peptide-4 (1-31) the HGEGTFTSDLSKQMEEAVR LFIEWLKNGGPY (SEQ ID NO:81) of Heloderma suspectum of aminoacid sequence.In addition, the present invention also comprises the analog through modifying of exciting peptide-3 of Heloderma suspectum and exciting peptide-4 peptide of Heloderma suspectum.

GLP-1 fusion rotein or the conjugate that are used for the treatment of diabetes, prediabetes or obesity through modifying

Can improve overall stability in the body with merging through the GLP-1 and the heterologous molecule of modifying.The method reorganization of knowing by this area or covalently bound on heterologous molecule will be through the GLP-1 and the heterologous molecule fusion of modifying.Can will merge or covalently bound arriving through the GLP-1 that modifies as plasma protein such as serum albumin or transferrin, on immunoglobulin or its part such as the Fc domain.More preferably, be fused on transferrin, lactotransferrin or the melanocyte transferrin through modified polypeptides or peptide.The method that is used to prepare this fusion rotein is described in U.S. Patent application US10/378, and in 094, it is incorporated herein by reference fully at this.

The GLP-1 molecule can be connected to by the joint of different length on the heterologous protein, so that better physical separation and more space motility to be provided between the albumen that is merged, thereby makes the maximization of human cytokines accessibility, for example makes it can reach its associated receptor.The joint peptide can by flexibility or have more inflexible aminoacid and form.For example, operable joint such as polyglycine chain.Described joint can be less than about 50,40,30,20,10 or 5 amino acid residues.Joint can by covalently bound to or between heterologous protein and GLP-1.Preferably, joint can be a Ser residue, two Ser residues, peptide Ser-Ser-Gly, peptide PEAPTD, peptide (PEAPTD)₂, with the peptide PEAPTD of IgG hinge connexon combination, and with the peptide (PEAPTD) of IgG hinge connexon combination₂These joints can be used for GLP-1 is connected on the transferrin.

Can modify being connected to through the transferrin on modified polypeptides or the peptide.It can present degree of glycosylation and descend.Can be selected from through the transferrin polypeptide of modifying: single transferrin N domain, single transferrin C-structure territory, transferrin N domain and C-structure territory, two transferrin N domains and two transferrin C-structure territories.

As above discuss, GLP-1 activates and the intravital important endocrine hormone of regulation and control machine system, and plays crucial control action in glucose metabolism.Different with all other Remedies for diabetes on the market, GLP-1 works as the somatomedin of β cell, has recovery potential, thereby improve the ability of pancreatic secretion insulin, and make existing insulin level work more effectively by improving sensitivity and stablizing glucose level better.This has reduced the burden of monitoring glucose level every day, and may delay the serious long-term side-effects that caused by the blood glucose fluctuation that diabetes cause.In addition, GLP-1 can reduce appetite and reduce body weight.Obesity is the intrinsic result to glucose metabolism control difference, and diabetic conditions is worsened.

Because natural GLP-1 is degraded (half-life has only several minutes) rapidly in circulation, so the clinical practice of natural GLP-1 is restricted.In order to keep the level of treatment agent in the circulation, need to use pump or diaphragm device (patch device) to continue administered with high dose, this has increased the treatment cost.This is inconvenient for life-time service, particularly is used for treating diabetes and monitors G/W at ordinary times with collaborative the making of all other medicines treatments.Keep the activity of GLP-1 through the GLP-1 fusion rotein of modifying, but had long half-lift (14-17 days), stability and the bio distribution characteristic of transferrin.These characteristics can provide every month s.c. (subcutaneous) injection of a kind of low cost, small size, and this series products is that life-time service is required fully.

Also can be covalently bound to blood constituent through the GLP-1 that modifies, improve its stability.For example covalently bound on the Fc part of serum albumin, transferrin, immunoglobulin or immunoglobulin through the GLP-1 that modifies.In one embodiment, can will be connected on fatty acid or the derivative of fatty acid through the GLP-1 that modifies.In another embodiment, arrived in the affine complex of medicine (DAC) by through engineering approaches through the GLP-1 that modifies.As discussed previously, (2003, Diabetes 52 (3): 751) disclose the affine complex of GLP-1-albumin medicine for people such as Kim.People such as Kim prove, the natural GLP-1 of the link coupled DAC:GLP-1 simulation of albumin.People such as Kim provide a kind of new method that is used for activating for a long time the GLP-1R signal.

When carrying out subcutaneous administration, DAC: through the GLP-1 that modifies albumin-binding apace and selectively in vivo.Formed biological conjugate has therapeutic activity identical with endogenous GLP-1 and similar effect, and has and more approach albuminous pharmacokinetics pattern.

Through GLP-1 and the fusion rotein and the combination of other therapeutic agent of modifying

In one aspect of the invention, GLP-1 peptide and fusion rotein thereof through modifying of the present invention, for example, the GLP-1-Tf fusion rotein, be used in combination with at least a second therapeutic molecules and treat type ii diabetes, obesity and other and unusual relevant disease or the situation of glucose level, described second therapeutic molecules such as Glucophage  (metformin hydrochloride (metformin) tablet) or Glucophage  XR (the long release tablet formulations of metformin hydrochloride).

Glucophage  and Glucophage  XR are the oral antihyperglycemics that is used to control type ii diabetes.Glucophage  XR is the long release formulation of Glucophage.Therefore, because medicine slowly discharges, only need take Glucophage  XR every day one time from Glucophage  XR.Glucophage  helps body to generate less glucose from liver.Therefore, Glucophage  effectively controls the intravital blood sugar level of patient.Glucophage  can not make body produce more insulin, and therefore, it seldom causes hypoglycemia (hypoglycemia).

Glucophage  also helps to reduce fat composition, triglyceride and the cholesterol of blood, and these materials are normally high-caliber in suffering from the human body of type ii diabetes.Metformin has been proved to be able to reduce appetite, helps it to reduce some body weight when people begin to take this medicine.

Metformin has gone through to be used for the treatment of with sulfonylureas or insulin, perhaps as single therapy agent treatment (individually).Metformin has been proposed to be used in the various cardiovascular disease of treatment, the hypertension among the insulin resistance patient (WO 9112003-Upjohn) for example, be used for lysed blood grumeleuse (with the combination of t-PA-derivant) (WO 9108763, WO 9108766, WO 9108767 and WO9108765-Boehringer Mannheim), ischemic anoxia and histanoxia (EP 283369-Lipha), arteriosclerosis (DE 1936274-Brunnengraber ﹠amp; Co., DE 2357875-Hurka and U.S. Pat 4,205,087-ICI).In addition, advised using metformin combination prostaglandin analogue cyclopentane derivatives as the coronary artery expansion thing and be used to bring high blood pressure down (U.S. Pat 4,182,772-Hoechst).Metformin also is proposed and 2-hydroxyl-3,3,3-trifluoroacetic acid derivant (U.S. Pat 4,107,329-ICI), 1,2-diarylethene derivatives (U.S. Pat 4,061,772-Hoechst), the aryloxy-3,3 that replaces, 3-three fluoro-2-propanoic acid, ester and salt (U.S. Pat 4,055,595-ICI), hydroxyphenyl-piperidones of replacing (U.S. Pat 4,024,267-Hoechst) and partially hydrogenated 1H-indeno-[1,2B]-pyridine derivate (U.S. Pat 3,980,656-Hoechst) combination is used for cholesterol reducing.

People such as Montanari (Pharmacological Research, 25 (1), 1992) open dosage (b.i.d.) with twice 500mg every day uses metformin to increase blood flow behind the ischemia in the mode similar to three 850mg metformin every day (t.i.d.).People such as Sirtori (J.Cardiovas.Pharm., 6:914-923,1984) are open, and the metformin increase of three 850mg dosage every day (t.i.d) suffers from peripheral blood vessel patient's artery blood flow.

The invention provides the Therapeutic Method of various diseases, comprise GLP-1 or its fusion rotein and one or more for example metformin associatings of treatment reagent through modifying of the present invention.In one embodiment, with treating disease and the situation relevant, for example diabetes with abnormal blood glucose through GLP-1 or its fusion rotein combination metformin modified.Preferably, treat type ii diabetes or obesity with GLP-1/mTf fusion rotein combination metformin.

Can include, but are not limited to sulfonylureas and sulfonylureas sample preparation through the GLP-1 of modification and other therapeutic agent of fusion rotein combination thereof with of the present invention, thiazole diketone (thiazolidinedione), peroxisome proliferator activated receptor (Peroxisome Proliferator-Activated Receptor) is the γ instrumentality (PPAR), PPAR α instrumentality, Protein-tyrosine-phosphatase-1B inhibitor, the insulin receptor tyrosine kinase activator, the 11beta-Hydroxysteroid dehydrogenase inhibitor, glycogen phosphorylase inhibitors, glucokinase activators, β-3 2-adrenergic agonist components and glucagon receptor agonist.

The DPP-IV inhibitor

The inhibitor that has proved DPP-IV is very promising in the various situations by the DPP-IV mediation of treatment.For example, in the treatment of glucose tolerance and the situation relevant with hyperglycemia such as type ii diabetes or obesity, the inhibitor of DPP-IV is very promising scheme.And verified, DPP-IV is relevant with immunoreation, and for example (Transplantation 1997,63 (10): 1495-1500) for transplant rejection.Therefore, DPP-IV can be used for preventing transplant rejection.In addition, because the endotheliocyte DPP-IV of lung can promote cancer cell metastasis in conjunction with the fibronectin of cancerous cell, so the inhibitor of DPP-IV can be used for treating cancer, is used to prevent cancer cell metastasis (J.Biol.Chem.1998,273 (37:24207-24215).Think equally DPP-IV in the pathogeny of periodontitis, play an important role (Infect.Immun.2000,68 (2), 716-724), and responsible deactivation GLP-2, GLP-2 be the factor that promotes the recovery of intestinal after big excision (J.Surg.Res.1999,87 (1), 130-133).Therefore, the DPP-IV inhibitor also can be used for the recovery of intestinal.

WO 95/15309 discloses some peptide derivant, and they are inhibitor of DPP-IV, thereby can be used for handling the process that is mediated by DPP-IV in a large number.WO 95/13069 discloses some cyclic amine compound, and it can be used for stimulating the natural or endogenous growth hormone of release.European patent 555,824 discloses some benzimidazole based compound, and it prolongs thrombin time and Trombin inhibiting and serine associated protein enzyme.Archives of Biochemistry and Biophysics, the 323rd volume, the 1st phase, 148-154 (1995) discloses some aminoacyl pyrrolidine-2-nitrile, can be used as the DPP-IV inhibitor.Journal ofNeurochemistry, the 66th volume, 2105-2112 (1996) discloses some Fmoc-aminoacyl pyrrolidine-2-nitrile, can be used for suppressing prolyl oligopeptidase.Bulletin of the Chemical Society ofJapan, the 50th volume, the 7th phase, 1827-1830 (1977) discloses amino six peptides, i.e. and Z-Val-Val-lmPro-Gly-Phe-Phe-OMe's, and relevant ammonia peptide is synthetic.In addition, also detected the antibacterial characteristics of described chemical compound.WO 90/12005 discloses some amino-acid compound, and it suppresses the prolyl endopeptidase activity, therefore can be used for treating dementia or amnesia.Derwent Abstract95:302548 discloses the heterocyclic compound that some N-(aryl (alkyl) carbonyl) replaces, and it is the activator of cholinesterase, and the periphery selectivity strengthens, and can be used for treating because the situation that activity of cholinesterase descends and causes.Chemical Abstracts 84:177689 discloses some 1-acyl group-pyrrolidine-2-nitrile compound, can be used as to have angiotensin converting enzyme (ACE) suppresses the intermedium of active proline compounds.Chemical Abstracts 96:116353 discloses some 3-amino-2-mercapto phenyl formic-propyl group-proline compounds, and they are Ras farnesyl-inhibitors, can be used for treating various cancers or myeloid leukemia.WO95/34538 openly suppresses some pyrrolidine (pyrrolidide), phosphate, azetidine (azetidine), peptide and the azepine proline of DPP-IV, is used for the treatment of the situation that can suppress improvement by DPP-IV.WO 95/29190 discloses some chemical compound, is characterised in that to have many KPR type repeat patterns, and is entrained by peptide substrate, make them repeatedly be presented on enzyme DPP-IV before, and DPP-IV had affinity, this chemical compound can suppress HIV and enter cell.WO 91/16339 discloses some tetrapeptide boric acid, and they are inhibitor of DPP-IV, can be used for the situation for the treatment of autoimmune disease and being mediated by the IL-2 inhibition.WO 93/08259 discloses some polypeptide boric acid, and they are inhibitor of DPP-IV, can be used for the situation for the treatment of autoimmune disease and being mediated by the IL-2 inhibition.WO 95/11689 discloses some tetrapeptide boric acid, and they are inhibitor of DPP-IV, can be used for stoping HIV to enter cell.Deutsches Wirtschafts Patent 158109 discloses the peptidyl-hydroxamic acid and the Nitrobenzol formyl oxamides of some N-protected, and it can be used as the inhibitor of DPP-IV.Disclose some dipeptides proline phosphate (ester) among the WO 95/29691, they are inhibitor of DPP-IV, can be used for treating immune system abnormality.Deutsche Bundespatent DD 296075 discloses some amino acid amide compound, and it suppresses DPP-IV.Biochimica et BiophysicaActa, the 1293rd volume, 147-153 discloses the preparation of some dipeptides and tripeptides p-nitroaniline (nitroanilide), with the modification of research side chain to the influence of their DPP-IV and PEP catalyzing hydrolysis.Bioorganic and Medicinal Chemistry Letters, the 6th volume, the 10th phase, 1163-1166 (1996) discloses some 2-Cyanopyrolidine, and they are inhibitor of DPP-IV.J.Med.Chem., the 39th volume, 2087-2094 (1996) discloses some and has contained the dipeptides of proline boric acid, and they are inhibitor of DPP-IV.Diabetes, the 44th volume, 126-1131 (in JIUYUE, 96) relates to these research, and this studies have shown that when the GLP-1 amide is administered to diabetics or ND by subcutaneous or intravenous path and is degraded rapidly.

U.S. Pat 6,727,261 provide new DPP-IV inhibitor pyrrole [2, the 1-a] isoquinilone derivatives of trembling, and can be used for treating and/or preventing disease relevant with DPP-IV and impaired glucose tolerance.The disease relevant with DPP-IV be diabetes for example, particularly noninsulin dependent diabetes, and these chemical compounds also can be used for treating and/or preventing Bowl disease, ulcerative colitis, Morbus Crohn, obesity (obesity) and/or metabolism syndrome.

U.S. Pat 6,716,843 provide alpha-amino acid sulfuryl compounds, can be used as the inhibitor of DPP-IV.

U.S. Pat 6,645,995 disclose the unsaturated heterocyclic compound that 2-replaces, and wherein assorted ring nitrogen is connected on aminoacid or the amino acid derivativges by amido link or peptide bond.These chemical compounds are inhibitor of DPP-IV effectively and optionally, and can be effective to treat can DPP-IV regulates and control or the situation of normalization by suppressing.

U.S. Pat 6,617,340 disclose N-(glycyl of replacement)-pyrrolidine, and described chemical compound is in the purposes that suppresses dipeptidyl peptidase-IV.U.S. Pat 6124,305 discloses N-(glycyl of replacement)-2-Cyanopyrolidine, and it suppresses DPP-IV.These chemical compounds can be used for treating the situation that is mediated by DPP-IV.

Give 93 patients that suffer from type ii diabetes (average HbA_1cBe 7.4%) use Novartis chemical compound 1-[[[2-[(5-cyanopyrimidine-2-yl) amino] ethyl] amino] acetyl group]-2-cyano group-(S)-pyrrolidine (NVP DPP728) 4 weeks, during the research in this 4 week, plasma glucose, insulin and HbA_1cLevel descend (referring to Diabetes Care 2002,25 (5): 869-875).

Use the combined therapy of DPP-IV inhibitor

An aspect the invention provides the transferrin fusion proteins and one or more DPP-IV inhibitor that comprise human cytokines, polypeptide or peptide and makes up the purposes that is used for the treatment of various situations.The invention provides pharmaceutical composition, it comprises transferrin fusion proteins and one or more DPP-IV inhibitor.As U.S. Patent application US10/378,094 is disclosed, and it is incorporated herein by reference fully at this, the transferrin that can modification be connected with human cytokines, polypeptide or peptide.It can present degree of glycosylation and descend.Can be selected from through the transferrin polypeptide of modifying: single transferrin N domain, single transferrin C-structure territory, transferrin N and C-structure territory, two transferrin N domains and two transferrin C-structure territories.Therapeutical peptide that is connected with transferrin or peptide can be its native form or the form through modifying.Preferably, transferrin fusion proteins comprises the GLP-1 as therapeutic peptide, and this GLP-1 is connected to through on the transferrin molecule of modifying, and is as U.S. Patent application US10/378, disclosed in 094.And combined therapy of the present invention comprises the GLP-1/mTf fusion rotein, one or more DPP-IV inhibitor, and another kind of therapeutic molecules.This molecule can be Glucophage  or Glucophage  XR.

Another aspect the invention provides the purposes that makes up through the albumen modified or peptide or its fusion rotein and one or more DPP-IV inhibitor that two peptidyl protease crackings is had resistance.The invention discloses pharmaceutical composition, it comprises through the albumen of modifying or peptide or its fusion rotein and the combination of one or more DPP-IV inhibitor.Preferably, be the GLP-1 that process is modified through the peptide of modifying, and fusion rotein is the GLP-1/mTf albumen through modifying.In addition, combined therapy of the present invention comprises through the GLP-1 that modifies or through GLP-1/mTf albumen, one or more DPP-IV inhibitor and other therapeutic molecules modified for example Glucophage  or Glucophage  XR.

The DPP-IV inhibitor can be used in the method for the present invention, to treat any relevant disease.For example; GLP-1-transferrin fusion proteins disclosed herein can be used for the treatment of the symptom of prediabetes (prediabetes), diabetes, obesity or diabetes, described DPP-IV inhibitor such as 1-[[[2-[(5-cyanopyrimidine-2-yl with the combination of DPP-IV inhibitor) amino] ethyl] amino] acetyl group]-2-cyano group-(S)-pyrrolidine (NVP DPP728).Described therapeutic agent can be used or use simultaneously in proper order.

Transferrin fusion proteins can comprise GLP-1 (7-37) peptide (SEQ ID NO:32) or GLP-1 (7-36) peptide (amino acid/11-30 of SEQ ID NO:32).More preferably, GLP-1 (7-37) peptide or GLP-1 (7-36) peptide comprise A8 are sported G and K34 is sported A.Transferrin albumen also can comprise joint between GLP-1 (7-37) peptide or GLP-1 (7-36) peptide and transferrin molecule.Preferably, this joint is (PEAPTD)₂Peptide.

The combined therapy of use endopeptidase inhibitor strengthens the pharmacokinetics and the pharmacodynamics of fusion rotein

The present invention also provides the combined therapy that uses neutral endopeptidase (NEP) inhibitor and transferrin fusion proteins.And, the present invention includes the combined therapy that uses NEP and DPP-IV inhibitor and transferrin fusion proteins.The inhibitor of NEP and DPPIV can be used simultaneously or sequentially.And described inhibitor and transferrin fusion proteins can be used simultaneously or sequentially.Can before or after using transferrin fusion proteins, use inhibitor.

Transferrin fusion proteins comprises GLP-1 (7-37) peptide (SEQ ID NO:32) or GLP-1 (7-36) peptide (amino acid/11-30 of SEQ ID NO:32).More preferably, GLP-1 (7-37) peptide or GLP-1 (7-36) peptide comprise A8 are sported G and K34 is sported A.Transferrin albumen can also comprise joint between GLP-1 (7-37) peptide or GLP-1 (7-36) peptide and transferrin molecule.Preferably, this joint is (PEAPTD)₂Peptide.

Neutral endopeptidase (NEP) also is called enkephalin, neutral lyase (neprilysin) and atrium peptidase, be that the membrane conjugated zinc that is present in many tissues refers to Zinc metalloproteinase, these tissues comprise brain, kidney, lung, gastrointestinal tract, heart and peripheral vascular system.NEP plays a major role in the removing of natriuretic peptide by degraded circulation natriuretic peptide (natriuretic peptide), thereby hinders the effect of natriuretic peptide to vasodilation, blood pressure and blood volume.NEP passes through degraded and deactivation natriuretic peptide, thereby relevant with hypertension, heart failure and renal failure.

Except degraded circulation natriuretic peptide, NEP also degrades, and other causes vasodilative material, comprises the circulation Kallidin I; Adrenomedullin, kidney vasodilation peptide and short natruresis-diuretic hormone (natriuretic-diuretic peptide); And/or the ANP of kidney form urine relaxin (urodilatin).

NEP also participates in the degraded of vasoconstriction thing Endothelin isotype ET-1, and also may relevant with the formation of ET-1 (people such as Brunner-La Rocca, Cardiovascular Research 51 (2001) 510-520).The NEP Angiotensin II of also degrading, a kind of effective vasoconstriction thing, endorphins, and a large amount of peptide relevant Kallidin I for example with metabolism, GLP-1 (Hupe-Sodmann, K., McGregor, G P., Brideribaugh, people Regulatory Peptide 1995 such as R; 58:149-56, Hupe-Sodmann, K, Goeke, R., Goeke, people Peptide 1997 such as B; 18:625-32), PYY (Medeiros MD, Turner AJ., Endocrinology.1994 May; 134 (5): 2088-94) and glucagon (people Am J Physiol Endocrinol Metab.2004 JIUYUE such as Trebbien R; 287 (3): E431-8).

For example phosphodolophine (phosphoramidon) or NEP/ACE inhibitor (compriseU.S. Pat 5 to disclose a large amount of nep inhibitors in the document, 508, disclosed omapatrilat,U.S. Pat 5 in 272,552, disclosed gempatrilat,U.S. Pat 5 in 397, disclosed sampatrilat and MDL 100240 in 430,145), be used for independent treatment (monotherapeutic treatment) as hypertension and heart failure.People such as Nathisuwan, " A Review of Vasopeptidase Inhibitor:ANew Modality in the Treatment of Hypertension and Chronic Heart Failure ", Pharmacotherapy, 22 (1), 27-42 (2002).Candoxatril and ecadotril are the nep inhibitors of two kinds of high specials, are just carrying out clinical experiment at present, are the future drugs of heart failure.These two kinds of medicines are the prodrugs that can be metabolised to active congener in vivo.Candoxatril is activated in liver and is candoxatrilat, and ecadotril is converted into its active congener S-Tetramethylene sulfide (S-thiorphan).

The example of some ACE/NEP inhibitor is disclosed in U.S. Pat 5,508, and 272, US5,362,727, US5,366,973, US5,225,401, US4,722,810, US5,223,516, US5,552,397, US4,749,688, US5,504,080, US5,612,359 and US5,525,723, and European patent application EP 0481,522, EP 0534363A2, EP 534,396 and EP534,492.

The invention provides combination (associating) treatment that comprises transferrin fusion proteins and DPP-IV inhibitor and ACE/NEP inhibitor, be used for the treatment of various diseases or situation.This disease or situation include, but are not limited to diabetes, are preferably type ii diabetes, congestive heart failure, obesity, hypertension and irritable bowel syndrome (irritable bowel syndrome).

Transgenic animal

One embodiment of the invention comprise genetically modified non-human animal's production, and this animal expression is protected and avoids the process modified polypeptides or the peptide of the active effect of DPP.In certain embodiments, comprise the genetically modified non-human animal of expressed fusion protein, this fusion rotein comprises through modified polypeptides or peptide and stability raising.

Described in a large amount of patents and patent application and successfully produced genetically modified non-human animal, for example U.S. Pat 6,291,740 (mandates on the 18th of calendar year 2001 JIUYUE); U.S. Pat 6,281,408 (mandates on August 28 calendar year 2001); And U.S. Pat 6,271,436 (mandate on August 7 calendar year 2001), its content is incorporated herein by reference fully at this.

The ability that changes the genetic constitution of animal makes them can carry out a large amount of commercial application, and described animal is for example comprised cow, pig, goat, horse, cattle and sheep by the animal of handling.These application comprise the production of animal, the a large amount of forms of collecting easily of this animal expression (for example, express in milk or the blood) extrinsic protein, weight increase, feed conversion rate height, meat are formed (carcass composition) improvement, milk yield or milk composition height, animal disease-resistant and that infection has resistance to specified microorganisms, produce fast growth or the strong animal of reproductive performance.The animal that contains exogenous DNA array in genome is called transgenic animal.

Be widely used in the method for producing transgenic animal the most and be DNA microinjection (people such as Wall, J.Cell.Biochem.49:113[1992]) in fertilization embryo's protokaryon.Other method that is used to produce transgenic animal comprises with retrovirus or retroviral vector infecting embryo.Reported before implanting with the retroviral infection of wild type or reorganization or the mice embryonic after implanting (Janenich, Proc.Natl.Acad.Sci.USA 73:1260[1976]; People such as Janenich, Cell 24:519[1981]; People such as Stuhlmann, Proc.Natl.Acad.Sci.USA 81:7151[1984]; People such as Jahner, Proc.Natl.Acad.Sci.USA 82:6927[1985]; People such as Van der Putten, Proc.Natl.Acad.Sci.USA 82:6148-6152[1985]; People such as Stewart, EMBOJ.6:383-388[1987]).

With retroviral infection embryo's alternative is with virus or the injection cell of producing virus in the segmentation cavity of mice embryonic (Jahner, people such as D., Nature 298:623[1982]).Be reported that and adopt intrauterine retroviral infection mice embryonic mid trimester of pregnancy, transgenic is imported in the sexual cell of mice (people such as Jahner, with above [1982]).Reported that the embryo with retrovirus or retroviral vector infected cattle and sheep generates transgenic animal.These schemes comprise that microinjection retroviral particle or growth stop the cell of (promptly splitting rhzomorph C with silk handles), this cell with retroviral particle be discharged to germ cell or body early embryo yolk week space (pct international patent application WO 90/08832[1990]; And Haskell and Bowen, Mol.Reprod.Dev., 40:386[1995].Pct international patent application WO 90/08832 describes the yolk week space that wild type feline leukaemia virus B is expelled to the sheep embryo of 2-8 cell stage.The fetus that the embryo who injects from process obtains is proved and contains a plurality of integration sites.

U.S. Pat 6,291, the retroviral vector (for example deriving from the carrier of murine leukemia virus [MLV]) that uses the transduction noble cells is described in 740 (mandates on the 18th of calendar year 2001 JIUYUE), foreign DNA is imported in immature oocyte and the sophisticated unfertilized oocyte (being progamous oocyte) produce transgenic animal.This patent has also been described and has been used for the method and composition that the initial and mouse mammary tumor LTR of cytomegalovirus promoter expresses various recombiant proteins.

U.S. Pat 6,281,408 (mandates on August 28 calendar year 2001) have been described the method for producing transgenic animal with embryonic stem cell.In brief, embryonic stem cell and morula are used for cell mixing to be cultivated altogether, generates transgenic animal.Before cultivating altogether, present, exogenous genetic material is imported in the embryonic stem cell by for example electroporation, microinjection or retrovirus.By selected marker such as neomycin, the embryonic stem cell of transfection is by this way selected the cell of selecting producer to integrate.

U.S. Pat 6,271, the production of transgenic animal is described in 436 (mandates on August 7 calendar year 2001), this method comprises separates primary sexual cell, cultivate these cells and produce the cell line in primary sexual cell source, transform primary sexual cell and cultured cells system of institute, produced transgenic animal by cell transformed and cell line with these.The efficient that generates transgenic animal is greatly improved, thereby makes can adopt homologous recombination when producing the non-rodent kind of transgenic.

Gene therapy

One embodiment of the invention comprise that polypeptide of the present invention or peptide construction are used for the purposes of gene therapy.By coming modified polypeptide or peptide, make it avoid the active effect of DPP at the one or more additional aminoacid of the terminal interpolation of N-.For example, provide to be coded in the nucleic acid construct that its N-end comprises the GLP-1 of additional His residue, be used for gene therapy.In addition, provide the nucleic acid construct of the GLP-1/ transferrin fusion proteins of coding, be used for gene therapy through modifying.GLP-1 construct through modification of the present invention is protected and is not subjected to the active effect of DPP, and more stable; Therefore, they are ideally suited and are used for gene therapy.

In brief, in people such as Ijima (June 10 calendar year 2001) Human Gene Therapy (U.S.) 12/9:1063-77, confirmed recently and can carry out gene therapy by the injection adenovirus vector, this carrier contains the gene of the soluble fusion rotein of encoding, and this fusion rotein partly is made up of the Fc of cytotoxic lymphocyte antigen 4 (CTLA4) and immunoglobulin G while 1.In this gene therapy application, successfully treat the II Collagen Type VI by this carrier of intra-articular injection and induce arthritic Muridae model.

In many United States Patent (USP)s, also described gene therapy, comprised U.S. Pat 6,225,290 (mandates on May 1 calendar year 2001); U.S. Pat 6,187,305 (mandates on February 13 calendar year 2001); With U.S. Pat 6,140,111 (mandates on October 31st, 2000).

U.S. Pat 6,225,290 provide method and construction, thereby make the enterocyte of mammalian subject be expressed the proteic gene with expectation therapeutical effect by hereditary change operationally to insert.Transform intestinal cell by using the preparation of mainly forming, and Orally administered this DNA has finished described conversion by naked DNA.Use the path in oral cavity or other the gastrointestinal simple application process is provided, and the complication relevant with using viral vector avoided in the use of naked nucleic acid, and realize gene therapy.Expressed protein direct secretion obtains the protein of curative blood levels in gastrointestinal tract and/or blood flow, thereby treatment needs this proteic patient.The epithelial cell that is transformed provides short-term or secular disease treatment scheme, and described disease is relevant with the defective of specific protein, perhaps is fit to treat by this proteic overexpression.

U.S. Pat 6,187,305 are provided at the method for carrying out gene or DNA target practice in the particularly mammiferous cell in vertebrates source.In brief, by homologous recombination or the target practice of DNA, DNA is imported in the primary cell and progeny cell in vertebrates source, described DNA is directed on the site of selecting in advance in the genomic DNA of the primary cell in vertebrates source and progeny cell.

U.S. Pat 6,140,111 (mandates on October 31st, 2000) have been described reverse transcription virus gene treatment carrier.Disclosed retroviral vector comprises the insertion site of gene of interest, and its can a large amount of various by the cell transformed type in high level expression derive from the albumen of this interested gene.But also disclose the retroviral vector of shortage selected marker, thereby made them be adapted at carrying out the human gene therapy in the treatment of various disease states, and not coexpression marked product, for example antibiotic.These retroviral vectors are particularly suitable in some package cell line.The genomic ability that the reverse transcription carrier inserts mammalian cell makes them become promising especially material standed in the gene therapy of heredopathia of treatment humans and animals.Gene therapy generally includes (1) to be added hereditary material in patient's cells in vivo to, or (2) take out patient's cell from body, new hereditary material is added in the cell, and they are introduced in the body again, be i.e. outer-gene treatment.The discussion of carrying out gene therapy with retroviral vector to how in various kinds of cell is found in, in JIUYUE, 1989 U.S. Pat 4 of authorizing in 19th for example, 868,116, US4 with the mandate on the 25th of nineteen ninety December, 980,286 (epithelial cells), Augusts 10 in 1989 disclosed WO 89/07136 (hepatocyte), July 25 nineteen ninety disclosed EP 378,576 (fibroblasts), and on June 15th, 1989 disclosed WO 89/05345 and nineteen ninety disclosed WO/90/06997 on June 28 (endotheliocyte), its disclosure is hereby incorporated by.

Can believe, need not to further describe that utilize previous description and following illustrative embodiment, those of ordinary skills can implement and utilize claimed the present invention.Therefore, following work embodiment points out the preferred embodiments of the invention especially, limits disclosed other parts by any way and be not interpreted as.The application's all articles of mentioning, publication, patent and file in full is incorporated herein by reference fully at this.

Embodiment

Embodiment 1: the GLP-1 through modifying with DPP IV protection

This embodiment describes the GLP-1 peptide through modifying of avoiding the DPP-IV active function.The following peptide of solid phase Fmoc chemosynthesis of employing standard, and carry out purification by reversed-phase HPLC with the C18 post, and quantize by the absorbance of 220nm.Analyze the peptide of purification by mass spectrography (MALDI-TOF):

GLP-1

NH₂-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-

Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH (amino acid/11-30 of SEQ ID NO:32)

GLP-1(A8G)

NH₂-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-

Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH(SEQ ID NO：90)

H-GLP-1

NH₂-His-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-

Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH(SEQ ID NO：91)

H-GLP-1(A8G)

NH₂-His-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-

Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH(SEQ ID NO：92)

HH-GLP-1

NH₂-His-His-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-

Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH(SEQ ID NO：93)

G-GLP-1

NH₂-Gly-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-

Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-COOH(SEQ ID NO：94)

The exciting peptide-4 of H-Heloderma suspectum

NH₂-His-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-

Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-

Ser-COOH(SEQ ID NO：95)

Dipeptidyl peptidase-IV is handled

Deng the various peptides (6 μ M) of mole number concentration 2 μ g recombined human DPP-IV (1 μ g/ μ L, the R﹠amp that is dissolved among the 25mM Tris-Cl (pH 8.0); D Systems, Minneapolis MN) handles.Simultaneously to the parallel control reaction of carrying out except DPP-IV of various peptides.Room temperature incubation digest 2 hours, in this time, reaction is with having added 1mM 3-isobutyl-1-methylxanthine (IBMX, Calbiochem, San Diego, Krebs-Ringer buffer (Biosource International CA), Camarillo, CA) dilution is 10 times.Following discussion is analyzed peptide, to determine the activating activities of residue GLP-1 receptor.

Ring AMP stimulates analysis

The previous day of handling is with 2 * 10⁴The density of cells/well, in the RPMI/10%FBS culture fluid of four 96-hole tissue culture plate, implant the CHO-GLPlR cell (people 1997 J.Biol.Chem.272 such as Montrose-Rafizadeh, 21201-21206).Second day, cell was with degree of the converging uniform distribution of about 60-80%.At second day that implants culture plate, with Krebs-Ringer buffer (KRB) washed cell twice, then in KRB, cultivated 1 hour for 37 ℃, reduce intracellular cAMP level.Then in KRB/IBMX, cultivate 10 minutes to suppress the enzyme of intracellular decomposition cAMP.The diluent of the various test compounds of preparation in KRB/IBMX, 37 ℃, three hole CHO-GLPlR cells were handled whole 20 minutes with every hole 50 μ l test compounds.Come termination twice with ice-cold phosphate-buffered salt water washing culture.Room temperature was added 0.1ml dissolving buffer 1B (Amersham BiosciencescAMP Biotrak EIA test kit) 10 minutes, the preparation pyrolysis product.Use cAMP BiotrakEnzyme Immunoassay System (Amersham Biosciences Corporation, Piscataway, NJ, production number RPN225), according to the test kit explanation, the various cell extracts of full volumetric are analyzed, determine cAMP concentration.Find that peptide of the present invention is higher than the not form through modifying to the resistance of DPP-IV.

The special ELISA of active GLP-1

Alternately, with (glucagon-like-peptide-1 [Active] ELISA test kit [the Linco Research of ELISA system, Inc., St.Charles, MO]) analyze the degraded of DPP-IV to GLP-1 of the present invention and GLP-1 derivant, this system is specific to complete active GLP-1, but two aminoacid of nonrecognition N-end are because the effect of DPP-IV and removed GLP-1, i.e. GLP-1 (9-36 or 9-37).GLP-1 and H-GLP-1 (1200pM) recombined human DPP-IV (200ng/ μ L, the R﹠amp that is dissolved in 25mM Tris-Cl (pH 8.0) Deng mole number concentration; D Systems, Minneapolis MN) handles, and dilutes cessation reaction with the analysis buffer that provides in the test kit, and this test kit contains protease inhibitor.

This test kit comprises by the 96-hole microtitration flat board of monoclonal antibody of anti-GLP-1.Washing dull and stereotyped (at dull and stereotyped cleaning machine, among the ThermoLabsystems Ultrawash Plus, with 25mM borate buffer saline x4 washing), room temperature is with peptide sample incubation 3 hours (300pM and 10 times of serial dilutions are put into flat board) then.After washing as mentioned above, room temperature was with the link coupled anti-GLP antibody of alkali phosphatase (promptly using in the test kit composition) incubation flat board 2 hours.After the washing, with 4-methyl umbrella shape based phosphates (4-Methyllumbelliferyl Phosphate) (MUP) substrate (at 50mM borate pH9.5 with dilution in 1: 200) add to institute porose in, room temperature isincubation 30 minutes in the dark.At 355nm excitation wavelength and 460nm emission wavelength, in SpectraMax Gemini EM fluorescence plate reader, flat board is carried out reading.Because H-GLP-1 is not easy in conjunction with monoclonal antibody than GLP-1, therefore the concentration of remaining active H-GLP-1 is determined with the H-GLP-1 standard curve after DPP-IV handles.Fig. 8 shows that H-GLP-1 is higher than GLP-1 in fact to the resistance of DPP-IV effect.

Embodiment 2: through the GLP-1 fusion rotein of modifying

This embodiment describes and comprises the fusion rotein through the GLP-1 that modifies that is fused to through on the transferrin molecule of modifying, and is protected and avoids the active effect of DPP-IV through the GLP-1 that modifies.

In order to make up the sequence of coding transferrin secretion targeting sequencing, design following overlapping primer, described targeting sequencing back is the N-end portion of GLP-1 and transferrin:

P0236-

TTCCCATACAAACTTAAGAGTCCAATTAGCTTCATCGCCA(SEQ ID NO：96)

P0237-

GGTTTAGCTTGTTTTTTTATTGGCGATGAAGCTAATTGGACTCTTAAGTTTGTAT

GGGAA(SEQ ID NO：97)

P0244-

ATAAAAAAACAAGCTAAACCTAATTCTAACAAGCAAAGATGAGGCTCGCCGTG

GGAGCCC(SEQ ID NO：98)

P0245-

CAGGACGGCGCAGACCAGCAGGGCTCCCACGGCGAGCCTCATCTTTGCTTGTTA

GAATTA(SEQ ID NO：99)

P0248-

TGCTGGTCTGCGCCGTCCTGGGGCTGTGTCTGGCGCATGCTGAAGGTACTTTTA

CTTCTGATGTTTCTTC(SEQ ID NO：100)

P0249-

AATTCTTTAGCAGCTTGACCTTCCAAATAAGAAGAAACATCAGAAGTAAAAGT

ACCTTCAGCATGCGCCAGACACAGCCC(SEQ ID NO：101)

P0250-

TTATTTGGAAGGTCAAGCTGCTAAAGAATTTATTGCTTGGTTGGTTAAAGGTAG

GGTACCTGATAAAACT(SEQ ID NO：102)

P0251-

AGTTTTATCAGGTACCCTACCTTTAACCAACCAAGCAATA(SEQ ID NO：103)

The position display of these primers is as follows.

AflII

-+----

>>...........P0236............>>

721 ccaatgttac gtcccgttat attggagttc ttcccataca aacttaagag tccaattagc

ggttacaatg cagggcaata taacctcaag aagggtatgt ttgaattctc aggttaatcg

<<...........P0237............<<

>>P0236.>> >>.....................P0244........................>>

781 ttcatcgcca ataaaaaaac aagctaaacc taattctaac aagcaaagat gaggctcgcc

aagtagcggt tatttttttg ttcgatttgg attaagattg ttcgtttcta ctccgagcgg

<<...........P0237............<< <<...........P0245............<<

>>...nL.....>

m r l a

>>P0244.>> >>.....................P0248........................>>

841 gtgggagccc tgctggtctg cgccgtcctg gggctgtgtc tggcgcatgc tgaaggtact

caccctcggg acgaccagac gcggcaggac cccgacacag accgcgtacg acttccatga

<<...........P0245............<< <<...........P0249............<<

>......................nL......................>>

v g a l l v c a v l. g l c l a

>>...GLP-1.....>

h a e g t

>>.....P0248.......>> >>...............P0250...................>>

901 tttacttctg atgtttcttc ttatttggaa ggtcaagctg ctaaagaatt tattgcttgg

aaatgaagac tacaaagaag aataaacctt ccagttcgac gatttcttaa ataacgaacc

<<...................P0249..........................<< <<.P0251<<

>.............................GLP-1.............................>

f t s d v s s y l e g q a a k e f i a w

KpnI

-----+

>>.........P0250..............>>

961 ttggttaaag gtagggtacc tgataaaact gtgagatggt gtgcagtgtc ggagcatgag

aaccaatttc catcccatgg actattttga cactctacca cacgtcacag cctcgtactc

<<...........P0251............<<

>....GLP-1....>>

l v k g r

>>.....................mTf......................>

v p d k t v r w c a v s e h e

(SEQ ID NO:104 is a coding strand; SEQ ID NO:105 is coded protein.)

Add primer (8 μ L 20pmol conc.) and be heated to 65℃ 5 minutes, carry out annealing reaction then temperature slowly be reduced to room temperature.

The T4 dna ligase is being added in the annealing reaction, and following incubation 2 hours, taking out 1 μ l reactant, be used for the increase complete insert of (outer primers) P0236 that has outer primer and P0251 of PCR reaction in room temperature.The PCR reaction condition is as follows:

94℃ 5 minutes

25 circulations: 94 ℃ 30 seconds

50 ℃ 30 seconds

72℃ 1 minute

72 ℃ 7 minutes

4 ℃ of placements

The PCR product that is generated with AflII and KpnI digestion, and the pREX0094 that is connected to earlier with AflII and KpnI digestion goes up (Fig. 1).Use the junctional complex transformed into escherichia coli.DNA from the clone who is generated is checked order, select to meet the clone of AflII/KpnI insert length, and called after pREX0198 (Fig. 2).Then, pREX0198 is with NotI and PvuI digestion and be inserted among the pSAC35 (Fig. 3) generation pREX0240 (Fig. 4).

In order to generate the natural transferrin of coding secretion targeting sequencing, to be the plasmid that is fused to through the H-GLP-1 (7-36) on the transferrin of modifying (mTf) afterwards, design overlapping primer P0424 and P0425, add the terminal histidine of extra N-to pREX0198 coded sequence.

P04245 ' is to 3 '

CTGTGTCTGGCGCATCATGCTGAAG(SEQ ID NO：106)

P04255 ' is to 3 '

CTTCAGCATGATGCGCCAGACACAG(SEQ ID NO：107)

PREX0198 is used for initial PCR reaction as template, uses two overlapping mutant primers and two outer primers in each reaction, and promptly P0424 adds P0012, and P0425 adds P0025.Then, for they are linked together, take turns among the PCR second, the product of these reactions is used as template, and only uses outer primer, and promptly P0012 adds P0025.The reaction condition of two-wheeled PCR be 1 * 94℃ 1 minute, 20 * 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ of 1 minute and 1 * 72 ℃ 7 minutes are to finishing.

PCR product from final reaction digests with AflII and KpnI, and is connected among the pREX0052 (Fig. 5) that digests with AflII/KpnI, generates pREX0367 (Fig. 6).Construct is carried out dna sequencing, confirmed to insert the codon of coding additional set propylhomoserin.

Use NotI and PvuI (latter destroys ampicillin resistance gene) digestion pREX0367 then, and be connected in advance on the pSAC35 with NotI digestion, generate pREX0368 (Fig. 7).

By electroporation pREX0368 is transformed in host's Wine brewing yeast strain, on buffered minimum culture fluid (minimal medium) flat board, selects the clone who is transformed according to leucine prototroph (leucine prototrophy).After selecting single clone, yeast transformant leaves in 40% trehalose, and-70 ℃ of storages.Determine to express by growth in the minimum culture fluid of the liquid of adjusting to pH6.5, and by SDS-PAGE, western trace and elisa assay supernatant.

Described in the U.S. Patent application of submitting on March 4th, 2,003 10/378,094, make up the plasmid of coding GLP-1/mTf (pREX0 100) and H-GLP-1/mTf, this patent application is incorporated herein by reference fully at this.In order to prepare the GLP-1/mTf fusion rotein, can use the aminoacid sequence of GLP-1 (7-36) and GLP-1 (7-37).

Haegtftsdvssylegqaakefiawlvkgr (amino acid/11-30 of SEQ ID NO:32)

haegtftsdvssylegqaakefiawlvkgrg(SEQ ID NO：32)

For example, the peptide sequence with GLP-1 (7-36) can instead be translated into DNA again, and optimizing codon is used for yeast expression:

catgctgaaggtacttttacttctgatgtttcttcttatttggaaggtcaagctgctaaagaa

h a e g t f t s d v s s y l e g q a a k e

tttattgcttggttggttaaaggtaga(SEQ ID NO：117)

F i a w l v k g r (amino acid/11-30 of SEQ ID NO:32)

Design primer specifically, after annealing, forming 5 ' XbaI and 3 ' KpnI sticky end, and make it possible to be directly connected on the pREX0052 of XbaI/KpnI cutting, just in time be positioned at the N-end of 5 of targeting sequencing ' end and mTf.Alternately, through engineering approaches prepares other sticky end to be connected on other carrier.

XbaI

-+-----

1 aggtctctag agaaaaggca tgctgaagg tacttttactt ctgatgtttc ttcttatttg

tccagagatc tcttttccgt acgacttcc atgaaaatgaa gactacaaag aagaataaac

>>......FL.......>>

r s l e k r

>>..................GLP-1....................>

h a e g t f t s d v s s y l

KpnI

------+

61 gaaggtcaag ctgctaaaga atttattgct tggttggtta aaggtagggt acctgata

cttccagttc gacgatttct taaataacga accaaccaat ttccatccca tggactat

>......................GLP-1......................>>

e g q a a k e f i a w l v k g r

>>..mTf..>>

v p d

SEQ ID NOs:118 and 119

Annealing be connected after, the clone is checked order, to verify correct insertion.This carrier is named as pREX0094.From pREX0094, cut out expression cassette with NotI, and sub-clone prepares pREX0100 in the yeast vector pSAC35 of NotI enzyme action.

Then with the plasmid electroporation in host's Wine brewing yeast strain, on minimum culture fluid flat board, select the anauxotrophic transformant of leucine.Determine to express by growth in the minimum culture fluid of liquid, and by SDS-PAGE, western trace and elisa assay supernatant.

GLP-1/mTf and H-GLP-1/mTf are expressed, and by cation exchange and anion-exchange chromatography purification from fermentation culture medium (described culture is grown under standard conditions).

Dipeptidyl peptidase-IV is handled

Deng the GLP-1/mTf of mole number concentration and recombined human DPP-IV (1 μ g/ μ L, the R﹠amp among H-GLP/1-mTF (2 μ M) the usefulness 25mM Tris-Cl (pH 8.0); D Systems) handles.Simultaneously to the parallel control reaction of carrying out except DPP-IV of various fusion rotein.The incubation digest is 2 hours under the room temperature, and in this time, reaction is diluted 20 times with the Krebs-Ringer buffer (Biosource International) that has added 1mM IBMX (Calbiochem).

Ring AMP stimulates analysis

The previous day of handling is with 1 * 10⁵The density of cells/well is implanted the CHO-GLPlR cell in the RPMI/10%FBS culture fluid in tissue culture plate (24-hole).Second day, cell was with degree of the converging uniform distribution of about 60-80%.After cell is implanted culture plate second day, with Krebs-Ringer buffer (KRB) washed cell twice, then 37 ℃ ofincubations 1 hour in KRB reduce intracellular cAMP level.Then in KRB/IBMX incubation 10 minutes to suppress the enzyme of intracellular decomposition cAMP.The diluent of the various test compounds of preparation in KRB/IBMX, and at 37 ℃, three hole CHO-GLPlR cells were handled whole 50 minutes with every hole 0.15ml test compounds.Come termination twice with ice-cold phosphate-buffered salt water washing culture.Room temperature was added 0.2ml dissolving buffer 1B (Amersham Biosciences cAMP Biotrak EIA test kit) 10 minutes, the preparation pyrolysis product, use cAMP Biotrak Enzyme Immunoassay System (AmershamBiosciences) then, according to the test kit explanation, analyze 100 each cell extract of μ l, determine cAMP concentration.

Embodiment 3: the GLP-1/mTf through modifying that is used for the treatment of diabetes

In this embodiment, the GLP-1/mTf through modification of the present invention treats diabetes as therapeutic agent.To be administered to the Zucker rat through the GLP-1/mTf that modifies, the Zucker rat is the standard animal model of type ii diabetes.The blood sugar level of Zucker rat is high unusually.Verified, handle these animals with GLP-1 and induce insulin secretion, and blood sugar lowering.

Zucker rat overnight fasting is handled with H-GLP-1 or the H-GLP-1 (H-GLP-1/mTf) that is fused on the transferrin then.After 30 minutes, animal carries out glucose tolerance test (GTT) at subcutaneous injection H-GLP-1 or H-GLP-1/mTf.For this test, give the fasting animal glucose solution (1.5mg/g body weight) of feeding, and with reasonable time interval measurement blood glucose.Soon, the blood sugar level of not processed animal raises, and slowly drops to baseline after glucose administration, and has injected the animal of H-GLP-1 or H-GLP-1/mTf because the short islets of langerhans effect demonstration of GLP-1 returns to normal blood sugar level sooner.

In another experiment, be used to make the high-caliber fasting glucose of Zucker rat to return to normal level through H-GLP-1 or the H-GLP-1/mTf that modifies, this Zucker rat is glucose administration not.And in not processed animal, blood sugar level is still very high, and in the animal that the H-GLP-1/mTf that modifies with H-GLP-1 or process handles, blood glucose significantly descends.

Embodiment 4: the glucagon through modifying with DPP IV protective effect

This embodiment has described the glucagon molecule through modifying of avoiding the DPP-IV active function through protection.

With the following peptide of solid phase Fmoc chemosynthesis of standard, and with the C18 post by the reversed-phase HPLC purification, and quantize by the absorbance of 220nm.(MALDI-TOF) analyzes purified peptide by mass spectral analysis:

Glucagon

NH₂-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-COOH(SEQ ID NO：35)

The H-glucagon

NH₂-His-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Val-Leu-Asp-Ser-Arg-Arg-

Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH(SEQ ID NO：108)

As mentioned above, the DPP-IV pretreatment of this peptide is analyzed the ability that it activates glucagon receptor with the recombinant cell lines of expressing the glucagon receptor of being cloned then.

Embodiment 5: the GIP through modifying with DPP IV protective effect

This embodiment provides and has been protected and avoids the GIP molecule through modifying of DPP-IV active function.

With the following peptide of solid phase Fmoc chemosynthesis of standard, and with the C18 post by the reversed-phase HPLC purification, and quantize by the absorbance of 220nm.(MALDI-TOF) analyzes purified peptide by mass spectral analysis:

GIP

NH₂-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-

Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-

Ile-Thr-Gln-COOH(SEQ ID NO：31)

Y-GIP

NH₂-Tyr-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-

Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-

Ile-Thr-Gln-COOH(SEQ ID NO：109)

As mentioned above, the DPP-IV pretreatment of this peptide is analyzed the ability that it activates gip receptor with the recombinant cell lines of expressing the gip receptor of being cloned then.

Should be appreciated that previous discussion and embodiment only provide some detailed description of the preferred embodiments.Therefore, for a person skilled in the art, obviously can carry out various improvement and be equal to alternatively, and not break away from spirit of the present invention and protection domain.All magazines of mentioning in present patent application, other list of references, patent and patent application are incorporated herein by reference in full.

Sequence table

＜110〉Shenyang XinSong robot automation Co., Ltd (BIOREXIS PHARMACEUTICAL CORPORATION)

Sadeghi，Homayoun

Prior，Christopher P.

Ballance，David J.

＜120〉use comprises the combined therapy of the transferrin fusion proteins of GLP-1

<130>054710-5012-WO

<150>US60/598,031

<151>2004-08-03

<160>119

<170>PatentIn version 3.2

<210>1

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>1

Ala Pro Val Arg Ser

1 5

<210>2

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>2

Ala Pro Thr Ser Ser

1 5

<210>3

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>3

Ile Pro Thr Glu Ile

1 5

<210>4

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>4

Val Pro Pro Gly Glu

1 5

<210>5

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>5

Ser Pro Gly Gln Gly

1 5

<210>6

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>6

Ser Pro Gly Pro Val

1 5

<210>7

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>7

Lys Pro Arg Leu Leu

1 5

<210>8

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>8

Gly Pro Val Ser Ala

1 5

<210>9

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>9

Ala Pro Ala Arg Ser

1 5

<210>10

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>10

Thr Pro Leu Gly Pro

1 5

<210>11

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>11

Ala Pro Pro Arg Leu

1 5

<210>12

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>12

Phe Pro Thr Ile Pro

1 5

<210>13

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>13

Val Pro Leu Ser Arg

1 5

<210>14

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>14

Met Pro Ile Thr Arg

1 5

<210>15

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>15

Met Pro Val Glu Arg

1 5

<210>16

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>16

Gly Pro Glu Thr Leu

1 5

<210>17

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>17

Ala Pro Leu Ala Thr

1 5

<210>18

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>18

Met Pro Met Phe Ile

1 5

<210>19

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>19

Glu Pro Asp Ala Ile

1 5

<210>20

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>20

Tyr Pro Ser Lys Pro

1 5

<210>21

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>21

Ala Pro Leu Glu Pro

1 5

<210>22

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>22

Tyr Pro Ile Lys Pro

1 5

<210>23

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>23

Leu Pro Ile Cys Pro

1 5

<210>24

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>24

Ser Pro Tyr Ser Ser

1 5

<210>25

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>25

Arg Pro Lys Pro Gln

1 5

<210>26

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>26

Ser Pro Ala Pro Pro

1 5

<210>27

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>27

Met Pro Gly Met Ile

1 5

<210>28

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>28

Met Pro Ser Cys Ser

1 5

<210>29

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>29

Leu Pro Gly Val Leu

1 5

<210>30

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>30

Ala Pro Met Ala Glu

1 5

<210>31

<211>42

<212>PRT

＜213〉people (Homo sapiens)

<400>31

Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

1 5 10 15

Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys

20 25 30

Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210>32

<211>31

<212>PRT

＜213〉people (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 31st X=G or-NH2.

<400>32

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa

20 25 30

<210>33

<211>33

<212>PRT

＜213〉people (Homo sapiens)

<400>33

His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn

1 5 10 15

Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr

20 25 30

Asp

<210>34

<211>44

<212>PRT

＜213〉people (Homo sapiens)

<400>34

Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly

20 25 30

Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu

35 40

<210>35

<211>29

<212>PRT

＜213〉people (Homo sapiens)

<400>35

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210>36

<211>27

<212>PRT

＜213〉people (Homo sapiens)

<400>36

His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln

1 5 10 15

Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met

20 25

<210>37

<211>103

<212>PRT

＜213〉people (Homo sapiens)

<400>37

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

20 25 30

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

35 40 45

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

50 55 60

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

65 70 75 80

Met Pro Lys Ala Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly

85 90 95

Ser Ala Gly Asn Lys Thr Tyr

100

<210>38

<211>9

<212>PRT

＜213〉people (Homo sapiens)

<400>38

Arg Pro Pro Gly Phe Ser Pro Phe Arg

1 5

<210>39

<211>11

<212>PRT

＜213〉people (Homo sapiens)

<400>39

Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met

1 5 10

<210>40

<211>22

<212>PRT

＜213〉people (Homo sapiens)

<400>40

Arg Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala Glu

1 5 10 15

Ala Phe Pro Leu Glu Phe

20

<210>41

<211>36

<212>PRT

＜213〉people (Homo sapiens)

<400>41

Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp

1 5 10 15

Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr

20 25 30

Arg Gln Arg Tyr

35

<210>42

<211>36

<212>PRT

＜213〉people (Homo sapiens)

<400>42

Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu

1 5 10 15

Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr

20 25 30

Arg Gln Arg Tyr

35

<210>43

<211>199

<212>PRT

＜213〉people (Homo sapiens)

<400>43

Leu Pro Ile Cys Pro Gly Gly Ala Ala Arg Cys Gln Val Thr Leu Arg

1 5 10 15

Asp Leu Phe Asp Arg Ala Val Val Leu Ser His Tyr Ile His Asn Leu

20 25 30

Ser Ser Glu Met Phe Ser Glu Phe Asp Lys Arg Tyr Thr His Gly Arg

35 40 45

Gly Phe Ile Thr Lys Ala Ile Asn Ser Cys His Thr Ser Ser Leu Ala

50 55 60

Thr Pro Glu Asp Lys Glu Gln Ala Gln Gln Met Asn Gln Lys Asp Phe

65 70 75 80

Leu Ser Leu Ile Val Ser Ile Leu Arg Ser Trp Asn Glu Pro Leu Tyr

85 90 95

His Leu Val Thr Glu Val Arg Gly Met Gln Glu Ala Pro Glu Ala Ile

100 105 110

Leu Ser Lys Ala Val Glu Ile Glu Glu Gln Thr Lys Arg Leu Leu Glu

115 120 125

Gly Met Glu Leu Ile Val Ser Gln Val His Pro Glu Thr Lys Glu Asn

130 135 140

Glu Ile Tyr Pro Val Trp Ser Gly Leu Pro Ser Leu Gln Met Ala Asp

145 150 155 160

Glu Glu Ser Arg Leu Ser Ala Tyr Tyr Asn Leu Leu His Cys Leu Arg

165 170 175

Arg Asp Ser His Lys Ile Asp Asn Tyr Leu Lys Leu Leu Lys Cys Arg

180 185 190

Ile Ile His Asn Asn Asn Cys

195

<210>44

<211>92

<212>PRT

＜213〉people (Homo sapiens)

<400>44

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asp Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asp His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>45

<211>145

<212>PRT

＜213〉people (Homo sapiens)

<400>45

Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu

1 5 10 15

Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr

20 25 30

Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val

35 40 45

Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asn Val Arg Phe

50 55 60

Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val

65 70 75 80

Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser

85 90 95

Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp

100 105 110

Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu

115 120 125

Pro Ser Pro Ser Arg Leu Pro Lys Pro Ser Asp Thr Pro Ile Leu Pro

130 135 140

Gln

145

<210>46

<211>5

<212>PRT

＜213〉people (Homo sapiens)

<400>46

Ala Pro Gly Pro Arg

1 5

<210>47

<211>27

<212>PRT

＜213〉people (Homo sapiens)

<400>47

Val Pro Leu Pro Ala Gly Gly Gly Thr Val Leu Thr Lys Met Tyr Pro

1 5 10 15

Arg Gly Asn His Trp Ala Val Gly His Leu Met

20 25

<210>48

<211>133

<212>PRT

＜213〉people (Homo sapiens)

<400>48

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210>49

<211>153

<212>PRT

＜213〉people (Homo sapiens)

<400>49

Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys

1 5 10 15

Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Leu His Leu Gln

20 25 30

Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met Ser Phe Val Gln

35 40 45

Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu Gly Leu Lys Glu

50 55 60

Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu

65 70 75 80

Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Met Glu

85 90 95

Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn Lys Leu Glu Phe

100 105 110

Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Glu

115 120 125

Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly Gln Asp Ile Thr

130 135 140

Asp Phe Thr Met Gln Phe Val Ser Ser

145 150

<210>50

<211>4

<212>PRT

＜213〉people (Homo sapiens)

<400>50

Tyr Pro Phe Phe

1

<210>51

<211>4

<212>PRT

＜213〉people (Homo sapiens)

<400>51

Tyr Pro Leu Gly

1

<210>52

<211>58

<212>PRT

＜213〉people (Homo sapiens)

<400>52

Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala

1 5 10 15

Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr

20 25 30

Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala

35 40 45

Glu Asp Cys Met Arg Thr Cys Gly Gly Ala

50 55

<210>53

<211>68

<212>PRT

＜213〉people (Homo sapiens)

<400>53

Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala

1 5 10 15

Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly

20 25 30

Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln

35 40 45

Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser

50 55 60

Leu Glu Met Ser

65

<210>54

<211>246

<212>PRT

＜213〉people (Homo sapiens)

<400>54

Asn Pro Leu Leu Ile Leu Thr Phe Val Ala Ala Ala Leu Ala Ala Pro

1 5 10 15

Phe Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn

20 25 30

Ser Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe Cys Gly

35 40 45

Gly Ser Leu Ile Asn Glu Gln Trp Val Val Ser Ala Gly His Cys Tyr

50 55 60

Lys Ser Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Glu Val Leu

65 70 75 80

Glu Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro

85 90 95

Gln Tyr Asp Arg Lys Thr Leu Asn Asn Asp Ile Met Leu Ile Lys Leu

100 105 110

Ser Ser Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro

115 120 125

Thr Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly

130 135 140

Asn Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln Cys Leu

145 150 155 160

Asp Ala Pro Val Leu Ser Gln Ala Lys Cys Glu Ala Ser Tyr Pro Gly

165 170 175

Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe Leu Glu Gly Gly Lys

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn Gly Gln

195 200 205

Leu Gln Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gln Lys Asn Lys

210 215 220

Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val Lys Trp Ile Lys Asn

225 230 235 240

Thr Ile Ala Ala Asn Ser

245

<210>55

<211>184

<212>PRT

＜213〉people (Homo sapiens)

<400>55

Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln Glu Asn Phe

1 5 10 15

Asn Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala Ile Gly Ser

20 25 30

Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr Val Ser Thr

35 40 45

Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser Met Thr Ser

50 55 60

Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly Ala Tyr Glu

65 70 75 80

Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser Lys Trp Asn

85 90 95

Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp Glu Tyr Ala

100 105 110

Ile Phe Leu Thr Lys Lys Phe Ser Arg His His Gly Pro Thr Ile Thr

115 120 125

Ala Lys Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr Leu Leu Gln

130 135 140

Asp Phe Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu Asp Ser Ile

145 150 155 160

Phe Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu Gln Glu Pro

165 170 175

Glu Pro Ile Leu Ile Pro Arg Val

180

<210>56

<211>77

<212>PRT

＜213〉people (Homo sapiens)

<400>56

Val Pro Leu Ser Arg Thr Val Arg Cys Thr Cys Ile Ser Ile Ser Asn

1 5 10 15

Gln Pro Val Asn Pro Arg Ser Leu Glu Lys Leu Glu Ile Ile Pro Ala

20 25 30

Ser Gln Phe Cys Pro Arg Val Glu Ile Ile Ala Thr Met Lys Lys Lys

35 40 45

Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser Lys Ala Ile Lys Asn Leu

50 55 60

Leu Lys Ala Val Ser Lys Glu Arg Ser Lys Arg Ser Pro

65 70 75

<210>57

<211>74

<212>PRT

＜213〉people (Homo sapiens)

<400>57

Gly Pro Ala Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala Asn Arg

1 5 10 15

Lys Ile Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile Thr Ser Gly

20 25 30

Lys Cys Pro Gln Lys Ala Val Ile Phe Leu Thr Lys Leu Ala Lys Asp

35 40 45

Ile Cys Ala Asp Pro Lys Lys Lys Tyr Val Gln Asp Ser Met Lys Tyr

50 55 60

Leu Asp Gln Lys Ser Pro Thr Pro Lys Pro

65 70

<210>58

<211>74

<212>PRT

＜213〉people (Homo sapiens)

<400>58

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro

65 70

<210>59

<211>76

<212>PRT

＜213〉people (Homo sapiens)

<400>59

Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile

1 5 10 15

Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20 25 30

Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly

35 40 45

Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met

50 55 60

Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro

65 70 75

<210>60

<211>76

<212>PRT

＜213〉people (Homo sapiens)

<400>60

Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe Ile

1 5 10 15

Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr

20 25 30

Ser Ser His Cys Pro Arg Glu Ala Val Ile Phe Lys Thr Lys Leu Asp

35 40 45

Lys Glu Ile Cys Ala Asp Pro Thr Gln Lys Trp Val Gln Asp Phe Met

50 55 60

Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu

65 70 75

<210>61

<211>77

<212>PRT

＜213〉people (Homo sapiens)

<400>61

Gly Pro Val Ser Ala Val Leu Thr Glu Leu Arg Cys Thr Cys Leu Arg

1 5 10 15

Val Thr Leu Arg Val Asn Pro Lys Thr Ile Gly Lys Leu Gln Val Phe

20 25 30

Pro Ala Gly Pro Gln Cys Ser Lys Val Glu Val Val Ala Ser Leu Lys

35 40 45

Asn Gly Lys Gln Val Cys Leu Asp Pro Glu Ala Pro Phe Leu Lys Lys

50 55 60

Val Ile Gln Lys Ile Leu Asp Ser Gly Asn Lys Lys Asn

65 70 75

<210>62

<211>68

<212>PRT

＜213〉people (Homo sapiens)

<400>62

Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60

Ala Leu Asn Lys

65

<210>63

<211>72

<212>PRT

＜213〉people (Homo sapiens)

<400>63

Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser

1 5 10 15

His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro

20 25 30

Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln

35 40 45

Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

50 55 60

Ala Leu Asn Lys Arg Phe Lys Met

65 70

<210>64

<211>69

<212>PRT

＜213〉people (Homo sapiens)

<400>64

Gly Pro Tyr Gly Ala Asn Met Glu Asp Ser Val Cys Cys Arg Asp Tyr

1 5 10 15

Val Arg Tyr Arg Leu Pro Leu Arg Val Val Lys His Phe Tyr Trp Thr

20 25 30

Ser Asp Ser Cys Pro Arg Pro Gly Val Val Leu Leu Thr Phe Arg Asp

35 40 45

Lys Glu Ile Cys Ala Asp Pro Arg Val Pro Trp Val Lys Met Ile Leu

50 55 60

Asn Lys Leu Ser Gln

65

<210>65

<211>7

<212>PRT

＜213〉people (Homo sapiens)

<400>65

Tyr Pro Phe Val Glu Pro Ile

1 5

<210>66

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>66

Ala Pro Gly Pro Arg Gly Ile Ile Ile Asn Leu Glu Asn Gly Glu Leu

1 5 10 15

Cys Met Asn Ser Ala Gln Cys Lys Ser Asn Cys Cys Gln His Ser Ser

20 25 30

Ala Leu Gly Leu Ala Arg Cys Thr Ser Met Ala Ser Glu Asn Ser Glu

35 40 45

Cys Ser Val Lys Thr Leu Tyr Gly Ile Tyr Tyr Lys Cys Pro Cys Glu

50 55 60

Arg Gly Leu Thr Cys Glu Gly Asp Lys Thr Ile Val Gly Ser Ile Thr

65 70 75 80

Asn Thr Asn Phe Gly Ile Cys His Asp Ala Gly Arg Ser Lys Gln

85 90 95

<210>67

<211>8

<212>PRT

＜213〉people (Homo sapiens)

<400>67

His Ser Asp Ala Val Phe Thr Asp

1 5

<210>68

<211>6

<212>PRT

＜213〉people (Homo sapiens)

<400>68

His Ser Asp Gly Ile Phe

1 5

<210>69

<211>8

<212>PRT

＜213〉people (Homo sapiens)

<400>69

His Ser Gln Gly Thr Phe Thr Ser

1 5

<210>70

<211>8

<212>PRT

＜213〉people (Homo sapiens)

<400>70

Phe Pro Thr Ile Pro Leu Ser Arg

1 5

<210>71

<211>8

<212>PRT

＜213〉people (Homo sapiens)

<400>71

His Ser Asp Gly Thr Phe Thr Ser

1 5

<210>72

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 2nd X=V or S; The 3rd X=Gln, D-Gln or Asp; The 10th X

=T or G; The 12nd X=K or A.

<400>72

His Xaa Xaa Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210>73

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 20th X=G, S, C, T, N, Q, Y, A, V, I, L, M, F, A,

D-Lys or K; The 28th X=G, S, C, T, N, Q, Y, A, V, I, L,

M, F, A, d-Lys or K; The 30th X=G, S, C, T, N, Q, Y, A,

V, I, L, M, F, K, R, d-Arg or OH.

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 29th X=G or OH; The 31st X=G or OH.

<400>73

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Xaa Xaa Xaa

20 25 30

<210>74

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 25th X=antioxidative aminoacid; The 29th X=G or OH;

The 30th X=R or OH; The 31st X=G or OH.

<400>74

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Xaa Xaa Xaa Xaa

20 25 30

<210>75

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 10th X=Y or V; The 12nd X=K or S; The 15th X

=D or E; The 16th X=S or G; The 17th X=R or Q;

The 18th X=R or A; The 20th X=Q or K.

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 29th X=G or OH; The 30th X=R or OH; The 31st X

=G or OH.

<400>75

His Ala Glu Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Tyr Leu Xaa Xaa

1 5 10 15

Xaa Xaa Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Xaa Xaa Xaa

20 25 30

<210>76

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 2nd X=G or S or C or A; The 3rd X=D or G or S

Or C or T or N or Q or Y or A or V or I or L or M or F or E; The 4th X

=S or C or T or N or Q or Y or A or V or I or L or M or F or G;

The 9th X=E or D.

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 29th X=G or OH; The 30th X=R or OH; The 31st X

=G or OH.

<400>76

His Xaa Xaa Xaa Thr Phe Thr Ser Xaa Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Xaa Xaa Xaa

20 25 30

<210>77

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 1st X=G or S or C or T or N or Q or Y or A or V or I

Or L or M or F or d-His or alkylation His or acidylate His; The 29th X

=G or OH; The 30th X=R or OH; The 31st X=G or OH.

<400>77

Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Xaa Xaa Xaa

20 25 30

<210>78

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Qu Daiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 1st X=1-or d-His, deaminizating-His, 2-amino-His,

Beta-hydroxy-His, same histidine (homohistidine), α-methyl fluoride-His, and Alpha-Methyl-His;

The 2nd X=A, G, V, T, I or Alpha-Methyl-Ala;

The 15th and the 21st 's X=E, Q, A, T, S or G.

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉the 31st X=Gly or NH2.

<400>78

Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly

1 5 10 15

Gln Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa

20 25 30

<210>79

<211>39

<212>PRT

＜213〉people (Homo sapiens)

<400>79

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210>80

<211>39

<212>PRT

＜213〉people (Homo sapiens)

<400>80

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210>81

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉the exciting peptide (exendin)-4 of the Heloderma suspectum of modifying

<400>81

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Tyr

20 25 30

<210>82

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>82

His His Ala Glu

1

<210>83

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>83

His His Gly Glu

1

<210>84

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>84

His His Ser Glu

1

<210>85

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>85

Gly His Ala Glu

1

<210>86

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>86

Gly His Gly Glu

1

<210>87

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the GLP-1N-end of Xiu Shiing

<400>87

Gly His Ser Glu

1

<210>88

<211>32

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Xiu Shiing

<400>88

His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

1 5 10 15

Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210>89

<211>32

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of Xiu Shiing

<220>

<221>MISC_FEATURE

<222>(1)..(31)

＜223〉aminoacid of X=except that 1-or d-Lys

<400>89

His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

1 5 10 15

Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly

20 25 30

<210>90

<211>30

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 of A8G modification

<400>90

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210>91

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 that modifies with the terminal His of extra N-

<400>91

His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

1 5 10 15

Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210>92

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 that modifies with terminal His of extra N-and A8G

<400>92

His His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

1 5 10 15

Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210>93

<211>32

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 that modifies with 2 terminal His residues of extra N-

<400>93

His His His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu

1 5 10 15

Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210>94

<211>31

<212>PRT

＜213〉artificial

<220>

＜223〉GLP-1 that modifies with the terminal Gly of extra N-

<400>94

Gly His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu

1 5 10 15

Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210>95

<211>40

<212>PRT

＜213〉artificial

<220>

＜223〉the exciting peptide of modifying with the terminal His of extra N--4 of Heloderma suspectum

<400>95

His His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu

1 5 10 15

G1u Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser

35 40

<210>96

<211>40

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>96

ttcccataca aacttaagag tccaattagc ttcatcgcca 40

<210>97

<211>60

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>97

ggtttagctt gtttttttat tggcgatgaa gctaattgga ctcttaagtt tgtatgggaa 60

<210>98

<211>60

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>98

ataaaaaaac aagctaaacc taattctaac aagcaaagat gaggctcgcc gtgggagccc 60

<210>99

<211>60

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>99

caggacggcg cagaccagca gggctcccac ggcgagcctc atctttgctt gttagaatta 60

<210>100

<211>70

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>100

tgctggtctg cgccgtcctg gggctgtgtc tggcgcatgc tgaaggtact tttacttctg 60

atgtttcttc 70

<210>101

<211>80

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>101

aattctttag cagcttgacc ttccaaataa gaagaaacat cagaagtaaa agtaccttca 60

gcatgcgcca gacacagccc 80

<210>102

<211>70

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>102

ttatttggaa ggtcaagctg ctaaagaatt tattgcttgg ttggttaaag gtagggtacc 60

tgataaaact 70

<210>103

<211>40

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of the Tf fusion rotein that the GLP-1-of modification modifies

<400>103

agttttatca ggtaccctac ctttaaccaa ccaagcaata 40

<210>104

<211>300

<212>DNA

＜213〉artificial

<220>

＜223〉sequence of the Tf fusion rotein of the GLP-1-modification of coding modification

<220>

<221>CDS

<222>(109)..(300)

<400>104

ccaatgttac gtcccgttat attggagttc ttcccataca aacttaagag tccaattagc 60

ttcatcgcca ataaaaaaac aagctaaacc taattctaac aagcaaag atg agg ctc 117

Met Arg Leu

1

gcc gtg gga gcc ctg ctg gtc tgc gcc gtc ctg ggg ctg tgt ctg gcg 165

Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu Cys Leu Ala

5 10 15

cat gct gaa ggt act ttt act tct gat gtt tct tct tat ttg gaa ggt 213

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

20 25 30 35

caa gct gct aaa gaa ttt att gct tgg ttg gtt aaa ggt agg gta cct 261

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Val Pro

40 45 50

gat aaa act gtg aga tgg tgt gca gtg tcg gag cat gag 300

Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu

55 60

<210>105

<211>64

<212>PRT

＜213〉artificial

<220>

＜223〉sequence of the Tf fusion rotein of the GLP-1-modification of coding modification

<400>105

Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu

1 5 10 15

Cys Leu Ala His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr

20 25 30

Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly

35 40 45

Arg Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu

50 55 60

<210>106

<211>25

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of plasmid with natural Tf secretion signal

<400>106

ctgtgtctgg cgcatcatgc tgaag 25

<210>107

<211>25

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer of plasmid with natural Tf secretion signal

<400>107

cttcagcatg atgcgccaga cacag 25

<210>108

<211>30

<212>PRT

＜213〉artificial

<220>

＜223〉glucagon of modifying with the terminal His of extra N-

<400>108

His His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Val Leu Asp

1 5 10 15

Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25 30

<210>109

<211>43

<212>PRT

＜213〉artificial

<220>

＜223〉GIP that modifies with the terminal Tyr of extra N-

<400>109

Tyr Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp

1 5 10 15

Lys Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly

20 25 30

Lys Lys Asn Asp Trp Lys His Asn Ile Thr Gln

35 40

<210>110

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉dipeptidyl peptidase serine protease motif

<400>110

Gly Trp Ser Tyr Gly

1 5

<210>111

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉transferrin secretory signal sequence

<400>111

Arg Ser Leu Asp Lys Arg

1 5

<210>112

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉transferrin secretory signal sequence

<400>112

Arg Ser Leu Asp Arg Arg

1 5

<210>113

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉transferrin secretory signal sequence

<400>113

Arg Ser Leu Glu Lys Arg

1 5

<210>114

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉transferrin secretory signal sequence

<400>114

Arg Ser Leu Glu Arg Arg

1 5

<210>115

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the N-end of DPP-resistance

<400>115

His His Ala Glu

1

<210>116

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉the N-end of DPP-resistance

<400>116

Gly His Ala Glu

1

<210>117

<211>90

<212>DNA

＜213〉artificial

<220>

＜223〉DNA sequence of coding GLP-1 (7-36)

<400>117

catgctgaag gtacttttac ttctgatgtt tcttcttatt tggaaggtca agctgctaaa 60

gaatttattg cttggttggt taaaggtaga 90

<210>118

<211>118

<212>DNA

＜213〉artificial

<220>

＜223〉has the pREX0052 site that GLP-1 inserts

<220>

<221>CDS

<222>(1)..(117)

<400>118

agg tct cta gag aaa agg cat gct gaa ggt act ttt act tct gat gtt 48

Arg Ser Leu Glu Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val

1 5 10 15

tct tct tat ttg gaa ggt caa gct gct aaa gaa ttt att gct tgg ttg 96

Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu

20 25 30

gtt aaa ggt agg gta cct gat a 118

Val Lys Gly Arg Val Pro Asp

35

<210>119

<211>39

<212>PRT

＜213〉artificial

<220>

＜223〉has the pREX0052 site that GLP-1 inserts

<400>119

Arg Ser Leu Glu Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val

1 5 10 15

Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu

20 25 30

Val Lys Gly Arg Val Pro Asp

35

Claims (52)

1. the disease among the treatment patient or the method for situation comprise transferrin (Tf) fusion rotein and at least a second reagent of using effective dose.

2. the process of claim 1 wherein that second reagent is selected from: DPP-IV inhibitor and neutral endopeptidase (NEP) inhibitor.

3. the process of claim 1 wherein that the Tf fusion rotein is subject to DPP-IV and removes.

4. the process of claim 1 wherein that the Tf fusion rotein is responsive to the DPP-IV removing, have resistance or have partial resistance.

5. the process of claim 1 wherein that the Tf fusion rotein comprises the GLP-1 peptide on the one or more Tf of being fused to molecules.

6. the method for claim 5, wherein the Tf fusion rotein also comprises joint.

7. the method for claim 6, its center tap are PEAPTD or (PEAPTD)₂

8. the method for claim 5, wherein the GLP-1 peptide is positioned at the N-end of fusion rotein.

9. the method for claim 5, wherein the GLP-1 peptide is GLP-1 (7-37) or the GLP-1 (7-36) (amino acid/11-30 of SEQ ID NO:32) with SEQ ID NO:32.

10. the method for claim 9, wherein the GLP-1 peptide is modified by A8 is sported G.

11. the method for claim 9, wherein the GLP-1 peptide is modified by K34 is sported A.

12. the method for claim 9, wherein the GLP-1 peptide is modified by A8 being sported G and K34 being sported A.

13. the method for claim 12, wherein the GLP-1 peptide is by joint (PEAPTD)₂Be connected on the Tf molecule.

14. the method for claim 5, wherein the Tf molecule is modified, and descends to compare its degree of glycosylation with complete glycosylated Tf molecule.

15. the method for claim 5, wherein the Tf molecule is modified not show glycosylation.

16. the method for claim 5, wherein the Tf molecule is modified to comprise at least one and is prevented glycosylated sudden change.

17. the method for claim 5, wherein the Tf molecule is modified, and reduces to compare its affinity to transferrin receptor (TfR) with wild type Tf molecule.

18. the method for claim 5, wherein the Tf molecule is modified with not in conjunction with TfR.

19. the method for claim 5, wherein the Tf molecule is modified, and descends to compare its affinity to ferrum with wild type Tf molecule.

20. the method for claim 5, wherein the Tf molecule is modified with not in conjunction with ferrum.

21. the method for claim 5, wherein the Tf molecule is lactotransferrin or melanocyte transferrin.

22. the process of claim 1 wherein that this disease is a metabolic disease.

23. the method for claim 22, wherein metabolic disease is prediabetes, diabetes or obesity.

24. the method for claim 23, wherein diabetes are type ii diabetes.

25. the process of claim 1 wherein that disease is congestive heart failure.

26. the process of claim 1 wherein that disease is irritable bowel syndrome or dyspepsia.

27. the process of claim 1 wherein two kind of second reagent.

28. the method for claim 27, wherein two kind of second reagent is DPP-IV inhibitor and nep inhibitor.

29. the method for claim 29, wherein two kind of second reagent is used simultaneously.

30. the method for claim 1 or 29, wherein transferrin fusion proteins and one or more second reagent are used in proper order.

31. the method for claim 1 or 29, wherein transferrin fusion proteins and one or more second reagent are used simultaneously.

32. a compositions, it comprises transferrin (Tf) fusion rotein and at least a second reagent.

33. the compositions of claim 32, wherein second reagent is selected from: DPP-IV inhibitor and neutral endopeptidase (NEP) inhibitor.

34. the compositions of claim 32, wherein the Tf fusion rotein is subject to the DPP-IV removing.

35. the compositions of claim 32, wherein the Tf fusion rotein is removed sensitivity, is had resistance or have partial resistance DPP-IV.

36. the compositions of claim 32, wherein the Tf fusion rotein comprises the GLP-1 peptide on the one or more Tf of being fused to molecules.

37. the compositions of claim 36, wherein the Tf fusion rotein also comprises joint.

38. the compositions of claim 37, its center tap are PEAPTD or (PEAPTD)₂

39. the compositions of claim 36, wherein the GLP-1 peptide is positioned at the N-end of fusion rotein.

40. the compositions of claim 36, wherein the GLP-1 peptide is GLP-1 (7-37) or the GLP-1 (7-36) (amino acid/11-30 of SEQ ID NO:32) with SEQ ID NO:32.

41. the compositions of claim 40, wherein the GLP-1 peptide is modified by A8 is sported G.

42. the compositions of claim 40, wherein the GLP-1 peptide is modified by K34 is sported A.

43. the compositions of claim 40, wherein the GLP-1 peptide is modified by A8 being sported G and K34 being sported A.

44. the compositions of claim 43, wherein the GLP-1 peptide is by joint (PEAPTD)₂Be connected on the Tf molecule.

45. the compositions of claim 32, wherein the Tf molecule is modified, and descends to compare its degree of glycosylation with complete glycosylated Tf molecule.

46. the compositions of claim 32, wherein the Tf molecule is modified not show glycosylation.

47. the compositions of claim 32, wherein the Tf molecule is modified to comprise at least one and is prevented glycosylated sudden change.

48. the compositions of claim 32, wherein the Tf molecule is modified, and reduces to compare its affinity to transferrin receptor (TfR) with wild type Tf molecule.

49. the compositions of claim 32, wherein the Tf molecule is modified with not in conjunction with TfR.

50. the compositions of claim 32, wherein the Tf molecule is modified, and descends to compare its affinity to ferrum with wild type Tf molecule.

51. the compositions of claim 32, wherein the Tf molecule is modified with not in conjunction with ferrum.

52. the compositions of claim 32, wherein the Tf molecule is lactotransferrin or melanocyte transferrin.

Images