CN101426519A - Human GLP-1 mimetibodies, compositions, methods and uses - Google Patents

Abstract

The present invention relates to at least one novel human GLP-1 mimetibody or specified portion or variant, including isolated nucleic acids that encode at least one GLP-1 mimetibody or specified portion or variant, GLP-1 mimetibody or specified portion or variants, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

Description

Human glp-1 mimetibodies, compositions, method and purposes

Background of invention

Invention field

The present invention relates to having bioactive albumen, fragment or part to have specific mammal GLP-1 analogue body (mimetibody), specific part or variant, GLP-1 analogue body code nucleic acid and complementary nucleic acid, host cell, and preparation and using method, comprise the treatment preparation, use and install.

Background technology

Recombiant protein is the emerging therapeutic agent of a class.This recombinant therapy is obtaining progress aspect protein formulation and the chemical modification.This modification can strengthen the treatment effectiveness of therapeutic protein potentially, for example by prolong half-life (for example, being exposed to proteolytic enzyme by hindering it), strengthens biological activity or reduces harmful side effect.A kind of such modification is to utilize to be merged to receptor protein, as the immunoglobulin fragment of enteracept.People also once utilized the Fc district to make up therapeutic protein, attempted the half-life that provides long or integrated such as Fc receptors bind, A protein binding and the bonded function of complement.

Diabetes are a kind of epidemic diseases that developing, and infect to its estimation in 2025 to surpass 300,000,000 people, also do not have effectivedrug treatment.Type 2 diabetes mellitus accounts for the 90-95% of all cases.The complication that the blood sugar level that continues to improve causes comprises cardiovascular diseases, nephropathy, neuropathy and retinopathy.In addition, in the later stage oftype 2 diabetes mellitus, therefore the beta cell death of pancreas stop excreting insulin.Interrelate for treatment of diabetes and various side effect now, described side effect comprises hypoglycemia and weight increase.In addition, now can not cure diseases for the treatment oftype 2 diabetes mellitus, but simply time expand till patient needs insulin treatment.

Glucagon-like-peptide-1 (GLP-1) be a kind of after oral glucose stimulates 37 the amino acid whose peptides that contain by intestinal L-emiocytosis.Endogenous cracking between the 6th and the 7th has subsequently produced bioactive GLP-1 (7-37) peptide.GLP-1 (7-37) peptide sequence can be divided into two domains.The amino-terminal end domain of peptide participates in signal activity, and the remainder of peptide appears to combine with GLP-1 receptor born of the same parents outer shroud with helical conformation.Glucose made reply, the GLP-1 receptors bind of active GLP-1 and pancreas, and cause the increase (insulinotropic activity) of insulin secretion.In addition, proved that GLP-1 reduces gastric emptying, its minimizing is discharged into the amount of the glucose in the circulation, and may reduce food intake.These effects combine and have just reduced blood sugar level.Proved that also GLP-1 suppresses apoptosis, promoted in the pancreas-propagation of cell.Therefore, GLP-1 is the attractive therapeutic agent of diabetics blood sugar lowering and protection pancreas-cell.In addition, the activity of GLP-1 is subjected to the control of blood sugar level.When blood sugar level reduces to a certain threshold level, the GLP-1 non-activity.Therefore, there is not to take place the danger of the hypoglycemia relevant with the GLP-1 treatment.

The feasibility of verified clinically GLP-1 treatment.The GLP-1 injection in six weeks has reducedtype 2 diabetes mellitus patient fasting blood glucose level and 8 hourly average blood sugar levels effectively.The GLP-1 treatment also causes the improvement of beta cell function.Exenatide is a kind of GLP-1 analog in clinical trial at present.At first identify Exenatide in Gila monster (gila monster) saliva, itself and GLP-1 have 53% homology.Exenatide can be in conjunction with the GLP-1 receptor, and the signal transduction cascade of the initial responsible various active that is caused by GLP-1 (7-37).Up to now, it has been proved the HbA1c level and the serum fructosamine level that can reduce thetype 2 diabetes mellitus patient.In addition, it suppresses the healthy gastric emptying of being examined the volunteer, and suppresses their food intake.

But GLP-1 is in vivo by the rapid inactivation of protease DPP IV (DPP-IV) institute.Therefore, the purposes that relates to the treatment of GLP-1 peptide has been subjected to their rapid removing and short-decayed restriction.For example, the serum half-life of GLP-1 (7-37) only is 3-5 minute.When subcutaneous administration, be about 50 minutes the action time of GLP-1 (7-36) amide.Even the cracked analog of anti-endogenous protease and derivant do not have the sufficiently long half-life to avoid repeat administration in 24 hours time period yet.For example, the anti-DPP-IV of Exenatide, however because its short-half-life in pharmacokinetics and significant variability in vivo, still need twice of the every day of dosed administration before the meal.At the another kind of compound N N2211 that clinical trial is generally used, be a kind of GLP-1 analog that is rich in lipid.It is considered to dosed administration once a day.

In the time need keeping the high blood levels of therapeutic agent in long-time, the rapid removing of therapeutic agent is inconvenient, because essential repeat administration.In addition, long-acting chemical compound includes only the diabetics particular importance of oral drugs for the therapeutic scheme in past.These patients have the very time transition of difficulty for the scheme that comprises repeatedly the medicine injection usually.Have growth half-life GLP-1 treatment relatively GLP-1 peptide and the chemical compound in other exploitation have remarkable advantages.

Therefore, need provide the improvement and/or the modification type of GLP-1 therapeutic protein, overcoming an above-mentioned one or more difficult problem, and other difficult problem known in the art.The analogue body technology provides new delivery platform for peptide therapy.The GLP-1 analogue body just can provide the method for sending the GLP-1 peptide in the mode that continues, and the improvement that the GLP-1 peptide in the present exploitation is carried out is provided.In addition, based on its dimeric structure and tissue distribution characteristic, the GLP-1 analogue body preserves for insulin secretion, beta cell and food intake has the feature that can distinguish.

Summary of the invention

The content that place like this is described and/or realized, and in conjunction with content known in the art, the invention provides human glp-1 mimetibodies, the immunoglobulin, pyrolysis product and other specific part and the variant thereof that comprise modification, GLP-1 analogue body compositions, coding or complementary nucleic acid, carrier, host cell, compositions, preparation, device, transgenic animal, transgenic plant also are provided, and methods for making and using same.

The present invention also provides place like this to describe and/or at least a separation GLP-1 analogue body known in the art or specific part or variant.This GLP-1 analogue body can randomly comprise at least one CH3 district, this CH3 district directly links to each other with at least one CH2 district, this CH2 district directly links to each other with at least one hinge region (H) or its fragment, this hinge region or its fragment directly link to each other with at least one part variable region (V), this part variable region directly links to each other with any joint sequence (L), and this joint sequence directly links to each other with at least one GLP-1 therapeutic peptide (P).

In a kind of preferred embodiment of a pair of CH3-CH2-hinge-part of V region sequence-joint-therapeutic peptide sequence, this randomly connects by association or covalent bond sequence, for example, but be not limited only at least one Cys-Cys disulfide bond and connect or at least one CH4 or other immunoglobulin sequences.In a kind of embodiment, the GLP-1 analogue body comprises molecular formula (I): a. (P (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t),

Wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby provide the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently, simulate dissimilar immunoglobulin molecules, for example, but be not limited only to IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE etc., or its subclass etc., or its combination.

The variable region of antibody sequence can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:47-55, its fragment perhaps as shown in table 1, at least one displacement, insertion or the disappearance that further selectively comprise the corresponding SEQ ID NO:1-9 shown in open text WO05/05604 (PCT US04/19898) Fig. 1-9 of PCT, this application was submitted on June 24th, 2004, and is open on January 20th, 2005.CH2 district, CH3 district and hinge region can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:56-64, its fragment perhaps as shown in table 1, further selectively comprise at least one displacement, insertion or disappearance as the corresponding SEQ IDNO:32-40 of the open text WO 05/05604 (PCT US04/19898) of PCT, this application was submitted on June 24th, 2004, and is open on January 20th, 2005.

Therefore, GLP-1 analogue body of the present invention is under the situation that keeps inherent character and function, simulated at least one part of antibody or immunoglobulin structure or function, also provide simultaneously a kind of GLP-1 therapeutic peptide and in intrinsic or external, the body that obtains or original position characteristic or activity.Adopt method described herein, and, can change a plurality of parts of antibody of the present invention and the therapeutic peptide part of GLP-1 analogue body in conjunction with methods known in the art.

The present invention also provides at least a separation GLP-1 analogue body described herein and/or known in the art or specific part or variant, wherein GLP-1 analogue body or specific part or variant have at least a activity, for example, but be not limited only to the GLP-1 peptide of at least a biologically active corresponding or the known organism activity of polypeptide, as described in this or known in the art with the P of molecular formula (I) part.

One aspect of the present invention provides at least a isolating human glp-1 mimetibodies, and it has comprised at least a aminoacid sequence among the SEQ ID NO:1, or selectively has one or more displacement described herein or known in the art, disappearance or insertion.On the other hand, at least a GLP-1 analogue body among the present invention or specific part or variant have been simulated combining corresponding at least a GLP-1 peptide of the analogue body P in the molecular formula (I) part or polypeptide and at least a epi-position, this epi-position has comprised the aminoacid of 1-3 at least of at least a part to complete amino acid sequence, this part for example, but be not limited only to GLP-1 receptor or its fragment, wherein part combines with at least a portion of SEQ ID NO:1, perhaps selectively with as described herein or have one or more displacements as known in the art, disappearance or insertion.Above-mentioned at least a GLP-1 analogue body can be randomly and the GLP-1 receptors bind, and this combination has and is selected from least 10^-9M, at least 1^-10M, at least 10^-11M or at least 10^-12The affinity of one of M.Therefore, can screen the GLP-1 analogue body according to corresponding activity, for example, but be not limited only to and receptor or its segmental activity that combines with known method.

The present invention further provides at least a anti-hereditary sexual type antibody of at least a GLP-1 analogue body of the present invention.Anti-hereditary sexual type antibody or fragment combine with at least a GLP-1 analogue body of the present invention specifically.Should comprise any molecule that contains albumen or peptide by anti-hereditary sexual type antibody, this molecule comprises at least a portion of immunoglobulin molecules, described part can be, but be not limited only at least a complementary determining region (CDR) or its ligand binding moiety, heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework region or its arbitrary portion of heavy chain or light chain, its competitiveness is in conjunction with the GLP-1 ligand binding domain of at least a GLP-1 analogue body of the present invention.Of the present inventionly should any mammal be can comprise or derive from by heredity sexual type antibody, for example, but people, mice, rabbit, rat, rodent, primate etc. were not limited only to.

One aspect of the present invention provides the isolated nucleic acid molecule that comprises specific polynucleotide, with the complementary isolated nucleic acid molecule of these specific polynucleotide, with these specific polynucleotide have remarkable homogeneity isolated nucleic acid molecule or with the isolated nucleic acid molecule of its hybridization, wherein at least one GLP-1 analogue body of this specific polynucleotide encoding or GLP-1 analogue body resist hereditary sexual type antibody or its specific part or variant, and comprise its at least one particular sequence, zone, part or variant.The present invention further provides the coding nucleic acid molecule that comprises at least a above-mentioned separation GLP-1 analogue body or GLP-1 analogue body anti-id AB recombinant vector, contain the host cell of above-mentioned nucleic acid and/or recombinant vector and the preparation and/or the application process of nucleic acid, carrier and/or the host cell of above-mentioned GLP-1 analogue body or GLP-1 analogue body anti-id AB.

The present invention also provides the isolating nucleic acid of encode at least a separation mammal GLP-1 analogue body or the anti-hereditary sexual type antibody of GLP-1 analogue body; Comprise the isolating nucleic acid carrier of this isolating nucleic acid, and/or comprise the protokaryon or the eukaryotic host cell of this isolating nucleic acid.This host cell can randomly be at least and be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,653, SP2/0,293, HeLa, myeloma or lymphoma cell, or the cell of one of the deriving arbitrarily of above-mentioned cell, immortalization or transformant.

The present invention also provides at least a method that is used for expressing at host cell at least a GLP-1 analogue body or the anti-hereditary sexual type antibody of GLP-1 analogue body, this method comprises cultivates host cell described herein and/or known in the art under given conditions, under this condition, anti-hereditary sexual type antibody of at least a GLP-1 analogue body or GPL-1 analogue body or specific part or variant are can detect and/or callable amount is expressed.A kind of method that generates at least a GLP-1 analogue body or the anti-hereditary sexual type antibody of GLP-1 analogue body also is provided, be included in external, the body or under the original position condition, the code nucleic acid of the anti-hereditary sexual type antibody of translation GLP-1 analogue body or GLP-1 analogue body makes the anti-hereditary sexual type antibody of GLP-1 analogue body or GLP-1 analogue body can detect or callable quantity is expressed.

The present invention also provides a kind of method for preparing at least a isolating human glp-1 mimetibodies or the anti-hereditary sexual type antibody of GLP-1, this method comprises provides a kind of host cell or transgenic animal or transgenic plant, and described host cell or transgenic animal or transgenic plant can reach GLP-1 analogue body or GLP-1 anti-id AB with callable scale.

The present invention further provides at least a GLP-1 analogue body in order to the preparation of top method.

The present invention also provides at least a compositions, and said composition comprises (a) isolating GLP-1 analogue body described herein or specific part or variant code nucleic acid and/or GLP-1 analogue body; (b) a kind of suitable carriers or diluent.According to known mode, this carrier or diluent may optionally be pharmacopedics can accept carrier or diluent.Said composition can randomly further comprise at least a other chemical compound, protein or compositions.

The present invention also provides a kind of compositions, and said composition comprises at least a isolating human glp-1 mimetibodies and at least a medicine acceptable carrier or diluent.Said composition further contains at least a chemical compound or the albumen of effective dose, and this chemical compound or albumen are selected from detectable label or reporter molecule, anti-infectives, diabetes or insulin metabolism related drugs, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomic nervous system (ANS) medicine, respiratory drugs, gastrointestinal (GI) tract drug, hormonal medicaments, the medicine that is used for balance body fluid or electrolyte, blood substance, antitumor drug, immunoregulation medicament, eye, ear or nose medication, local application, nutritional drugs, the TNF antagonist, antirheumatic, muscle relaxant, anesthetis, on-steroidal anti-inflammatory medicaments (NTHE), analgesic, anesthetics, tranquilizer, local anesthetic, neuromuscular blocking agents, antibacterial, antipsoriatic, corticosteroid, anabolic steroid, erythropoietin, immunity, immunoglobulin, immunosuppressant, growth hormone, the hormone replacement medicine, radiopharmaceutical, antidepressants, psychosis, analeptic, the asthma medicine, beta-agonists, suck steroid, epinephrine or analog, at least a in cytokine or the cytokine antagonist.

The present invention also provides at least a treatment or the GLP-1 analogue body at least a of the present invention of prevention effective dose or compositions, device and/or the method for specific part or variant of passing that be used to send.

At least a GLP-1 analogue body method or compositions have been the present invention further provides, when applying the treatment effective dose, it is conditions associated to can be used at least a GLP-1 that regulates or treat among cell, tissue, organ, animal or the patient, and/or as known in the art and/or described herein be applied to conditions associated before, afterwards or during.

The method or the compositions of at least a GLP-1 analogue body, specific fragment or variant have been the present invention further provides, when applying the treatment effective dose, be used for regulating, treating or alleviate cell, tissue, organ, animal or patient's at least a metabolism, immunity, cardiovascular, infectiousness, pernicious and/or sacred disease symptom, and/or according to multiple different situations, for example, but be not limited only to before relevant disease known in the art or the treatment condition, afterwards or during needs and use.

The present invention further provides at least a GLP-1 analogue body that is used for method or compositions, specific fragment or variant, when applying the treatment effective dose, be used for regulating, treatment or alleviate at least a diabetes or insulin metabolism associated disorders, bone and joint disorders, cardiovascular disorder, tooth or oral cavity disorder, the dermatosis disorder, ear, nose or larynx disorder, endocrine or metabolism disorder, gastrointestinal disturbance, gynecological's disorder, liver or gallbladder disorder, obstetrics' disorder, the blood disorder, immunity or allergic disorder, infectious disease, the disorder of flesh skeleton, the tumor disorder, neurological disorders, the nutrition disorder, the vision disorder, the department of pediatrics disorder, it is disorderly to poison, abalienation, the kidney disorder, the lung disorder, or any other known disorder (is seen, for example, Merck Manual, 17th ed., MerckResearch Laboratories, Merck and Co., Whitehouse Station, NJ (1999), be hereby incorporated by with its integral body), according to multiple different situations, for example, but be not limited only to before relevant disease known in the art or treatment condition, afterwards or during needs and use.

The present invention also provides at least a compositions of at least a GLP-1 analogue body of the present invention, the method for installing and/or sending, and diagnosable GLP-1 is conditions associated.

The method or the compositions of at least a GLP-1 analogue body have been the present invention further provides, at least a GLP-1 among diagnosable cell, tissue, organ, animal or the patient is conditions associated, and/or as known in the art and/or described herein be applied to conditions associated before, afterwards or during.

The present invention also provides the method for the disease symptoms that is used for diagnosing or treat cell, tissue, organ or animal, comprising: (a) make cell, tissue, organ or animal contact or it is used the compositions of at least a isolating human glp-1 mimetibodies of the present invention that comprises effective dose.This method can comprise further randomly that employing every kilogram of cell, tissue, organ or animal weight in every 0-24 hour, 1-7 days, 1-52 week, 1-24 month, 1-30 or any scope described herein or value use the effective dose of 0.001-50mg.This method can randomly further comprise by at least a parenteral that is selected from, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical, medicine group, vagina, rectum, cheek, the Sublingual, the mode of intranasal or percutaneous contacts or uses.This method can randomly further be included in before (a) contact or use, simultaneously or use at least a compositions afterwards, said composition contains at least a specific compound or the protein of effective dose, and this chemical compound or protein are selected from least a detectable label or reporter molecule, anti-infectives, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomic nervous system (ANS) medicine, respiratory drugs, gastrointestinal (GI) tract drug, hormonal medicaments, the medicine that is used for body fluid or electrolyte balance, blood substance, antitumor, immunoregulation medicament, eye, ear or nose medication, local application, nutritional drugs, the TNF antagonistic, antirheumatic, muscle relaxant, anesthetis, on-steroidal anti-inflammatory medicaments (NSAID), analgesic, anesthetics, tranquilizer, local anesthetic, neuromuscular blocking agents, antibacterial, antipsoriatic, corticosteroid, anabolic steroid, erythropoietin, immunity, immunoglobulin, immunosuppressant, growth hormone, the hormone replacement medicine, radiopharmaceutical, antidepressants, psychosis, analeptic, the asthma medicine, beta-agonists, suck steroid, epinephrine or analog, cytokine or cytokine antagonist.

The present invention also provides the medical apparatus that comprises at least a isolating human glp-1 mimetibodies of the present invention, and wherein this device is fit to by at least a parenteral that is selected from, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical, medicine group, vagina, rectum, cheek, the Sublingual, the mode of intranasal or percutaneous contacts or uses at least a human glp-1 mimetibodies.

The present invention also provides the goods that are used for people's medicine or diagnostic uses, comprises at least a container that separates human glp-1 mimetibodies of packaging material and the present invention who contains solution or lyophilized form.These goods can randomly comprise container as parenteral, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical, medicine group, vagina, rectum, cheek, the Sublingual, an ingredient of intranasal or percutaneous delivery device or system.

The present invention further provides any invention described herein.

Description of drawings

Fig. 1 has illustrated nucleotide and the peptide sequence of GLP-1 MMB in the IgG1 skeleton that shows the critical function territory.

Fig. 2 A-2C has illustrated that the FACS of GLP-1 MMB is in conjunction with test.Fig. 2 A has shown that GLP-1 MMB combines with the HEK293 cell of expressing GLP-1R excessively.Gray area: GLP-1MMB but do not have two anti-; Gray line: only have two to resist; Dotted line: negative contrast MMB and two resists; Black line: GLP-1 MMB and two resists.Fig. 2 B has shown that GLP-1 MMB does not combine with the HEK293 cell of contrast.Gray area: GLP-1 MMB but do not have two anti-; Black line: only have two to resist; Gray line: GLP-1 MMB and two resists.Fig. 2 C has shown that GLP-1 peptide analogues (A2S) can combine the HEK293 cell of expressing GLP-1R with GLP-1MMB competitiveness.Gray area: GLP-1 MMB but do not have two anti-; Black line: GLP-1 MMB and two resists; Geosutures: GLP-1 MMB, 0.2nM competition thing, two resists; Dotted line: GLP-1 MMB, 20nM competition thing, two resists; Gray line: GLP-1 MMB, 100nM competition thing, two resists).

Fig. 3 A-3E has illustrated the cAMP test of GLP-1 MMB.Wild type GLP-1 MMB in Fig. 3 A:IgG1 support; Fig. 3 B:GLP-1 peptide; GLP-1 (A2G) MMB in Fig. 3 C:IgG4 (Ala/Ala, Ser-〉Pro) support; GLP-1 (A2S) MMB in Fig. 3 D:IgG4 (Ala/Ala, Ser-〉Pro) support; Wild type GLP-1 MMB in Fig. 3 E:IgG4 (Ala/Ala, Ser-〉Pro) support.

Fig. 4 has illustrated that GLP-1 MMB is to the cracked resistance of DPP-IV.

Fig. 5 has illustrated the stability of GLP-1 MMB in serum.

Fig. 6 represents that GLP-1 MMB causes the insulin secretion of RINm cell.Fig. 6 A shows that GLP-1 (7-36) peptide and exendin-4 peptide stimulate the insulin secretion of RINm cell.Fig. 6 B is presented at GLP-1 (A2S) MMB in IgG1 or IgG4 (Ala/Ala, Ser-〉Pro) support or is stimulating at GLP-1 (A2G) MMB in IgG4 (Ala/Ala, Ser-〉Pro) support in the insulin secretion of RINm cell and can work.

Fig. 7 represents that GLP-1 MMB reduces glucose (Fig. 7 A) in dose-dependent mode (Fig. 7 B).

Fig. 8 shows four kinds of GLP-1 MMB (A2G, A2S, Ex-cap and wild type) pharmacokinetics figure of machin.

Fig. 9 shows the effect of GLP-1 MMB in the oral glucose tolerance test of diabetic mice.

Figure 10 shows that GLP-1 MMB is to the effect of fasting glucose in the diabetic mice of long term administration.

Figure 11 shows the effect of carrying out GLP-1MMB in the oral glucose tolerance test after the diabetic mice long term administration.

GLP-1 MMB was to reducing the effect of HbA1c after Figure 12 showed the diabetic mice long term administration.

Figure 13 shows that GLP-1 MMB is to the effect of blood glucose (Figure 13 A) and insulin (Figure 13 B) level in the oral glucose tolerance test of normal machin.

After Figure 14 was presented at single-dose, GLP-1 MMB was to the painted effect of the insulin of diabetic mice islets of langerhans.

Figure 15 illustrates that GLP-1 MMB postpones gastric emptying to normal Canis familiaris L..

Figure 16 illustrates GLP-1 MMB blood sugar lowering in the oral glucose tolerance test of the fat Mus of diet induced.

Figure 17 illustrates GLP-1 MMB blood sugar lowering (Figure 17 A) and reduction insulin level (Figure 17 B) in the intraperitoneal glucose tolerance test of diabetic mice.

Detailed Description Of The Invention

The invention provides separation, restructuring and/or synthetic analogue body or specific part or change Body, and composition and the coding nucleic acid molecule that comprises at least a polynucleotides, these are specific many At least a GLP-1 analogue body of nucleotide coding. The invention described above analogue body or specific part or Variant has comprised specific GLP-1 analogue body sequence, zone, fragment and specific variants thereof, and The present invention also provides preparation and the application side of above-mentioned nucleic acid and analogue body or specific part or variant Method comprises therapeutic composition, method and apparatus.

It is described herein and/or at least a separation known in the art that the present invention also provides GLP-1 analogue body or specific part or variant. This GLP-1 analogue body optionally comprises at least A CH3 district, this CH3 district directly links to each other with at least one CH2 district, this CH2 district with extremely A few hinge area or its fragment (H) directly link to each other, this hinge area or its fragment (H) and extremely A few part variable region (V) directly links to each other, this part variable region (V) and any joint order Row (L) directly link to each other, this any joint sequence (L) and at least one therapeutic peptide (P) Directly link to each other.

In a kind of preferred embodiment, the GLP-1 analogue body comprises molecular formula (I):

((P (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), Wherein P has bioactive GLP-1 polypeptide at least one, and L is at least one joint order Row, it is for by making analogue body have optional orientation and binding characteristic, thereby provides structure gentle The property polypeptide, V is at least one part of immune globulin variable region C-end, H be the immunity At least one part of the variable hinge area of globulin, CH2 is immunoglobulin (Ig) CH2 constant region At least one part, CH3 is at least one part of immunoglobulin (Ig) CH3 constant region, m, N, o, p, q, r, s and t can be the integer among the 0-10, simulate dissimilar immune globulins White molecule, for example, but be not limited only to IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE or its subclass etc., or its combination.

Therefore, GLP-1 analogue body of the present invention has been simulated and has been had the anti-of antibody inherent characteristic and function Body structure, provide simultaneously a kind of therapeutic peptide and in intrinsic or external, the body that obtains or Original position characteristic or activity. In the preferred embodiment when t=1, monomer CH3-CH2-hinge area-Part J sequence-joint sequence-therapeutic peptide can be by association or covalent bond and other monomer phase Connect, for example, but be not limited only to the Cys-Cys disulfide bond. Adopt method described herein, and In conjunction with methods known in the art, can change a plurality of parts of antibody of the present invention and at least a The therapeutic peptide part of GLP-1 analogue body.

If keep and the combination of remedying acceptor, the part of CH3-CH2-hinge area can be according to this Invention is formed variant by a large amount of the modification. In such acceptor, can remove one or more Natural site, these sites provide non-fusion molecule of the present invention required architectural feature or function Active. Can replace or the disappearance residue by for example, residue is inserted this site, or clip The part that comprises this site removes this site. The residue that inserts or replace also can change Amino acid, for example peptide analogue body or D-amino acid. The variant of CH3-CH2-hinge area may lack One or more natural sites or residue, its impact or participation: (1) disulfide bond forms, (2) Incompatible with selected host cell, the heterogeneity that express in selected host cell (3), (4) glycosylation, the interaction of (5) and complement, (6) are with except remedying acceptor The combination of Fc acceptor, or (7) ADCC (ADCC). Typically CH3-CH2-hinge area variant comprises following molecule and sequence: participate in the position that disulfide bond forms 1. Point has been removed. It is thin with the host for generation of molecule of the present invention that such removal can be avoided Other of cellular expression contains the protein phase reaction of cysteine. For this purpose, cysteine is residual Base can be lacked or be replaced (for example: alanyl, seryl-) with other amino acid. Even when cysteine residues is lacked, strand CH3-CH2-hinge area domain still can shape One-tenth is with the non-covalent dimerization CH3-CH2-hinge area domain that combines; 2.CH3-CH2-Hinge area has been modified, so that it is compatible with selected host cell. For example, when thin on bacterium Born of the same parents, for example among the E.Coli recombinant expressed this minute the period of the day from 11 p.m. to 1 a.m, can remove the PA sequence of hinge, this order Being listed in may be by digestive ferment among the E.Coli, and for example proline iminopeptidase is identified; 3. when selecting When expressing in the fixed host cell, the part of hinge area is by disappearance or with other amino acid institute It is heterogeneous that displacement is avoided; 4. one or more glycosylation sites are removed. Typical case's glycosylation (example As: residue asparagine) participates in cytolytic reaction. Such residue can be lacked or be used Other non-glycosylated residue (for example: alanine) is replaced; 5. participate in the position with complementary reaction Point, for example the C1q binding site has been removed. Complement is raised may be to the molecule among the present invention not Profit is so can eliminate such variant; 6. impact and Fc receptors bind, but not remedy the acceptor knot The site of closing is removed. The CH3-CH2-hinge area may have and specific leucocyte interaction The site, these sites are neither fusion molecule of the present invention needed, so can be removed; 7.ADCC the site has been removed. The ADCC site is well known in the art, sees, for example, Molec.Immunol.29 (5): 633-9 (1992) is about the ADCC site among the IgG1. These sites are neither fusion molecule of the present invention needed, so can be removed.

The joint polypeptide finishes thereby provide by making analogue body have optional orientation and binding characteristic The structure flexibility. When occurring, its chemical constitution is not crucial. Joint is preferentially logical by amino acid Crossing peptide bond links together and forms. Accordingly, in a preferred embodiment, joint is by 1 to 20 Amino acid links together by peptide bond and forms, and wherein amino acid is naturally occurring from 20 kinds Choose in the amino acid. In these amino acid some can be glycosylated, and it is at this The field is known. In a preferred embodiment, 1 to 20 amino acid is from following Choose in the amino acid, described amino acid is: glycine, alanine, serine, Proline, asparagine, glutamine and lysine. More preferably, joint is by most of skies Between upper uncrossed amino acid and forming, for example glycine and alanine. Therefore, preferred Joint is poly (Gly-Ser), and polyglycine (especially, (Gly)₄，(Gly)₅), Poly (Gly-Ala) and Polyalanine. The particular instance of other joint is: (Gly)₃Lys (Gly)₄(SEQ ID NO：65)，(Gly)₃AsnGlySer(Gly)₂(SEQ ID NO：66)，(Gly)₃Cys(Gly)₄(SEQ ID NO:67) and GlyProAsnGlyGly (SEQ ID NO:68).

In order to explain above term, for example, (Gly)₃Lys(Gly)₄The meaning be Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. The also assembly of preferred Gly and Ala. Herein Shown joint can imitate; Joint within the scope of the present invention may be longer, also may Comprise other residue.

Also allow non-peptide linker. For example, the alkyl joint, as-NH-(CH₂) s-C (O)-, Wherein used s=2-20. These alkyl connect and may further be substituted by non-steric hindrance group, Such as short-chain alkyl (for example: C₁-C₆), short chain acyl, halogen (for example: Cl, Br), CN, NH2, phenyl etc. A kind of typical non-peptide linker is the PEG joint, and it has 100 To the molecular weight of 5000kD, preferred 100 to 500kD. As previously mentioned, may be with same Mode changes peptide linker and forms derivative.

" GLP-1 peptide ", " GLP-1 peptide, variant or derivative " used herein can be At least a GLP-1 peptide, GLP-1 fragment, GLP-1 homologue, GLP-1 analog or The GLP-1 derivative. The GLP-1 peptide have from about 25 to about 45 natural existence or non-day The right amino acid that exists, itself and natural GLP-1 (7-37) have enough homologys, to such an extent as to Its by with the beta cell of pancreas on the GLP-1 receptors bind, shown insulinotropic activity. GLP-1 (7-37) has the amino acid sequence of SEQ ID NO:15:

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。

The GLP-1 fragment is at the N-end from GLP-1 (7-37) or its analog or derivative And/or the C-end is clipped the resulting polypeptide of one or more amino acid. The GLP-1 homologue is with one Or a plurality of amino acid add to the N-of GLP-1 (7-37) or its fragment or analog terminal and/or The peptide of C-end. The GLP-1 analog is that one or more amino acid with GLP-1 (7-37) advances The peptide that row is modified and/or replaced. GLP-1 analog and GLP-1 (7-37) or GLP-1 (7-37) fragment has significant homology, to such an extent as to analog has insulinotropic activity. GLP-1 Derivative is defined as such molecule, and it has GLP-1 peptide or its homologue or analog Amino acid sequence, but have its one or more amino acid side groups, alpha-carbon atom, terminal amino group Or the chemical modification of terminal carboxyl group.

A lot of active GLP-1 fragments, analog and derivative be by known in the art, any this A little analogs and derivative also can be the parts of GLP-1 analogue body among the present invention. This area institute More known GLP-1 analogs and GLP-1 fragment are disclosed in U.S. Patent number 5,118, 666,5,977,071 and 5,545,618 and Adelhorst, etc., J.Biol.Chem.269: 6275 (1994). example include but not limited to GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Gln9-GLP-1 (7-37), D-Gln9-GLP-1 (7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-1 (7-37).

" GLP-1 analogue body ", " GLP-1 analogue body part " or " GLP-1 analogue body Fragment " and/or " GLP-1 analogue body variant " and similar analogies, all simulation or imitation are at least At least a biologically active of a kind of GLP-1 peptide, variant or derivative, for example, but not only for Ligand-binding activity in external, original position and/or preferred body, this GLP-1 peptide, variant or Derivative is such as but not limited at least a SEQ ID NO:1. For example, suitable GLP-At least a GLP-1 also can be regulated, be improved, change, activate to 1 analogue body, specific part or variant But the signal of acceptor or other can be measured or detection of active.

In the inventive method and composition useful GLP-1 analogue body be characterised in that have with The suitable compatibility of protein ligands (for example GLP-1 acceptor) combination, and optional and preferably Has hypotoxicity. Particularly, it is in the present invention equal to have a GLP-1 analogue body of following situation Useful, i.e. wherein independent component is such as variable region, constant region (no CH1 part) and frame Frame part, or its arbitrary portion (part in the J of Weight variable or light chain, D or V district for example; At least a portion of at least one hinge area, constant heavy chain or light chain etc.) independent and/or common appointing Choosing and preferably have a reduced immunogenicity. Can be applicable to the optional feature of analogue body of the present invention exists But have the long-term treatment patient in it, and can be from well to relief of symptoms capitally, and toxicity is low Ability. Reduced immunogenicity and/or high-affinity, and other not bright characteristic all may can to it The result for the treatment of that realizes works. " reduced immunogenicity " is defined in herein and is less than approximately 75%, or preferably be less than about 50,45,40,35,30,25,20,15,10,9, 8, occur among 7,6,5,4,3,2 and/or 1% the patient through treatment significant HAMA, HACA or NAHA reply, and/or the low titre of appearance (adopts in through the patient for the treatment of Two antigen enzyme immunoassays are determined as and are less than approximately 300, preferably are less than about 100) (see, Such as: Elliott etc., Lancet 344:1125-1127 (1994)).

Effectiveness. Isolating nucleic acid of the present invention can be used to generate at least a GLP-1 analogue body, Fragment or its specific variants, this GLP-1 analogue body, fragment or its specific variants can be used to Cell, tissue, organ or animal were worked in (comprising mammal and people), with regulate, Treatment, to alleviate at least a protein conditions associated, helps its morbidity of prevention, or reduce its disease Shape, this protein is conditions associated to be selected from, but is not limited only to, at least a diabetes related disorder, Insulin related disorder, immune disorder or disease, cardiovascular disorder or disease, infectious disease, evil Venereal disease and/or nervous disorder or disease, and other known or specific protein is conditions associated.

The method can comprise to the above-mentioned adjusting of needs, treatment, alleviation, prevention or reduce symptom, Cell, tissue, organ, animal or the patient of impact or mechanism uses the particular combination of effective dose Thing or pharmaceutical composition, said composition comprise at least a GLP-1 analogue body or specific part or Variant. This effective dose can comprise that single or multiple uses the amount of about 0.0001-500mg/kg, or Single or multiple is used the amount that makes serum-concentration reach 0.01-5000ug/ml serum, or arbitrarily effectively Scope or numerical value wherein, it is used with method for measurement of concentration and is described herein or relevant neck The method that the territory is known.

Quote as proof. Whole publications cited herein or patent are owing to embodied the while with the present invention This area present situation, and/or provide description of the invention and implementation method, thereby drawn by complete Enter as a reference. Publication refers to any science or patent publications, or any media form Any data of other that is provided comprises all records, electronics or layout. Following ginseng Examining document all is incorporated herein by reference by complete: Ausubel, etc., ed., Current Protocols In Molecular Biology, John Wiley ﹠Sons, Inc., NY, NY (1987-2003); Sambrook, etc., Molecular Cloning:A Laboratory Mannual, 2 nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, etc., eds., Current Protocols in Immunology, John Wiley ﹠Sons, Inc., NY (1994-2003); Colligan etc., Current Protocols in Protein Science, John Wiley ﹠Sons, NY, NY, (1997-2003).

Analogue body of the present invention. The GLP-1 analogue body can comprise at least one CH3 district, this CH3 The district directly links to each other with at least one CH2 district, this CH2 district and at least one hinge area or its fragment (H) directly link to each other, for example comprise at least one core hinge area, this hinge area or its fragment (H) directly link to each other with at least one part variable region (V), this part variable region (V) with An optional joint sequence (L) directly links to each other, this optional joint sequence and at least one GLP-1 therapeutic peptide (P) directly links to each other. In a kind of preferred embodiment, a pair of CH3-CH2-H-V-L-P is by associating or covalent bond, for example, connecting into but be not limited only to the Cys-Cys disulfide bond Right. Therefore, GLP-1 analogue body of the present invention has been simulated and has been had antibody inherent characteristic and function Antibody structure, a kind of therapeutic peptide is provided simultaneously and in external, body or original position consolidate The characteristic that has or obtain or activity. Adopt method described herein, and in conjunction with known in the art Method can change a plurality of parts of antibody of the present invention and the treatment of at least a GLP-1 analogue body The property peptide moiety.

Analogue body of the present invention is compared with known protein at least a proper characteristics, example is provided As, but be not limited only to, the half-life of prolongation, the activity of raising, more special activity, carry The clearance rate of high affinity, raising or reduction, elite or more suitable active hypotype, During the quality that little immunogenicity, at least a expection result for the treatment of are enhanced or prolongation lasting Between, at least a in the less characteristics such as side effect.

The fragment of the analogue body of molecular formula (I) can be by known in the art and/or described herein Enzymatic lysis, synthetic or recombinant technique preparation. Utilize antibody gene also can prepare multiple clipped form Analogue body, one or more terminator codons have been imported into natural termination in this antibody gene The upstream in site. A plurality of parts of analogue body can be linked together by chemistry by routine techniques, Perhaps by utilizing genetic engineering technology to be prepared as in abutting connection with albumen. For example, can express encoding human The nucleic acid of at least one constant region in the antibody chain can be applicable in the analogue body of the present invention with generation In abutting connection with albumen. See, for example, Ladner etc., U.S. Patent number 4,946,778 and Bird, R.E. etc., Science, 242:423-426 (1988), the part of relevant single-chain antibody.

Term used herein " human simulation body " refers to a kind of antibody, wherein every part of this protein (for example GLP-1 simulating peptide, C_HDistrict (C for example_H2、C_H3), hinge, V) Basically all be expected among the people basic non-immunogenicity, and only have less sequence variation or Variation. For people's antibody or analogue body of the present invention of unmodified, this variation or variation are appointed Choosing also preferably keeps in the people or has reduced immunogenicity. Therefore, people's antibody and originally corresponding Invention GLP-1 analogue body and chimeric or humanized antibody is completely different. Be to be noted that GLP-1 Analogue body can be by inhuman animal or Hemapoiesis, and the animal that this is inhuman or cell can be expressed the people Immunoglobulin (Ig) (for example: heavy chain and/or light chain) gene.

For suitable part, the GLP-1 acceptor that for example separates, or its part (comprises and closing Become molecule, for example synthetic peptide), can design the people who is specific to its at least a protein ligands Analogue body. Utilization can identify and characterize at least a protein or its part ligand binding domain or The known technology of sequence can prepare above-mentioned analogue body.

In a kind of preferred embodiment, at least a GLP-1 analogue body of the present invention or particular portion Divide or variant by at least a clone, cell mixing system, immortalized cell, or unlimited The clonal population of propagationization and/or cultured cell generates. Adopt suitable method just can obtain infinitely The protein production cell of propagationization. Preferably, this at least a GLP-1 analogue body or specific Part or variant be by providing specific nucleic acid or carrier to be prepared, this nucleic acid or carrier Contain to be derived from the DNA at least one human immunoglobulin(HIg) site, perhaps have with this at least one Individual's immunoglobulin locus is similar sequence, the wherein immune ball of above-mentioned at least one individual basically Protein loci is reset by function or acceptable function is reset, and above-mentioned nucleic acid or carrier can further wrap Contain analogue body structure described herein, for example, but be not limited only to molecular formula (I), wherein as Known in the art, the terminal variable region of C-part can be used as V, and hinge area can be used as H, CH2 can be used as CH2, and CH3 can be used as CH3.

Term used herein " function rearrangement " refers to derive from the nuclear in specific immunoglobulins site Acid fragment, wherein this fragment has experienced V (D) J restructuring, thereby has generated the coding immune globulin The immunoglobulin gene of white chain (for example heavy chain) or its any part. The immunity that function is reset Globulin gene can obtain identifying suitable method by adopting appropriate method directly or indirectly For example, nucleotide sequencing, utilization can be annealed to the probe of the coding junction between the genetic fragment The hybridization of carrying out (for example: Southern trace, Northern trace) or employing can be annealed Primer to the coding junction between the genetic fragment expands the enzyme that immunoglobulin gene carries out (for example: polymerase chain reaction) increase. Whether cell has generated GLP-1 analogue body or its part Or variant also can be determined by suitable method, wherein this GLP-1 analogue body or its part Or variant has comprised specific variable region or (for example: at least one P sequence) has contained particular sequence The variable region.

Analogue body of the present invention, specific part and variant also can be by following method preparation, i.e. profits With at least a GLP-1 analogue body or specific part or variant code nucleic acid, to obtain transgenosis Animal or mammal, such as goat, milk cow, horse, sheep etc. generates it in milk Above-mentioned analogue body or specific part or variant. Adopt and the method class that is applied to antibody coding sequence Like known method just can obtain above-mentioned animal. See, for example, but be not limited only toU.S. Patent number 5, 827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489 etc., these patents all by complete introducing herein as a reference.

Analogue body of the present invention, specific part and variant also can be in addition by following method preparations, promptly utilize at least a GLP-1 analogue body or specific part or variant code nucleic acid, with the plant cell that obtains transgenic plant and cultivation (for example, but be not limited only to Nicotiana tabacum L. and Semen Maydis), make it at plant part or therefrom generate above-mentioned analogue body or specific part or variant in institute's cultured cells.A limiting examples, promptly the transgene tobacco leaf of express recombinant protein has been successfully used to the recombiant protein that provides a large amount of, for example, utilizes inducible promoter.See, for example Cramer etc., Curr.Top.Microbol.Immunol.240:95-118 (1999) and the list of references of quoting thereof.Equally, transgenic Semen Maydis or corn also have been used to express mammalian proteins matter with commercial production levels, and these proteic biologic activity are equal to by other recombination system generation or by natural origin purification gained protein.See, for example Hood etc., Adv.Exp.Med.Biol.464:127-147 (1999) and the list of references of quoting thereof.Comprise antibody fragment, for example strand analogue body (scFv ' s) is also generated from the transgenic plant seed that comprises tobacco seed and potato tubers in large quantities at interior antibody.See, for example Conrad etc., Plant Mol.Biol.38:101-109 (1998) and the list of references of quoting thereof.Therefore, analogue body of the present invention, specific part and variant also can be according to known methods, utilize transgenic plant and prepared.Also see, for example Fischer etc., Biotechnol.Appl.Biochem.30:99-108 (in October, 1999), Ma etc., Trends Biotechnol.13:522-7 (1995); Ma etc., Plant Physiol.109:341-6 (1995); Whitelam etc., Biochem.Soc.Trans.22:940-944 (1994); And the list of references of being quoted in the above-mentioned literary composition.Above-mentioned list of references all is introduced in this as a reference by complete.

Analogue body of the present invention is affinity (K widely_D) in conjunction with the human protein part.In a kind of preferred embodiment, at least a human glp-1 mimetibodies of the present invention can be randomly with high-affinity in conjunction with at least a protein ligands.For example, at least a GLP-1 analogue body of the present invention can be equal to or less than about 10^-7The K of M_D, or more preferred, be equal to or less than about 0.1-9.9 (or any range wherein or numerical value) x 10^-7, 10^-8, 10^-9, 10^-10, 10^-11, 10^-12Or 10^-13M, or the K of any range wherein or numerical value_DIn conjunction with at least a protein ligands.

The GLP-1 analogue body can be able to measuring by adopting any appropriate method to the affinity or the affinity of at least a protein ligands, for example is used to measure the method for antibody-antigen binding affinity or affinity.(see, Berzofsky for example, etc., at Fundamental Immunology, Paul, W.E., Ed., Raven Press:New York, " the Antibody-Antigen Interaction " among the NY (1984); Kuby, Janis Immunology, W.H.Freeman and Company:New York, NY (1992); And the method for describing).The affinity difference of specific GLP-1 analogue body-ligand interaction that different condition (for example salinity, pH) is measured down.Therefore, preferably adopt the standard solution of GLP-1 analogue body and part and all standard buffers as described herein or known in the art to measure affinity and other part incorporating parametric (K for example_D, K_a, K_d).

Nucleic acid molecules.Utilize data provided herein, adopt and describe or methods known in the art herein, can obtain nucleotide sequence such as at least one part of the continuous amino acid of 90-100% at least of at least one sequence and specific antibodies among the coding SEQ ID NO:1 and 6, wherein above-mentioned sequence is used as the P sequence of molecular formula (I) and inserts, can obtain GLP-1 analogue body of the present invention, further comprise its specific fragment, variant or concensus sequence, perhaps can obtain the nucleic acid molecules of the present invention that comprised the preservation carrier of at least a above-mentioned sequence and encoded at least a GLP-1 analogue body or specific part or variant.

Nucleic acid molecules of the present invention can be a rna form, such as mRN A, hnRNA, tRNA or other any form, or dna form, include but not limited to by clone or synthetic or their cDNA that combination obtained and genomic DNA.This DNA can be three chains, two strands or strand, or the combination in any of three kinds of chain forms.The arbitrary portion of at least one chain of DNA or RNA all can be coding strand, also is usually said sense strand, can be noncoding strand perhaps, is also referred to as antisense strand.

Isolated nucleic acid molecule of the present invention can comprise and contain a reading frame (ORF), and randomly has the nucleic acid molecules of one or more introns, contains the nucleic acid molecules of the coded sequence of GLP-1 analogue body or specific part or variant; With contain the nucleotide sequence different basically with above-mentioned nucleotide sequence, but because the degeneracy of genetic code, the nucleic acid molecules of at least a GLP-1 analogue body described herein and/or known in the art of still encoding.Certainly, this genetic code is well known in the art.Therefore, to those skilled in the art, the above-mentioned degeneracy nucleic acid variant that the preparation code book is invented specific GLP-1 analogue body or specific part or variant is routine work.See, for example above-mentioned Ausubel, etc., and above-mentioned nucleic acid variant also is included among the present invention.

So point out in the place, and the nucleic acid molecules of the present invention that comprises the nucleic acid of coding GLP-1 analogue body or specific part or variant can include but not limited to, the nucleic acid molecules of the segmental aminoacid sequence of self coding GLP-1 analogue body; The coded sequence of complete GLP-1 analogue body or its part; The coded sequence of GLP-1 analogue body, fragment or part, and other sequence, the coded sequence of or fusogenic peptide leading such as at least a signal, has or do not have above-mentioned other coded sequence, such as at least one intron,, include but are not limited to non-coding 5 ' and 3 ' sequence together with other non-coding sequence, such as at the non-translated sequence through transcribing of transcribing, playing a role in the mRNA processing, comprise montage and polyadenylation signal (for example-mRNA ribosome combination and stability); Other aminoacid of encoding is such as other functional amino acid whose other coded sequence can be provided.Therefore, the sequence of coding GLP-1 analogue body or specific part or variant can be merged to labelled sequence, and such as the sequence of coding particular peptide, this peptide helps purification to contain fusion GLP-1 analogue body or the specific part or the variant of GLP-1 analogue body fragment or part.

Polynucleotide with polynucleotide selective cross described herein.The invention provides can be under the selective cross condition with disclosed polynucleotide herein or comprise its specific variants or the isolating nucleic acid of other multi-nucleotide hybrid of part.Therefore, the polynucleotide of this embodiment can be used to separate, detect and/or quantitatively contain the nucleic acid of these polynucleotide.

Low or medium stringency hybridization condition typically but exclusively is not applied to compare with complementary series the sequence that sequence homogeneity is lowered.Medium and high stringency can randomly be used to the sequence of higher homogeneity.Low stringency can realize having the selective cross of the sequence of about 40-99% sequence homogeneity, and can be used to identify directly to homology or symbiosis homologous sequence.

Randomly, the coded GLP-1 analogue body of the polynucleotide described from here of polynucleotide codified of the present invention or at least one part of specific part or variant.Polynucleotide of the present invention have comprised the nucleotide sequence that can be used to invent with code book the polynucleotide selective cross of GLP-1 analogue body or specific part or variant.See, for example, above-mentioned Ausubel; Above-mentioned Colligan, all herein as a reference by complete introducing.

The structure of nucleic acid.Isolating nucleic acid of the present invention can adopt (a) well known in the art recombination method, (b) synthetic technology, (c) purification technique, or its combination and being prepared.

This nucleic acid can comprise the sequence except that polynucleotide of the present invention easily.For example, the multiple clone site that comprises one or more endonuclease restriction sites can be inserted in this nucleic acid, to help separating these polynucleotide.Equally, but translation sequences can be inserted in this nucleic acid, to help separating translation polynucleotide of the present invention.For example, six histidine mark sequences provide purification the present invention the proteinic approach that makes things convenient for.Nucleic acid of the present invention randomly can be used as the carrier, conjugant or the joint that are used to clone and/or express polynucleotide of the present invention except coded sequence.

Other sequence also can be added into above-mentioned clone and/or expressed sequence, optimizing it in the function aspect clone and/or the expression, to help separating polynucleotide of the present invention, or impels polynucleotide of the present invention to be imported into cell.The purposes of cloning vehicle, expression vector, conjugant and joint all is well known in the art.See for example above-mentioned Ausubel; Or above-mentioned Sambrook.

Make up the recombination method of nucleic acid.Utilize any kind of cloning process well known by persons skilled in the art, all can from biological origin, obtain isolating nucleic acid compositions of the present invention, such as RNA, cDNA, genomic DNA or its combination in any.In some embodiment, adopted under suitable stringency can with the oligonucleotide probe of polynucleotide selective cross of the present invention, in cDNA or genome dna library, to identify needed sequence.The structure of the separation of RNA, cDNA and genomic library all is that those of ordinary skills know.(see for example above-mentioned Ausubel; Or above-mentioned Sambrook).

Be used to make up the synthetic method of nucleic acid.Isolating nucleic acid of the present invention also can by the preparation of known direct chemical synthetic method (see, for example above-mentioned Ausubel, etc.).Chemosynthesis generates single stranded oligonucleotide usually, by with complementary sequence hybridization, or by being template, adopt archaeal dna polymerase to carry out polymerization with this strand, this single stranded oligonucleotide can be converted into double-stranded DNA.This area any technical staff all will appreciate that, though the chemosynthesis of DNA can be limited to about 100 or the sequence of polybase base more, longer sequence can obtain by the connection of shorter sequence.

Recombinant expression cassettes.The present invention further provides the recombinant expression cassettes that comprises nucleic acid of the present invention.Nucleotide sequence of the present invention, for example the cDNA or the genome sequence of code book invention GLP-1 analogue body or specific part or variant can be used to make up specific recombinant expression cassettes, and this box can be imported in a kind of at least ideal host cell.Recombinant expression cassettes will typically comprise can be operatively connected the polynucleotide of the present invention of transcribing starting adjusting sequence to specific, and wherein this is transcribed starting adjusting sequence and will guide polynucleotide of the present invention transcribing in specifying host cell.Allos and non-allos (promptly endogenous) promoter all can be used to guide expression of nucleic acids of the present invention.

In certain embodiments, the isolating nucleic acid that serves as promoter, enhancer or other element can be imported on the correct position of the non-allos form of the present invention polynucleotide (in upstream, downstream or the intron), regulates the expression of polynucleotide of the present invention with plus or minus.For example, as known in the art, endogenesis promoter can be by sudden change, disappearance and/or displacement in vivo or external being changed.Polynucleotide of the present invention can have justice or antisense orientation to be expressed by specified.Should be understood that, can directly influence observable feature the control that justice or antisense orientation gene expression are arranged.The another kind of method that suppresses is to have justice to suppress.The nucleic acid that importing is set to sense orientation has been proved to be to block a kind of effective way that target gene is transcribed.

Carrier and host cell.The present invention also relates to comprise the carrier of isolated nucleic acid molecule of the present invention, via the host cell of this recombinant vector genetic modification, and by the preparation of recombinant technique known in the art at least a GLP-1 analogue body or specific part or variant.See, for example above-mentioned Sambrook, etc.; Above-mentioned Ausubel, etc., all herein as a reference by complete introducing.

Polynucleotide of the present invention can randomly be connected to specific support, but this carrier contains the selected marker that is useful in host's internal breeding.Usually, adopt suitable known method, with similarly other known method is with the plasmid vector transfered cell, other known method comprises that utilization can be used as sedimentary carrier such as electroporation, as the calcium phosphate precipitation thing, or form the carrier of complex with electrically charged lipid.If this carrier is a virus, can adopts suitable package cell line with its external packing, and subsequently it be imported host cell.

DNA inserts should be connected to being operated property suitable promoter.This expression construct can further contain and optional be used at least a transcription initiation, termination, and is positioned at the site of transcriptional domain, and the ribosome binding site that is used to translate.The termination codon that the coded portion of the expressed mature transcript of this construct will preferably include the translation initiation codon of beginning and suitably be positioned at mRNA end to be translated (for example, UAA, UGA or UAG), UAA and UAG are preferred for mammal or eukaryotic cell expression.

Expression vector can be preferred, but but also can randomly comprise at least a selected marker.Such labelling comprises, for example, but is not limited only to methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Patent number 4,399,216 that eukaryotic cell is cultivated; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin (G418), mycophenolic acid or glutamine synthetase (GS, U.S. Patent number 5,122,464; 5,770,359; 5,827,739) tetracycline or the ampicillin resistance gene (above-mentioned patent all by complete introducing herein as a reference) cultivated at E.coli and other antibacterial or prokaryote of resistant gene and being used for.The suitable culture medium and the condition that are used for above-mentioned host cell are known in the art.Suitable carriers should be that those of skill in the art know.With the vector construction body import transfection that host cell can be by calcium phosphate transfection, the mediation of DEAE-glucosan, cation lipid mediation transfection, electroporation, transduce, infect or other known method is realized.This area is such as above-mentioned Sambrook, 1-4 and 16-18 chapter; Above-mentioned Ausubel has described these methods in 1,9,13,15,16 chapters.

At least a GLP-1 analogue body of the present invention or specific part or variant all can be expressed by modified forms, such as fused protein, and can not only comprise secretion signal, also comprise other allos functional areas.For example, the additional aminoacid in a zone, especially charge residue can be added into the N-terminal of GLP-1 analogue body or specific part or variant, thereby improves this GLP-1 analogue body or specific part or variant in host cell, during the purification or stability and persistency between subsequent treatment and storage life.Equally, GLP-1 analogue body of the present invention or specific part or variant also can add peptide moiety, thereby help its purification.Before final preparation GLP-1 analogue body or its at least one fragment, above-mentioned zone can be removed.Many standard laboratory handbooks, such as above-mentioned Sambrook, 17.29-17.42 and 18.1-18.74 chapter; Above-mentioned Ausubel has all described said method in 16,17 and 18 chapters.

Those of ordinary skill in the art knows and can be used for expressing many expression systems that code book is invented proteinic nucleic acid.

[101] be mammalian cell to the useful cell culture example of the preparation of analogue body of the present invention, specific part or its variant.The mammal cell line system is generally the cell monolayer form, but also can adopt mammalian cell suspension or bioreactor.But this area has been developed a large amount of suitable host cell lines that the The expressed glycosylated protein, comprise COS-1 (for example ATCCCRL-1650), COS-7 (for example ATCC CRL-1651), HEK293, BHK21 (for example ATCC CRL-10), (for example ATCC CRL 1610 for CHO, DG-44) and BSC-1 (for example ATCC CRL-26) cell line, the hepG2 cell, P3X63Ag8.653, SP2/0-Ag14,293 cells, HeLa cell etc., all can be easily from for example, AmericanType Culture Collection (U.S. typical case DSMZ), Manassas, Va obtains.Preferred host cell comprises the cell in lymph source, such as myeloma and lymphoma cell.Especially preferred host cell is P3X63Ag8.653 cell (ATCC numbers CRL-1580) and SP2/0-Ag14 cell (ATCC numbers CRL-1851).

The expression vector that is used for above-mentioned cell can comprise one or more following expression control sequencs, for example, but is not limited only to origin of replication; Promoter (for example latter stage or early stage SV40 promoter, CMV promoter (for example U.S. Patent number 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglyceric kinase) promoter, EF-1 α promoter (for example U.S. Patent number 5,266,491), at least a humen immunoglobulin storter); Enhancer and/or machining information site, for example ribosome binding site, RNA splice site, polyadenylation site (for example the big T Ag of SV40 polyadenylic acid adds the angle of striking) and tanscription termination subsequence.See, for example, above-mentioned Ausubel etc.; Above-mentioned Sambrook, etc..To nucleic acid of the present invention or other useful cell of proteinic preparation all is known, and/or can from, for example American TypeCulture Collection Catalogue of Cell Lines and Hybridomas (U.S. typical case DSMZ's cell line and hybridoma catalogue) (www.atcc.org) or other known or commercial source obtain.

When using eukaryotic host cell, be that polyadenylation or transcription terminator typically are integrated in the above-mentioned carrier.An example of terminator sequence is the polyadenylation sequence in bovine growth hormone gene source.Also can comprise the sequence that is used for accurate montage transcript.An example of montage sequence be SV40 source the VP1 intron (Sprague, etc., J.Virol.45:773-781 (1983)).In addition, as known in the art, the gene order of duplicating in the control host cell can be integrated in the above-mentioned carrier.

The purification of GLP-1 analogue body or specific part or its variant.GLP-1 analogue body or specific part or variant can be by the methods of knowing, comprise, but be not limited only to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and agglutinin chromatograph, from reconstitution cell, reclaimed and purification.Also can use high performance liquid chromatography (" HPLC ") and carry out purification.See Colligan for example, Current Protocols in Immunology, or Current Protocolsin Protein Science, John Wiley ﹠amp; Sons, NY, NY, (1997-2002), 1,4,6,8,9,10 chapters for example, all herein as a reference by complete introducing.

Analogue body of the present invention or specific part or variant comprise natural purification product, chemosynthesis operation product and by recombinant technique from eukaryotic cell, comprise the product that obtains in for example yeast, higher plant, insecticide and the mammalian cell.According to the host who uses in the reorganization preparation manipulation, GLP-1 analogue body of the present invention or specific part or variant can be glycosylations or nonglycosylated, and be preferably glycosylated.Many standard laboratory handbooks, such as above-mentioned Sambrook, the 17.37-17.42 joint; Above-mentioned Ausubel, 10,12,13,16,18 and 20 chapters, above-mentioned Colligan, Protein Science has all described said method in the 12-14 chapter, all by complete introducing herein as a reference.

Analogue body, specific fragment and/or variant.Separation simulation body of the present invention comprises GLP-1 analogue body or the specific part or the variant of any one polynucleotide encoding of the present invention of more complete argumentation from here, or the GLP-1 analogue body of any separation or preparation or specific part or its variant.

Preferred GLP-1 analogue body or ligand binding moiety or variant combine at least one GLP-1 protein ligands, thereby and provide respective egg white matter or its segmental at least a GLP-1 biologic activity.Multiple all is well known in the art at the protein that has an important function aspect treatment or the diagnosis, and this proteinic suitable assay method or biologic activity also are well known in the art.

For the purpose of the present invention, the limiting examples of suitable GLP-1 peptide, variant or derivant is as follows: SEQ ID NO:1:His-Xaa2-Xaa3-Gly-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xa a11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa2 0-Xaa21-Phe-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xa a30-Xaa31, wherein: Xaa2 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa3 is Glu, Asp, or Lys; Xaa5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Arg, His, Glu, Asp, or Lys; Xaa6 is Phe, His, Trp, or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa9 is Asp or Glu; Xaa10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Met, Tyr, Trp, His, Phe, Glu, Asp, or Lys; Xaa11 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp, or Lys; Xaa12 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp or Lys; Xaa13 is Tyr, Phe, Trp, Glu, Asp or Lys; Xaa14 is Leu, Ala, Met, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp or Lys; Xaa15 is Glu, Ala, Thr, Ser, Gly, Gln, Asp or Lys; Xaa16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Gln, Asn, Arg, Cys, Glu, Asp or Lys; Xaa17 is Gln, Asn, Arg, His, Glu, Asp or Lys; Xaa18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp or Lys; Xaa19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Met, Glu, Asp or Lys; Xaa20 is Lys, Arg, His, Gln, Trp, Tyr, Phe, Glu or Asp; Xaa21 is Glu, Leu, Ala, His, Phe, Tyr, Trp, Arg, Gln, Thr, Ser, Gly, Asp or Lys; Xaa23 is Ile, Ala, Val, Leu or Glu; Xaa24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, His, Glu, Asp or Lys; Xaa25 is Trp, Phe, Tyr, Glu, Asp or Lys; Xaa26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp or Lys; Xaa27 is Val, Leu, Gly, Ala, Ser, Thr, Ile, Arg, Glu, Asp or Lys; Xaa28 is Lys, Asn, Arg, His, Glu or Asp; Xaa29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Arg, Trp, Tyr, Phe, Pro, His, Glu, Asp or Lys; Xaa30 is Arg, His, Thr, Ser, Trp, Tyr, Phe, Glu, Asp or Lys; With Xaa31 be Gly, Ala, Ser, Thr, Leu, IIe, Val, Arg, Trp, Tyr, Phe, His, Glu, Asp, Lys.

Another example of organizing preferred GLP-1 peptide, variant or derivant is as follows: SEQ ID NO:6:His-Xaa2-Xaa3-Gly-Thr-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Ser-Xaa12-Tyr-Xaa14-Glu-Xaa16-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Ph e-Xaa23-Ala-Trp-Leu-Xaa27-Xaa28-Gly-Xaa30, wherein: Xaa2 is Ala, Gly, or Ser; Xaa3 is Glu or Asp; Xaa6 is Phe or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Thr or Ala; Xaa9 is Asp or Glu; Xaa10 is Val, Leu, Met or Ile; Xaa12 is Ser or Lys; Xaa14 is Leu, Ala or Met; Xaa16 is Gly, Ala, Glu or Asp; Xaa17 is Gln or Glu; Xaa18 is Ala or Lys; Xaa19 is Ala, Val, Ile, Leu or Met; Xaa21 is Glu or Leu; Xaa23 is Ile, Ala, Val, Leu or Glu; Xaa27 is Val or Lys; Xaa28 is Lys or Asn; With Xaa30 be Arg or Glu.

These peptides can be by open and/or methods known in the art preparation.In these sequences (and in the whole text in this description, in particular instance as do not have do not indicate in addition) Xaas comprise the specified amino acid residues or derivatives thereof, or its modified aminoacid.Because enzyme, DPP IV (DPP-IV) may be responsible for the interior inactivation of the rapid body that observes of GLP-1, so preferably be not subjected to GLP-1 peptide, homologue, analog and the derivant of DPP-IV activity influence in the analogue body scope.

GLP-1 analogue body or specific part or its variant part ground or at least a GLP-1 biological activity that preferably provides basically, it can be in conjunction with the GLP-1 part, therefore a kind of activity is provided, this activity is by making GLP-1 and at least a part, GLP-1 receptors bind for example, or rely on or mediation mechanism and being mediated in addition by other protein.Term used herein " GLP-1 analogue body activity " refers to according to measuring approximately 20-10,000%, preferably with about at least 60,70,80,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,250,300,350,400,450,500,550,600,700,800,900,1000,2000,3000,4000,5000,6000,7000,8000,9000% or regulate more at high proportion or cause that at least a GLP-1 relies on active GLP-1 analogue body.

GLP-1 analogue body or specific part or variant can provide at least a protein to rely on active ability and preferably be evaluated by at least a suitable protein biology mensuration described herein and/or known in the art.Human glp-1 mimetibodies of the present invention or specific part or variant can be similar to the immunoglobulin (IgG, IgA, IgM etc.) or the isotype of any kind of, and can comprise the k or the lambda light chain of at least one part.In a kind of embodiment, human glp-1 mimetibodies or specific part or variant have comprised IgG weight chain variable fragment, hinge region, CH2 and CH3, at least a isotype, for example, IgG1, IgG2, IgG3 or IgG4.

At least a GLP-1 analogue body of the present invention or specific part or variant combine at least a specific ligand, subunit, fragment, part or this combination in any of several.At least a GLP-1 peptide, variant or the derivant of at least a GLP-1 analogue body of the present invention, specific part or variant be at least one specific ligand epi-position of binding partner randomly.This can comprise the combination in any of at least a specific amino acids sequence in conjunction with epi-position, this specific amino acids sequence comprises the complete specific part of the aminoacid of 1-3 at least of protein ligands until the continuous amino acid of protein ligands sequence, and this protein ligands is such as one of GLP-1 receptor or its part.

This analogue body can be by following method preparation, promptly adopt known technology that the different piece of the GLP-1 analogue body of molecular formula (I) is linked together, by adopting at least a nucleic acid molecules of known recombinant DNA technology preparation and this GLP-1 analogue body of expression coding, or by using other any appropriate method, such as chemical synthesis.

Adopt appropriate method, such as phage display (Katsube, Y., Deng., Int.J Mol.Med, 863-868 (1998)) or the method for applying transgene animal known in the art and/or described herein 1 (5):, can prepare part with people GLP-1, receptors bind for example, and comprise the analogue body of determining heavy or variable region of light chain or its part.In the suitable host cell, just can express this GLP-1 analogue body, specific part or variant by utilizing GLP-1 analogue body, specific part or variant code nucleic acid or its part.

The present invention also relates to analogue body, part binding fragment and immunoglobulin chain, it comprises and the essentially identical aminoacid sequence of aminoacid sequence described herein.Preferably, this analogue body or its part binding fragment can with people GLP-1 part, for example receptor high-affinity ground is in conjunction with (for example, being less than or equal to about 10^-7The K of M_D).Comprise with the essentially identical aminoacid sequence of sequence described herein and to contain conservative amino acid replacement, and the sequence of aminoacid deletion and/or insertion.Conservative amino acid replacement refers to that first aminoacid is had second amino acid replacement of chemistry similar to it and/or physical characteristic (for example electric charge, structure, polarity, hydrophobicity/hydrophilic).Conservative substitution is included in down in the group aminoacid scope, and a seed amino acid is by another kind of amino acid replacement, that is: lysine (K), arginine (R) and histidine (H); Aspartic acid (D) and glutamic acid (E); Agedoite (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; Alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino acid code.Forming the aminoacid of analogue body of the present invention or specific part or variant is abridged usually.By indicating this aminoacid by amino acid whose single-letter code, trigram code, title or three nucleotide codons well known in the art, just can express this aminoacid title and (see Alberts, B., Deng., Molecular Biology of The Cell, Third Ed., GarlandPublishing, Ino., New York, 1994):

Single-letter code trigram name of code trinucleotide codon

A Ala alanine GCA, GCC, GCG, GCU

C Cys cysteine UGC, UGU

D Asp aspartic acid GAC, GAU

E Glu glutamic acid GAA, GAG

F Phe phenylalanine UUC, UUU

G Gly glycine GGA, GGC, GGG, GGU

H His histidine CAC, CAU

I Ile isoleucine AUA, AUC, AUU

K Lys lysine AAA, AAG

UUA，UUG，CUA，CUC，

L Leu leucine

CUG，CUU

M Met methionine AUG

N Asn agedoite AAC, AAU

P Pro proline CCA, CCC, CCG, CCU

Q Gln glutamine CAA, CAG

AGA，AGG，CGA，CGC，

R Arg arginine

CGG，CGU

AGC，AGU，UCA，UCC，

S Ser serine

UCG，UCU

T Thr threonine ACA, ACC, ACG, ACU

V Val valine GUA, GUC, GUG, GUU

W Trp tryptophan UGG

Y Tyr tyrosine UAC, UAU

GLP-1 analogue body of the present invention or specific part or variant can comprise from natural sudden change or manually-operated one or more amino acid replacements, disappearance or interpolation as illustrate herein.Use in the present invention such or other sequence comprise, but be not limited only to the following sequence shown in the table 1, as shown in it, its specific part with SEQ ID NOS:47-64 is corresponding, wherein the part variable region of antibody sequence can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:47-55, its fragment perhaps as shown in table 1, further selectively comprise at least one displacement of the corresponding SEQ ID NO:1-9 shown in open text WO05/05604 (PCT US04/19898) Fig. 1-9 of PCT, insert or disappearance, this application was submitted on June 24th, 2004, and is open on January 20th, 2005.CH2 district, CH3 district and hinge region can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:56-64, its fragment perhaps as shown in table 1, further selectively comprise at least one displacement, insertion or disappearance as the corresponding SEQID NO:32-40 of the open text WO05/05604 (PCT US04/19898) of PCT, this application was submitted on June 24th, 2004, and is open on January 20th, 2005.Certainly, the amino acid replacement number that those of skill in the art can carry out depends on many factors, comprises above-described those factors.In general, the amino acid replacement of at least a GLP-1 analogue body, insertion or disappearance number will be no more than 40,30,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1 aminoacid, as 1-30 or any range wherein or numerical value, as explanation herein.

Formula I of the present invention ((P (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t) in, V part according to formula I, the H part, the CH2 part, the CH3 part can be any suitable human sequence or the compatible sequence of people, for example, as shown in table 1, wherein the part variable region of antibody sequence can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:47-55, its fragment perhaps as shown in table 1, further selectively comprise at least one displacement of the corresponding SEQ ID NO:1-9 shown in open text WO 05/05604 (PCT US04/19898) Fig. 1-9 of PCT, insert or disappearance, this application was submitted on June 24th, 2004, and is open on January 20th, 2005.The CH2 district, CH3 district and hinge region can be, but be not limited only at least one part of at least a aminoacid sequence among the SEQ ID NO:56-64, its fragment perhaps as shown in table 1, further selectively comprise at least one displacement as the corresponding SEQ IDNO:32-40 of the open text WO 05/05604 (PCT US04/19898) of PCT, insert or disappearance, this application was submitted on June 24th, 2004, open on January 20th, 2005, or its combination or consensus sequence, or its fusion rotein, when being used for human body preferred people source or design immunogenicity is dropped to minimum.

P partly comprises at least a GLP-1 therapeutic peptide known in the art or described herein, for example but be not limited only to the sequence shown in the SEQ ID NO:1, or its combination or consensus sequence, or its fusion rotein.In a preferred embodiment, the P part can comprise at least a GLP-1 peptide, and it has at least a sequence of SEQ ID NO:6, or its combination or consensus sequence, or its fusion rotein.

Optionally joint sequence can be any suitable peptide linker known in the art.Preferred sequence comprises any combination of G and S, X1-X2-X3-X4-...-Xn for example, and wherein X can be G or S, n can be 5-30.Limiting examples comprises GS, GGS, GGGS (SEQID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ IDNO:20) etc.

Requisite aminoacid concerning function in GLP-1 analogue body of the present invention or specific part or the variant can be differentiated by methods known in the art, and for example rite-directed mutagenesis, alanine detect mutation (Ausubel for example, supra, Chapters8,15; Cunningham and Wells, Science 244:1081-1085 (1989)).A kind of method in back is carried out single alanine mutation on each residue in molecule.Measure the biological activity of the mutant molecule obtain then, for example but that be not limited only to describe in detail or at least a albumen related activity known in the art herein.To differentiating of GLP-1 analogue body or specific part or variant in conjunction with crucial site also possible constructions analysis, for example crystallization, nuclear magnetic resonance, NMR or photoaffinity labeling (Smith, Deng., J.Mol.Biol.224:899-904 (1992) and de Vos, Deng., Science 255:306-312 (1992)).

Analogue body of the present invention or specific part or variant can comprise the P part of molecular formula (I), for example but be not limited only at least one at least a in SEQ ID NOS:1 and 6 part.GLP-1 analogue body or specific part or variant can further comprise with SEQ ID NOS:1 and 6 at least a P part that at least one funtion part of at least one polypeptide of 90-100% homology is at least arranged as molecular formula (I).Can strengthen or keep at least a active non-limiting variant as listed above and comprise, but be not limited only to any above polypeptide, further comprise with at least a displacement, insertion or lack corresponding at least a sudden change, described displacement, insertion or lack the biological activity or the function that are fit to of the described GLP-1 analogue body of not obvious influence.

In a kind of embodiment, the corresponding aminoacid sequence of the appropriate section of at least one sequence has about 90-100% homogeneity (promptly 90,91,92,93,94,95,96,97,98,99,100 or any range wherein or numerical value) in P aminoacid sequence or its part and SEQ ID NO:1 and 6.Preferably, utilize suitable computerized algorithm known in the art to determine 90-100% aminoacid homogeneity (promptly 90,91,92,93,94,95,96,97,98,99,100 or any range or numerical value wherein).

Analogue body of the present invention or specific part or variant can comprise any amount continuous amino acid residue that derives from GLP-1 analogue body of the present invention or specific part or variant, and wherein this quantity is selected from by continuous one of group of integers of forming of the 10-100% of residue quantity in the GLP-1 analogue body.Randomly, the subsequence of continuous amino acid contains about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250 or amino acids more at least on length, or contains the aminoacid of any range wherein or numerical value.In addition, the quantity of this subsequence can be the arbitrary integer that is selected from 1-20, such as at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or bigger integer.

The technical staff it should be understood that GLP-1 analogue body of the present invention or specific part or the variant that the present invention includes at least a biologically active.The given activity of the analogue body of biologically active or specific part or variant is active at least 20%, 30% or 40% of natural (non-synthetic), endogenous or relevant and known insertion or fusion rotein or specific part or a variant, be preferably at least 50%, 60% or 70%, most preferably at least 80%, 90% or 95%-1000%.The method of analysis and quantitation of enzyme activity and substrate specificity is well known to those skilled in the art.

On the other hand, the present invention relates to described hereinly add organic moiety and adorned human simulation body and part binding fragment by covalency.This modification can produce and have the improvement pharmacokinetic properties GLP-1 analogue body or the part binding fragment of (for example serum half-life in the body of Yan Changing).Organic moiety can be line style or branch's hydrophilic polymeric group, fatty acid group or fatty acid ester group.In the particular, the hydrophilic polymeric group can have about 800-about 120,000 daltonian molecular weight, and can be polyalkane glycol (for example Polyethylene Glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinylpyrrolidone, and fatty acid or fatty acid ester group can comprise about 40 carbon atoms of about 8-.

Modification analogue body of the present invention and part binding fragment can comprise one or more and GLP-1 analogue body or specific part or the covalently bound directly or indirectly organic moiety of variant.With GLP-1 analogue body of the present invention or bonded each organic moiety of part binding fragment can independently be hydrophilic polymeric group, fatty acid group or fatty acid ester group all.Term " fatty acid " used herein " comprise mono carboxylic acid and two carboxylic acids.Term used herein " hydrophilic polymeric group " refers to organic polymer more easily molten in water than in octane.For example, polylysine is easier to be molten in water than in octane.Therefore, the present invention includes the GLP-1 analogue body of being modified by covalency interpolation polylysine.Be applicable to that the hydrophilic polymer of modifying analogue body of the present invention can be line style or branch-like, and comprise, for example the polymer (for example polylysine, poly arginine, poly-aspartate etc.) of polyalkane glycol (for example PEG, mono methoxy-Polyethylene Glycol (mPEG), PPG etc.), carbohydrate (for example glucosan, cellulose, oligosaccharide, polysaccharide etc.), hydrophilic amino acid, poly-epoxyalkane (for example poly(ethylene oxide), poly(propylene oxide) etc.) and polyvinylpyrrolidone.Preferably, when the hydrophilic polymer of modifying GLP-1 analogue body of the present invention is the unimolecule entity form, have about 800-about 150,000 daltonian molecular weight.For example, can adopt PEG₂₅₀₀, PEG₅₀₀₀, PEG₇₅₀₀, PEG₉₀₀₀, PEG₁₀₀₀₀, PEG₁₂₅₀₀, PEG₁₅₀₀₀And PEG_20,000,, wherein be designated as dalton's mean molecule quantity of this polymer down.

The hydrophilic polymeric group can be by 1 to about 6 alkyl, fatty acid or fatty acid ester group displacement.Can be by fatty acid or the metathetical hydrophilic polymer of fatty acid ester group by using suitable method preparation.For example, the polymer that comprises amine groups can be coupled on the carboxyl of fatty acid or fatty acid ester, and the activated carboxyl on fatty acid or the fatty acid ester (for example via N, the activation of N-carbonyl dimidazoles) can be coupled to the oh group on the polymer.

Be applicable to that fatty acid and the fatty acid ester of modifying analogue body of the present invention can be saturated one or more unsaturated units that maybe can contain.Be applicable to that the fatty acid of modifying analogue body of the present invention comprises, for example, n-dodecylic acid (C₁₂, lauric acid), n-four capric acid (C₁₄, myristic acid), n-eight capric acid (C₁₈, stearic acid), n-arachic acid (C₂₀, arachic acid), n-behenic acid (C₂₂, mountain Yu acid), n-melissic acid (C₃₀), n-tetracontane acid (C₄₀), suitable-Δ 9-octadecanoid acid (C₁₈, oleic acid), all suitable-Δs 5,8,11,14-nervonic acid methyl ester (C₂₀, arachidonic acid), suberic acid, four decanedioic acid, eight decanedioic acid, docosandioic acid etc.The suitable fatty acids ester comprises the monoesters of the dicarboxylic acids that contains line style or branch's low-grade alkyl group.Can comprise 1 to about 12 than low-grade alkyl group, preferred 1 to about 6 carbon atoms.

Human simulation body of modifying and part binding fragment can adopt such as, by with the appropriate method preparation of one or more dressing agents reactions.Term used herein " dressing agent " refers to comprise the suitable organic group (for example hydrophilic polymer, fatty acid, fatty acid ester) of activated group." activated group " refers to can be under proper condition and the reaction of second chemical group, thus between above-mentioned dressing agent and second chemical group chemical part or the functional group of formation covalent bond.For example, the reactive activated group of amine comprises electrophilic group, such as tosylate, methanesulfonates, halogenation (chlorine, bromine, fluorine, iodine), N-hydroxyl succinimide ester (NHS) etc.Can comprise with the activated group of thiol reactant, for example maleimide, iodacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid mercaptan (TNB-mercaptan) etc.Aldehyde functional group group can be coupled to contain amine-or the molecule of hydrazides on, and azido group can with the three valent phosphors radical reaction, thereby form phosphoramidate or phosphamide key.The appropriate method that activated group is imported molecule is (seeing Hermanson for example, G.T., Bioconjugate Techniques, AcademicPress:San Diego, CA (1996)) known in the art.Activated group can directly be bonded to organic group (for example hydrophilic polymer, fatty acid, fatty acid ester), or by blank area, for example bivalence C₁-C₁₂Group is realized combination, and wherein one or more carbon atoms can be by the hetero atom displacement such as oxygen, nitrogen or sulfur.Suitable blank area comprises, for example TEG ,-(CH₂)₃-,-NH-(CH₂)₆-NH-,-(CH₂)₂-NH-and-CH₂-O-CH₂-CH₂-O-CH₂-CH₂-O-CH-NH-.The dressing agent that comprises blank area can be by following method preparation, for example make single Boc (tert-butyl group)-alkyl diamine (for example single Boc-ethylene diamine, single Boc-diamino hexane) under the existence condition of 1-ethyl-3-(3-two methyl aminopropyls) carbodiimides (EDC) and fatty acid response, thereby between free amine and fatty acid carboxylate, form amido link.Can from product, remove the Boc blocking group by following method; promptly adopt trifluoroacetic acid (TFA) to handle; with expose can with described another carboxylate coupling; or can with the primary amine of maleic anhydride reaction, and make the obtained product cyclisation, generate the maleimide derivatives that this fatty acid is activated and (see; Thompson for example; Deng., WO 92/16221, and its complete content is incorporated herein by refere).

Modification analogue body of the present invention can be generated by making the reaction of human glp-1 mimetibodies or part binding fragment and dressing agent.For example, by using the reactive dressing agent of amine, for example the NHS ester of PEG can not have the locus specificity mode organic moiety is bonded to the GLP-1 analogue body.Human simulation body of modifying or part binding fragment also can be prepared by the disulfide bond (for example intrachain disulfide bond) of reduction GLP-1 analogue body or part binding fragment.Subsequently, can make the reaction of reductive GLP-1 analogue body or part binding fragment and thiol-reactive dressing agent, to obtain modification GLP-1 analogue body of the present invention.By adopting appropriate method, such as reverse Proteolytic enzyme (Fisch etc., Bioconjugate Chem., 3:147-153 (1992); Werlen etc., BioconjugateChem., 5:411-417 (1994); Kumaran etc., Protein Sci.6 (10): 2233-2241 (1997); Itoh etc., Bioorg.Chem., 24 (1): 59-68 (1996); Capellas etc., Biotechnol.Bioeng., 456-463 (1997)) and Hermanson 56 (4):, G..T., Bioconjugate Techniques, Academic Press:San Diego, the described method of CA (1996), can prepare the modification human simulation body and the part binding fragment that contain specific organic moiety, wherein this specific organic moiety is incorporated in to the specific site of GLP-1 analogue body of the present invention or specific part or variant.

GLP-1 analogue body compositions.The present invention also provides at least a GLP-1 analogue body or specific part or variant compositions, said composition has comprised described herein and/or known in the art, at least a, at least two kinds, at least three kinds, at least four kinds, at least five kinds, at least six kinds or more analogue bodies or specific part or its variant that exist compositions, mixture or form to be provided with non-natural.The ratio of said composition is by weight, volume, concentration, molar concentration, and the molar concentration of liquid known in the art or described herein or dry solution, mixture, suspension, Emulsion or colloidal form is represented.

Said composition can comprise the weight of 0.00001-99.9999%, volume, concentration, molar concentration liquid known in the art or described herein, gas or dry solution, mixture, suspension, the molar concentration of Emulsion or colloidal form, or any range wherein or numerical value, for example, but be not limited only to 0.00001,0.00003,0.00005,0.00009,0.0001,0.0003,0.0005,0.0009,0.001,0.003,0.005,0.009,0.01,0.02,0.03,0.05,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.3,4.5,4.6,4.7,4.8,4.9,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,99.1,99.2,99.3,99.4,99.5,99.6,99.7,99.8,99.9%.Therefore, said composition of the present invention include but not limited to 0.00001-100mg/ml and/or 0.00001-100mg/g.

Said composition can randomly further comprise and is selected from least a diabetes or insulin metabolism related drugs, anti-infectives, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomic nervous system (ANS) medicine, respiratory drugs, gastrointestinal (GI) tract drug, hormonal medicaments, the medicine that is used for body fluid or electrolyte balance, blood substance, antitumor, immunoregulation medicament, eye, ear or nose medication, local application, at least a chemical compound or the protein of effective amount of drug such as nutritional drugs.Said medicine all is well known in the art, comprises preparation, indication, the dosage of listed each medicine herein and uses and (see, for example Nursing 2001 Handbookof Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional ' s Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; PharmcotherapyHandbook, Wells etc., ed., Appleton; Lange, Stamford, CT, all herein as a reference) by complete introducing.

The medicine relevant with diabetes can be at least a in the following medicine: glitazone; insulin and derivant; sulfonylurea; meglitinides; biguanide; alpha-glucosidase inhibitor; Protein-tyrosine-phosphatase-1B; GSK3; the glyconeogenesis inhibitor; pyruvic dehydrogenase kinase (PDH) inhibitor; the steatolysis inhibitor; the fat oxidation inhibitor; carnitine palmitoyltransferase I and/or II inhibitor; β-3 adrenoreceptor effectual drug; sodium and glucose symporter (SGLT) inhibitor or have following one or more effects, the chemical compound of at least a effect: autoimmune suppresses; immunomodulating; activation; propagation; the function of the migration of T-cell and/or inhibition cell; the removal of the T cell of id reaction; cross over the minimizing of blood brain barrier; the equilibrated change of proinflammatory (Th1) and immunomodulatory (Th2) cytokines; the inhibition of matrix metallo-proteinase inhibitor; neuroprotective; the minimizing of gliosis; the promotion that myelin forms again.

Anti-infectives can be at least a medicine that is selected from amebacide or at least a antiprotozoal, anthelmintic, antifungal agent, antimalarial, antitubercular agent or at least a antileprotic, aminoglycoside medicine, penicillin, cephalosporin, tetracycline, sulfanilamide, fluoroquinolone, antiviral agents, macrolide anti-infective, the comprehensive anti-infective.The CV medicine can be at least a medicine that is selected from variable force medicine, antiarrhythmics, anti-angina pectoris medicine, antihypertensive, antilipemic, the comprehensive cardiovascular drugs.The CNS medicine can be at least a medicine that is selected from non-narcotic analgesics, or be selected from least a medicine in antipyretic, on-steroidal anti-inflammatory medicaments, the anesthetics, or at least a opium sample analgesic, calmness-hypnotic, anticonvulsant, antidepressants, antianxiety drugs, psychosis, central nervous system's stimulant, antiparkinsonism drug, comprehensive medicine for central nervous system.The ANS medicine can be at least a medicine that is selected from cholinergic drug (parasympathomimetic agent), anticholinergic, beta adrenergic agent (sympathomimetic), adrenergic blocking drug (sympatholytic), skeletal muscle relaxant, the neuromuscular blocking agents.Respiratory drugs can be at least a medicine that is selected from hydryllin, bronchodilatation medicine, the expectorant or at least a cough medicine, comprehensive respiratory medications.The GI tract drug can be at least a medicine that is selected from antacid, or at least a adsorbent, or at least a antiflatulent, digestive enzyme or at least a cholelithiasis solubilizing agent, diarrhea, laxative, antiemetic, antiulcerative.Hormonal medicaments can be at least a medicine that is selected from corticosteroids, the androgen, or at least a anabolic steroids, estrogen or at least a progesterone, promoting sexual gland hormone, antidiabetic medicine or at least a glucagon, thyroid hormones, thyroid hormones antagonist, pituitary hormone, class parathyroid gland medicine.The medicine that is used for balance body fluid and electrolyte can be at least a medicine or at least a substitutional solution, acidulant or at least a basifier that is selected from diuretic, electrolyte.Blood substance can be at least a medicine that is selected from hematonic, anticoagulant, blood derivatives, the streptokinase.Antitumor drug can be the antitumor drug that is selected from alkylation medicine, antimetabolite, antibiotics antitumor drug, can changes hormonal balance, at least a medicine in the comprehensive antitumor drug.Immunoregulation medicament can be at least a medicine that is selected from immunosuppressant, the vaccine, or at least a toxoid, antitoxin or at least a venom, immune serum, biological respinse modifier.Eye, ear and nose medication can be at least a medicine that is selected from ophthalmology anti-infective, ophthalmology anti-inflammatory agent, miotic, mydriatic, ocular angiogenesis contracting agent, comprehensive eye, ear, the nose medication.Local application can be at least a medicine that is selected from local anti-infective agent, the antiscabietic or at least a antipedicular agent, local corticosteroids.Nutritional drugs can be at least a medicine that is selected from vitamin, mineral or the caloic.See the content among for example above-mentioned Nursing 2001Drug Handbook.

Above-mentioned at least a amebacide or antiprotozoal can be at least a medicine that is selected from atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, hydrochloric acid metronidazole, the pentamidine isethionate.Above-mentioned at least a anthelmintic can be at least a medicine that is selected from mebendazole, pamoic acid quinoline-pyrimidine, the thiabendazole.Above-mentioned at least a antifungal can be at least a medicine that is selected from amphotericin B, amphotericin B cholesteryl sulphate complex, amphotericin B lipid complex, AM Bison, fluconazol, flucytosine, microsize griseofulvin, ultra micro small size griseofulvin, itraconazole, ketoconazole, nystatin, the terbinafine HCl.Above-mentioned at least a antimalarial can be and be selected from chloroquine hydrochloride, chloroquine phosphate, doxycycline, sulphuric acid hydroxylation chloroquine, hydrochloric acid mefloquine, primaquine phosphate, Pyrimethamine, contains at least a medicine in the Pyrimethamine of sulfamethoxine.Above-mentioned at least a antitubercular agent or antileprotic can be at least a medicine that is selected from clofazimine, cycloserine, dapsone, ebutol, isoniazid, pyrazinamide, rifabutin, rifampicin, rifapentine, the streptomycin sulfate.Above-mentioned at least a aminoglycoside medicaments can be at least a medicine that is selected from amikacin sulfate, sulfate gentamicin, polygynax, streptomycin sulfate, the tobramycin sulfate.Above-mentioned at least a penicillin can be and is selected from amoxicillin/potassium clavulanate, BRL-2333, ampicillin, sodium ampicillin, Ampicillin Trihydrate, sodium ampicillin/sulbactam sodium, methocillin-S, dichlorophenyl isoxazolyl penicillin sodium, mezlocillin sodium, sodium ethoxynaphthamidopenicillanate, oxacillin sodium, benzathine penicillin G, scotcil, aquacillin, penicillin G sodium, potassium v calcium, piperacillin sodium, piperacillin sodium/sodium-tazobactam, the ticarcillin disodium, at least a in ticarcillin disodium/potassium clavulanate.Above-mentioned at least a cephalosporin can be and is selected from least a cefaclor, cefadroxil, the cefonicid sodium element, cefdinir, cefepime hydrochloride, cefixime, cyanogen azoles methoxy cefonicid sodium element, cefonicid sodium, oxygen piperazine oxybenzene azoles head sodium, cefotaxime sodium, the Cefotetan disodium, thiophene methoxy cefonicid sodium element, Cefpodoxime Proxetil, cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, CEFUROXIME AXETIL, Cefuroxime Sodium, cefalexin hydrochloride, one hydration cefalexin, Cefradal, at least a in the Loracarbef.Above-mentioned at least a tetracycline can be and is selected from least a in demeclocycline hydrochloride, doxycycline calcium, doxycycline hydrochloride, doxycycline hyclate, a hydration doxycycline, minocycline hydrochloride, the quadracycline.Above-mentioned at least a sulfanilamide can be and is selected from least a in abactrim, sulfadiazine, sulfamethoxazole, sulfafurazole, the acetyl-sulfisoxazole.Above-mentioned at least a fluoroquinolone can be and is selected from least a in my trovafloxacin of methanesulfonic acid, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixan, norfloxacin, ofloxacin, Sparfloxacin, the trovafloxacin mesilate.Above-mentioned at least a antiviral drugs can be and is selected from abacavir sulfate, Acyclovir Sodium, amantadine hydrochloride, amprenavir, cidofovir, methanesulfonic acid dilazep dimension is fixed, didanosine, Yi Feiweilun, famciclovir, Fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/neat many furans pyridine, nelfinavir mesilate, interior Wella is flat, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, the methanesulfonic acid saquinavir, take charge of his furan pyridine, valaciclovir hydrochlordide, zalcitabine, Zha Namiwei, at least a in neat many furans pyridine.Above-mentioned at least a macrolide anti-infective can be and is selected from least a in azithromycin, clarithromycin, dirithromycin, erythromycin substrate, erythromycin propionate lauryl sulfate, SAI NENG SHA, erythromycin lactobionate, the erythromycin octadecanoate.Above-mentioned at least a comprehensive anti-infective can be and is selected from aztreonam, bacitracin, chloromycetin sodium succinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, imipenum.At least a with in Cilastatin Sodium, meropenem, nitrofurantoin megacryst, nitrofurantoin microcrystal, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, the Lyphocin (Fujisawa).(seeing, for example the 24-214 page or leaf among the Nursing 2001Drug Handbook).

Above-mentioned at least a variable force medicine can be and is selected from least a of the upright agricultural middle school of lactic acid AMR, digoxin, lactic acid rice.Above-mentioned at least a anti-arrhythmic can be and is selected from adenosine, Amiodarone Hydrochloride, atropine sulfate, the toluenesulfonic acid bretylium tosylate, the hydrochloric acid DILTIAZEM HCl, norpace (disopyramide), disopyramide phosphate, esmolol hydrochloride, the acetic acid flecainide, Ibolite fumarate, lidocaine hydrochloride, hydrochloric acid Mai Xileiding, Moracizine Hydrochloride, phenytoin, phenytoin Sodium, procamide, propafenone hydrochloride, propranolol hydrochloride, the bisulphate chinidine, the gluconic acid chinidine, the polygalacturonic acid chinidine, quinidine sulfate, Suo Daluo, tocainide hydrochloride, at least a in the hydrochloric acid verapamil.Above-mentioned at least a anti-angina pectoris medicine can be and is selected from least a in Amlodipine Besylate Tablet, amyl nitrite, bepridil hydrochloride, hydrochloric acid DILTIAZEM HCl, isosorbide dinitrate, an isosorbidi dinitras, nadolol, Licardipine Hydrochloride, nifedipine, nitroglycerin, phloride, verapamil, the hydrochloric acid verapamil.Above-mentioned at least a antihypertensive can be and is selected from Acebutolol Hydrochloride, benzenesulfonic acid A Muluohe, atenolol, benazepril hydrochloride, hydrochloric acid Beta Luo Er, the bisoprolol fumarate, candesartan Cilexetil, captopril, the hydrochloric acid galutedin, carvedilol, clonidine, dichloranilino imidazolin, diazoxide, the hydrochloric acid DILTIAZEM HCl, Carclura, enalaprilat, enalapril maleate, the methanesulfonic acid Eprosartan, felodipine, Fenoldopam Mesylate, fosinopril sodium, the acetic acid guanabenz, guanadrel sulfate, hydrochloric acid paddy ammonia process is new, hydralazine hydrochloride, irbesartan, isradipine, Labetalol Hydrochloride, lisinopril, Losartan Potassium, methyldopa, Aldomet Ester Hydrochloride salt, the succinic acid metoprolol, the tartaric acid metoprolol, Minoxidil, CI-925, nadolol, Licardipine Hydrochloride, nifedipine, nisin, Nitroprusside sodium, penbutolol sulfate, perindopril, the fragrant Chlorazene of methanesulfonic acid, pindolol, minipress, propranolol hydrochloride, quinapril hydrochloride, ramipril, telmisartan, terazosin hydrochloride, timolol maleate, trandolapril, valsartan, at least a in the hydrochloric acid verapamil.Above-mentioned at least a antilipemic can be and is selected from least a in Atorvastatin calcium, cerivastatin sodium, Cholestyramine, colestipol hydrochloride, fenofibrate (micronization), Fluvastatin Sodium, gemfibrozil, lovastatin, nicotinic acid, pravastatin sodium, the simvastatin.Above-mentioned at least a comprehensive CV medicine can be and is selected from abciximab., at least a (seeing, for example the 215-336 page or leaf among the Nursing 2001Drug Handbook) in the Alprostadil, hydrochloric acid arbutamine, cilostazol, two Clopidogrel Hydrogensulfates, dipyridamole, Eptifibatide, Midodrine Hydrochloride, pentoxifylline, ticlopidine hydrochloride, tirofiban hydrochloride.

Above-mentioned at least a non-narcotic analgesics or antipyretic can be and be selected from least a in acetaminophen, aspirin, Choline magnesium trisalicylate, diflunisal, the salicylic acid enzyme.Above-mentioned at least a on-steroidal anti-inflammatory medicaments can be and is selected from least a in celecoxib, Ciclofenaziae potassium, Ciclofenaziae sodium, Yi Tuoduo thunder, fenopron, flurbiprofen, ibuprofen, indomethacin, three hydration indomethacin sodium, ketone propanoic acid, ketorolac tromethamine, nabumetone, naproxen, naproxen sodium, promazine, piroxicam, rofecoxib, the sulindac.Above-mentioned at least a anesthetics or opium sample analgesic can be and be selected from Fentanyl, buprenorphin hydrochloride, butorphanol tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl is through dermal system, fentanyl is striden mucosa, dihydro-morphinone hydrochloride, pethidine, methadone hydrochloride, morphine hydrochloride, morphine sulfate, morphine tartrate, nalbuphlne hydrochloride, oxycodone hydrochloride, pectination 1,4-hydroxyl dihydrocodeinone, the hydrochloric acid oxymorphone, pentazocine hydrochloride, pentazocine hydrochloride and naloxone hydrochloride, pentazocine lactate, PROPOXY PHEN HCL, the LOMAR PWA EINECS 246-676-2 dextropropoxyphene, remifentanil hydrochloride, sufentanil citrate, at least a in the tramadol hydrochloride.Above-mentioned at least a analgesic-hypnotic can be and is selected from least a in chloral hydrate, triazole nitrogen, flurazepam hydrochloride, pentobarbital, pentobarbital sodium, sodium phenobarbital, barbose, dicoumarol, triazole benzene phenodiazine, zaleplon, the Zolpidemtar Trate.Above-mentioned at least a anticonvulsant can be and is selected from acetazolamide sodium, carbamazepine, clonazepam, chlorine nitrogen dipotassium, stable, divalproex sodium, ethosuximide, fosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital, sodium phenobarbital, phenytoin, phenytoin Sodium, phenytoin Sodium (dilution), primidone mysoline, hydrochloric acid and replaces and add at least a in guest, topiramate, sodium valproate, the valproic acid.Above-mentioned at least a antidepressants can be and are selected from amitriptyline hydrochloride, amitriptyline embonate, chlorine piperazine oxygen, bupropion hydrochloride, citalopram hydrobromate, Clomipramine Hydrochloride, the hydrochloric acid desipramine, adapin, fluoxetine Hydrochloride, imipramine hydrochloride, imipramine embonate, mirtazapine, nefazodone hydrochloride, psychostyl, paroxetine hydrochloride, W-1544a, sertraline hydrochloride, tranylcypromine sulfate, stangyl, at least a in the wanlafacine hydrochloride.Above-mentioned at least a antianxiety drugs can be and is selected from least a in alprazolam, buspirone hydrochloride, chlordiazepoxide, hydrochloric acid chlordiazepoxide, chlorine nitrogen dipotassium, stable, adapin, hydroxyzine pamoate, hydroxyzine hydrochloride, hydroxyzine pamoate, tavor, mephrobamate, midazolam hydrochloride, the oxazepam.Above-mentioned at least a psychosis can be and is selected from chlorpromazine hydrochloride, clozapine, the capric acid fluphenazine, the enanthic acid fluphenazine, the hydrochloric acid fluphenazine, haloperidol, haloperidol decanoate, the lactic acid haloperidol, the hydrochloric acid loxapine, loxitane, mesoridazine benzenesulfonate, the hydrochloric acid molindone, olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine fumarate, risperidone, mellaril, thiothixene, thiothixene hydrochloride, at least a in the hydrochloric acid trifluoperazine.Above-mentioned at least a central nervous system's stimulant can be and is selected from least a in amphetamine sulfate, caffeine, sulphuric acid dexamphetamine, hydrochloric acid doxapram, methamphetamine hydrochloride, hydrochloric acid methylphenidate, modafinil, pemoline, the phentermine hydrochloride.Above-mentioned at least a antiparkinsonism drug can be and is selected from least a in virofral, methanesulfonic acid benzetropine, biperiden hydrochloride, biperiden lactate, bromocriptine methanesulfonate, carbidopa and levodopa, entacapone, levodopa, pergolide mesylate, two hydrochloric acid pramipexoles, ropinirole hydrochloride, SelegilineHydrochloride, tolcapone, the Cyclodol.Above-mentioned at least a comprehensive medicine for central nervous system can be and is selected from bupropion hydrochloride, donepezil hydrochloride, Droperidol, fluvoxamine maleate, lithium carbonate, Lithium Citrate de, hydrochloric acid and receives at least a in thunder Qu Tan, methacrylic acid nicotine (a kind of active component of smoking give-up chewing gum), nicotine transdermal system, propofol, Lizakuputan benzoate, Sibutramine hydrochloride, Sumatriptan Succinate, hydrochloric acid tacrine, the Zolmitriptan.(seeing, for example the 337-530 page or leaf among the Nursing 2001 Drug Handbook).

Above-mentioned at least a cholinergic agent (for example parasympathomimetic agent) can be and is selected from least a in bethanecholchloride, edrophonium chloride, Neostigmine, neostigmine methylsulfate, physostigmine salicylate, bromination 3-formyl oxygen dimethylamino-1 picoline.Above-mentioned at least a anticholinergic can be and is selected from least a in atropine sulfate, bentrl hydrothloride, robinul, Daturine, sulphuric acid Daturine, probanthine, scopolamine, butyl bromination scopolamine, the hydrogen scotropin.Above-mentioned at least a beta adrenergic agent (sympathomimetic) can be and is selected from least a in dobutamine hydrochloride, dopamine hydrochloride, acid Metaraminol Bitartrate, acid Noradenaline Bitatrate, phenylephrine hydrochloride, pseudoephedrine hydrochloride, the pseudoephedrine sulfate.Above-mentioned at least a adrenergic blocking drug (sympatholytic) can be and is selected from least a in agit, gynergen, desernil, the propranolol hydrochloride.Above-mentioned at least a skeletal muscle relaxant can be and is selected from least a in baclofen, carisoprodol, benzoflex, cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol, the tizanidine hydrochloride.Above-mentioned at least a neuromuscular blocking agents can be and is selected from least a along in atracurium, doxacurium chloride, mivacurium chloride, pancuronium bromide, pipecuronium bromide, thunder storehouse bromine ammonium, Rocuronium Bromide, succinylcholine chloride, tubocurarine chloride, the vecuronium bromide of benzenesulfonic acid atracurium, benzenesulfonic acid.(seeing, for example the 531-84 page or leaf among Nuring 2001 DrugHandbook).

Above-mentioned at least a hydryllin can be and is selected from least a in brompheniramine maleate, cetirizine hydrochloride, chlorphenamine, fumaric acid clematine, cyproheptadine, diphhydramine hydrochloride, fexofenadine hydrochloride, loratadine, promethazine hydrochloride, promethazine teoclate, the triprolidine hydrochloride.Above-mentioned at least a bronchodilator can be and is selected from least a in salbutamol, Salbutamol, aminophylline, atropine sulfate, ephedrine sulfate, epinephrine, hydrogen tartrate epinephrine, adrenalin hydrochloride, ipratropium bromide, isoproterenol, hydrochloric acid isoproterenol, sulphuric acid isoproterenol, albuterol hydrochloride, metaproterenol sultate, Oxtriphylline, pirbuterol acetate, carbonaphthoic acid salmaterol, sulphuric acid Arubendol, the theophylline.Above-mentioned at least a expectorant or cough medicine can be and be selected from least a in benzonatate dew, codeine phosphate, codeine sulfate, dextromethorphan hydrobromide, diphhydramine hydrochloride, guaifenesin, the dihydro-morphinone hydrochloride.Above-mentioned at least a comprehensive respiratory medications can be and is selected from acetylcysteine, beclomethasone dipropionate, beractant, budesonide, calfactant, sodium cromoglicate, streptodornase α, prostacyclin sodium, 9-and goes at least a in fluorine fluocinonide, fluticasone propionate, Menglusitena, sodium nedocromil, palivizumab, the third scorching pine, zafirlukast, the zileuton.(seeing the 585-642 page or leaf among the Nursing 2001Drug Handbook).

Above-mentioned at least a antacid, adsorbent or antiflatulent can be and be selected from least a in aluminium carbonate, aluminium hydroxide, calcium carbonate, magaldrate, magnesium hydroxide, magnesium oxide, dimethicone, the sodium bicarbonate.Above-mentioned at least a digestive enzyme or cholelithiasis solubilizing agent can be and be selected from least a in pancreatin, pancreatic lipase, the ursodeoxycholic acid.Above-mentioned at least a diarrhea can be and is selected from least a in Attagel, basic bismuth salicylate, WL-140, diphenoxylate hydrochloride or atropine sulfate, loperamide, octreotide acetate, tinctura opii, the tinctura opii (containing Camphora).Above-mentioned at least a aperient can be and is selected from least a in bisocodyl, WL-140, cascara sagrada, cascara sagrada aromatic hydrocarbon fluid extract, cascara sagrada fluid extract, Oleum Ricini, calcium dioctyl sulfosuccinate, docusate sodium, glycerol, lactulose, magnesium citrate, magnesium hydroxide, magnesium sulfate, methylcellulose, mineral oil, Polyethylene Glycol or electrolyte solution, Semen Plantaginis, Folium Sennae, the sodium phosphate.Above-mentioned at least a antiemetic can be and is selected from least a in chlorpromazine hydrochloride, dimenhydrinate, mesyl dolasetron, dronabinol, Granisetron Hydrochloride, meclizine, metocloproamide hydrochloride, Ondansetron Hydrochloride, fluphenazine, prochlorperazine, prochlorperazine edisylate, anti-naus, promethazine hydrochloride, scopolamine, thiethylperazine dimaleate, the trimethobenzamide hydrochloride.Above-mentioned at least a antiulcerative can be and is selected from least a in Altramet, hydrochloric acid Altramet, famotidine, Pilus Caprae seu Ovis diene, misoprostol, p-aminophenyl, omeprazole, RABEPRAZOLE SODIUM, rantidine bismuthcitrate, hydrochloric acid ranitidine, the aluminum sulfate.(seeing, for example the 643-95 page or leaf among the Nursing 2001Drug Handbook).Above-mentioned at least a steroid can be and is selected from betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, the cyclopentyl propionic acid hydrocortisone, the sodium phosphate hydrocortisone, the hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, Methylprednisolone Sodium Succinate, Bo Nisonglong, predniso lone acetate, prednisolone sodium phosphate, prednisolonetebutate, prednisone, the fluorohydrocarbon andrographolide, Triamcinolone Acetonide, at least a in the triamcinolone diacetate.Above-mentioned at least a androgen or anabolic steroids can be at least a in the system that is selected from the different azoles of alkynes hydroxyl androstene, FL, methyltestosterone, abolon, nandrolone phenylpropionate, testosterone, cyclopentyl propionic acid testosterone, testosterone enanthatas, sterandryl, estosterone percutaneous.Above-mentioned at least a estrogen or corpus luteum hormones can be the estrogen that is selected from esterification, estradiol, estradiol cypionate, the system of estradiol/norethindrone acetate percutaneous, estradiol valerate, estrogen (link coupled), estrone sulfuric ester piperazine, ethinylestradiol, ethinylestradiol and desogestrel, ethinylestradiol and ethynodiol diacetate, ethinylestradiol and desogestrel, ethinylestradiol and ethynodiol diacetate, ethinylestradiol and levonorgestrel, ethinylestradiol and norethindrone, ethinylestradiol and norethindrone acetate, ethinylestradiol and norgestimate, ethinylestradiol and methylnorethindron, ethinylestradiol and norethindrone and acetate and ferrous fumarate, levonorgestrel, 6 α methyl, 17 α megestrol acetates, female pure methyl ether and norethindrone, norethindrone, norethindrone acetate, methylnorethindron, progesterone.Above-mentioned at least a gonadroptropin can be and is selected from least a among ganirelix acetate, Gonadorelin Acetate, Supprelin (Roberts)., the menotropins.Above-mentioned at least a antidiabetic drug or glucagon can be and be selected from least a in acarbose, chlorpropamide, glutathion, glipizide, glucagon, glyburide, insulin, hydrochloric acid metformin, miglitol, pyrrolidine hydrochloride row ketone, repaglinide, rosiglitazone maleate, the troglitazone.Above-mentioned at least a thyroxin can be and is selected from least a in levothyroxine sodium, Cynomel, liotrix, the dried thyroid.Above-mentioned at least a thyroxin antagonist can be and is selected from the thin imidazoles of first, potassium iodide, potassium iodide (saturated solution), propylthiouracil (PTU), radioiodine (sodium iodide¹³¹I), at least a in the Lugol's solution.Above-mentioned at least a pituitary hormone can be and is selected from least a in thyroliberin, cosyntropin, desmophressin acetate, the bright dried meat Li Te of acetic acid, long-acting thyroliberin, somatrem, growth hormone, the vassopressin.Above-mentioned at least one kind parathyroid gland medicine can be and is selected from least a in calcifediol, calcitonin (people), calcitonin (salmon), calitriol, dihydrotachysterol, the etidronate.(seeing, for example the 696-796 page or leaf among the Nursing 2001 Drug Handbook).

Above-mentioned at least a diuretic can be and is selected from least a in acetyl azoles (sulphur) amine, acetyl azoles (sulphur) amine sodium, hydrochloric acid amiloride, bumetanide, chlortalidone, ethacrynate sodium, acidum ethacrynicum, furosemide, Hydrochlorothiazide, indapamide, mannitol, Mei Latuo ancestor, spironolactone, torasemide, traimterene, the urea.Above-mentioned at least a electrolyte or substitutional solution can be and be selected from calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, calcium lactate, calcium phosphate (binary), calcium phosphate (ternary), glucosan (high molecular), dextran (low-molecular-weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, the RingerShi injection, RingersShi injection (lactic acidization), at least a in the sodium chloride.Above-mentioned at least a acidulant or basifier can be and be selected from least a in sodium bicarbonate, sodium lactate, the tromethane.(seeing, for example the 797-833 page or leaf among the Nursing 2001 Drug Handbook).

Above-mentioned at least a hematonic can be and is selected from least a in ferrous fumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (drying), iron dextran, Iron Sorbitex, polysaccharide-iron complex, the Ferrlecit.Above-mentioned at least a anticoagulant can be and is selected from least a in Ardeparin Sodium, dalteparin sodium, Danaparoid sodium, Enoxaparin Sodium, calciparine, heparin sodium, the tintorane.Above-mentioned at least a blood derivatives can be and is selected from least a inalbumin 5%,albumin 25%, antihemophilic factor, counter inhibitor coagulant complex, Antithrombin III (people), the IX factor (people), IX factor complex, the plasma protein fraction.Above-mentioned at least a thrombolytic can be and is selected from least a in alteplase, Ah Buddhist nun's chain enzyme, reteplase (reorganization), streptokinase, the urokinase.(seeing, for example the 834-66 page or leaf among Nursing 2001 DrugHandbook).

Above-mentioned at least a alkylation medicine can be and is selected from least a in busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine, mustine hydrochlcride, melphalan, hydrochloric acid melphalan, streptozotocin, temozolomide, the thiotepa.Above-mentioned at least a antimetabolite can be and is selected from least a in capecitabine, cladribine, cytosine arabinoside, fluorine glycosides, fludarabine phosphate, fluorouracil, hydroxyurea, purinethol, methotrexate, methotrexate sodium, the thioguanine.Above-mentioned at least a antibiotic antitumor drug can be and is selected from least a in bleomycin sulfate, actinomycin D, citric acid daunorubicin liposome, daunorubicin hydrochloride, doxorubicin hydrochloride, hydrochloric doxorubicin liposome, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, pentoside, plicamycin, the valrubicin.The above-mentioned at least a antitumor drug that changes hormonal balance can be and is selected from least a in Anastrozole, bicalutamide, estramustine phosphate sodium, exemestane, flutamide, acetic acid goserelin, letrozole, the bright dried meat Li Te of acetic acid, megestrol acetate, nilutamide, TAMOXIFEN CITRATE, testolactone, the Toremifene Citrate.Above-mentioned at least a comprehensive antitumor drug can be and is selected from asparaginase, bacillus Calmette-Guerin (BCG) (live body intravesical), dacarbazine, Docetaxel, etoposide, the phosphoric acid etoposide, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, paclitaxel, Pegaspargase, porfimer sodium, hydrazine under the hydrochloride methyl, Rituximab, teniposide, topotecan hydrochloride, Herceptin, retin-A, Vinblastine Sulfate, the sulphuric acid vincristin, at least a in the Vinorelbine tartrate.(seeing, for example the 867-963 page or leaf among the Nursing2001 Drug Handbook).

Above-mentioned at least a immunosuppressant can be and is selected from least a in azathioprine, basiliximab, ciclosporin, daclizumab, lymphocyte immune globulin, muromonab-CD3, mycophenolic acid morpholine ethyl ester, hydrochloric acid mycophenolic acid morpholine ethyl ester, sirolimus, the tacrolimus.Above-mentioned at least a vaccine or toxoid can be and be selected from BCG vaccine, cholera vaccine, diphtheria and tetanus toxoid (absorbent-type), diphtheria and tetanus toxoid and acellular pertussis vaccine absorbent-type, diphtheria and tetanus toxoid and whole cell pertussis vaccine, b type haemophilus combined vaccine, Hepatitis A Vaccine (deactivation), hepatitis B vaccine (reorganization), influenza virus vaccine 1999-2000 triple type A﹠amp; B (surface antigen of purification), influenza virus vaccine 1999-2000 triple type A﹠amp; B (the subvirus body of subvirus body or purification), influenza virus vaccine 1999-2000 triple type A﹠amp; B (totivirus body), Japanese encephalitis virus vaccine (deactivation), Lyme borrelia burgdorferi disease vaccine (reorganization OspA), measles and parotitis and rubella virus vaccine (work), measles and parotitis and rubella virus vaccine (deactivation), measles virus vaccines (deactivation), the meningococcus polysaccharide vaccine, mumps virus vaccine (work), pestilence vaccine, Pnu-Imune 23 (multi-joint), poliovirus vaccine (deactivation), poliovirus vaccine (is lived, oral, three), rabies vaccine (absorbent-type), rabies vaccine (human diploid cell), rubella and mumps virus vaccine (work), rubella virus vaccine (is lived, deactivation), tetanus toxoid (absorbent-type), tetanus toxoid (liquid), antityphoid vaccine (oral), antityphoid vaccine (parenteral administration), Typhoid Vi Polysaccharide Vaccine, Varivax, at least a in the yellow fever vaccine.Above-mentioned at least a antitoxin or venom can be and be selected from least a in latrodectus mactans's venom, Crotalidae venom (multi-joint), diphtheria antitoxin (horse), the golden yellow Corallium Japonicum Kishinouye calmette's serum.Above-mentioned at least a immune serum can be and is selected from least a in cytomegalovirus immune globulin (intravenous), hepatitis B immune globulin (people), immunoglobulin intramuscular, immunoglobulin intravenous, rabies immune globulin (people), respiratory syncytial virus immunoglobulin intravenous (people), Rho (D) immunoglobulin (people), Rho (D) immunoglobulin intravenous (people), tetanus immune globulin (people), the varicella-zoster immunoglobulin.Above-mentioned at least a biological respinse modifier can be and is selected from Ah Di Liujin, at least a according in pool Ai Ting α, Fei Lasiting, injection glatiramer acetate, interferon alfacon-1, Intederon Alpha-2a (reorganization), Interferon Alpha-2b (reorganization), interferon beta-1a, interferon beta-1b (reorganization), gamma interferon 1-b, levamisole hydrochloride, rHuIL-11, the Sargramostim.(seeing, for example the 964-1040 page or leaf among the Nursing 2001DrugHandbook).

Above-mentioned at least a ophthalmology anti-infective can be selected from least a in bacitracin, chloromycetin, ciprofloxacin, erythromycin, sulfate gentamicin, pyridine carboxylic acid 0.3%, aerosporin, sulphacetamide 10%, sulphacetamide 15%, sulphacetamide 30%, tobramycin, the vidarabine.Above-mentioned at least a ophthalmology anti-inflammatory agent can be and is selected from least a in dexamethasone, dexamethasone sodium phosphate, diclofenac sodium 0.1%, chloromethane dragon, flurbiprofen sodium, ketorolac tromethamine, predniso lone acetate (suspension), the prednisolone phosphate sodium (solution).Above-mentioned at least a miotic can be and is selected from least a in acetylcholine chloride, carbachol (ophthalmic), carbachol (part), ecostigmine, pilocarpine, pilocarpine hydrochloride, the pilocarpine nitrate.Above-mentioned at least a iridodilator can be and is selected from least a in atropine sulfate, cyclogyl hydrochloride, adrenalin hydrochloride, epinephrine borate, homatropine hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide, the N-ethyl-N-(.gamma.-picolyl)tropamide.Above-mentioned at least a eye vasoconstrictor can be and is selected from least a in naphazoline hydrochloride, oxymetazoline hydrochloride, the Visine.Above-mentioned at least a comprehensive medicament for the eyes can be and is selected from hydrochloric acid apraclonidine, betaxolol hydrochloride, brimonidine tartrate, hydrochloric acid galutedin, dipivefrine hydrochloride, hydrochloric acid and stops up beautiful at least a in Bu Lage, sodium chloride (height oozes), the timolol maleate of amide, fumaric acid emedastine, fluorescein sodium, Fumaric acid ketotifen, latanoprost, Levobunolol Hydrochorid, hydrochloric acid.Above-mentioned at least a ear medication can be and is selected from least a in boric acid, carbamide peroxide, chloromycetin, the triethanolamine oleate polypeptide-condensed fluid.Above-mentioned at least a nose medication can be and is selected from beclomethasone dipropionate, budesonide, ephedrine sulfate, adrenalin hydrochloride, 9-and goes at least a in fluorine fluocinonide, fluticasone propionate, naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrine hydrochloride, Visine, the third scorching pine, the xylometazoline hydrochloride.(seeing, for example the 1041-97 page or leaf among the Nursing 2001 Drug Handbook).

Above-mentioned at least a local anti-infective agent can be and is selected from acycloguanosine, amphotericin B, Azelaic Acid butterfat, bacitracin, Nitric acid butoconazole, the p chloromethylbenzoic acid cillimycin, clotrimazole, econazole nitrate, erythromycin, sulfate gentamicin, ketoconazole, the acetic acid marfanil, metronidazole (part), the mould anti-azoles of nitric acid, mupirocin, naftifine hydrochloride, polygynax, nitro sugar hydrazone, nystatin, silver sulfadiazine, the special Binet phenol of hydrochloric acid, terconazole (triaconazole), quadracycline, tioconazole, at least a during tineatonsurans is moved back.Above-mentioned at least a antiscabietic or antipedicular agent can be and be selected from least a in crotamiton, gamma hch, permethrin, the pyrethrin.Above-mentioned at least a topical corticosteroid can be selected from two betamethasone dipropionates, betamethasone valerate, CBP, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate. at least a in diflorasone diacetate, acetone fluocinonide, fluocinolone ester, flurandrenolide, fluticasone propionate, halcionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, valeric acid hydrocortisone, furancarboxylic acid Mo Meitasong, the third scorching pine.(seeing, for example the 1098-1136 page or leaf among the Nursing2001 Drug Handbook).

Above-mentioned at least a little living element or mineral can be and be selected from vitamin A, compound vitamin B, cyanocobalamin, folic acid, hydroxocobalamine, calcium leucovorin, nicotinic acid, nicotiamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D, cholecalciferol, ergocalciferol, novel vitamin D analogues, the degree ostelin, the sharp ostelin of handkerchief, vitamin E, the vitamin K analog, vitamin K1, sodium fluoride, sodium fluoride (part), trace element, chromium, copper, iodine, manganese, selenium, at least a in the zinc.Above-mentioned at least a caloic can be the aminoacid preserved material that is selected from aminoacid preserved material (crystalline), the dextrose, contain contain electrolytical aminoacid preserved material in electrolytical aminoacid preserved material, the dextrose, at the aminoacid preserved material of liver failure, at least a in the aminoacid preserved material of hypermetabolism pressure, the aminoacid preserved material, dextrose, fat emulsion, MCT Oil at renal failure.(seeing, for example the 1137-63 page or leaf among Nursing 2001 DrugHandbook).

The present invention also provides at least a any particular composition suitable and/or effective dose or the pharmaceutical composition, this particular composition or pharmaceutical composition have comprised at least a GLP-1 analogue body or specific part or variant, this particular composition or pharmaceutical composition have randomly further comprised and (for example have been selected from least a TNF antagonist, but be not limited only to TNF chemistry or protein antagonist, TNF monoclonal or polyclonal antibody or fragment, solvable TNF receptor (p55 for example, p70 or p85) or fragment, its fused polypeptide, or micromolecule TNF antagonist, for example conjugated protein I of TNF or II (TBP-1 or TBP-II), nerelimonmab, English monoclonal antibody of sharp former times, enteracept, CDP-571, CDP-870, Afelimomab, Lenercept etc.), antirheumatic (for example, methotrexate, AF, aurothioglucose, azathioprine, Embrel, Kidon (Ono), sulphuric acid hydroxyl chloroquine, leflunomide, sulfasalazine), muscle relaxant, anesthetics, on-steroidal anti-inflammatory medicaments (NSAID), analgesic, anesthetis, tranquilizer, local anesthetic, neuromuscular blocking agents, antibacterial (aminoglycoside for example, antifungal, antiparasitic, antiviral drugs, carbamyl, cephalosporin, fluoroquinolone, macrolide, penicillin, sulfanilamide, tetracycline, other antibacterial), antipsoriatic, corticosteroid, anabolic steroid, the diabetes related drugs, mineral, nutrient substance, the thyroid medicine, vitamin, the calcium associated hormone, diarrhea, cough medicine, antiemetic, antiulcerative, aperient, anticoagulant, erythropoietin (for example according to pool Ai Ting α), Fei Lasiting (G-CSF for example, Neupogen), Sargramostim (GM-CSF, Leukine), immunity, immunoglobulin, immunosuppressant (basiliximab for example, cyclocyto polypeptide, daclizumab), growth hormone, the hormone replacement medicine, the estrogen receptor instrumentality, iridodilator, cycloplegic, the alkylation medicine, antimetabolite, mitotic inhibitor, radiopharmaceutical, antidepressants, antimaniacal drugs, psychosis, antianxiety drugs, hypnotic, sympathomimetic, analeptic, donepezil, tacrine, asthma drug, beta-agonists, the induction type steroid, the leukotriene inhibitor, methylxanthine, cromoglicic acid, epinephrine or analog, streptodornase α (Pulmozyme), at least a in cytokine or the cytokine antagonist.The limiting examples of above-mentioned cytokine include but not limited to any one among the IL-1-IL-23.Suitable dose is well known in the art.See Wells etal. for example, eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia,Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, TarasconPublishing, Loma Linda, CA (2000), above-mentioned list of references all herein by complete introducing, as a reference.

Above-mentioned composition also can comprise with at least a antibody of the present invention or polypeptide associate, combine, collaborative preparation or the collaborative lps molecule of using.This toxin can randomly play selectivity and kill pathological cells or tissue.This pathological cells can be cancerous cell or other cell.This toxin can be, but purification or the recombinant toxin or the toxin fragment at least one functional cell toxin district of particular toxin have been not limited only to comprise, this particular toxin for example is selected from, one of at least a ricin, diphtheria toxin, diphtherotoxin, venom toxin or bacteriotoxin.The term toxin also comprises by any can cause the pathological condition that comprises toxic shock in people and other mammal, and the endotoxin and the extracellular toxin that can cause dead natural existence, sudden change or recombinant bacteria or virus to be generated.This toxin can include but not limited to and produce enterotoxication heat-labile enterotoxin of E, coli (LT), thermally-stabilised enterotoxin (ST), shigella dysenteriae cytotoxin, Aeromonas enterotoxin, toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), B (SEB) or C (SEC), streptococcus enterotoxin etc.Above-mentioned antibacterial comprises, but be not limited only to enterotoxigenic escherichia coli (ETEC), cause enterorrhagia escherichia coli (for example bacterial strain of serotype 0157:H7), staphylococcus kind (staphylococcus aureus for example, staphylococcus pyogenes), shigella dysenteriae kind (dysentery bacterium for example, the Fu Shi bacillus dysenteriae, Shigella boydii and bacterium sonnei), salmonella strain (Bacillus typhi for example, Salmonella choleraesuls, Salmonella enteritidis), clostruidium kind (clostridium perfringens for example, difficult bacillus, bacillus botulinus), bent stick strain (campylobacter jejuni for example, embryo's Campylobacter), screw rod strain (for example helicobacter pylori), Aeromonas kind (for example, Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Plesiomonas shigelloides, Yersinia enterocolitica, vibrio kind (vibrio cholera for example, vibrio parahaemolyticus), the klebsiella bacillus kind, Pseudomonas aeruginosa and streptococcus.See Stein for example, ed., INTERNALMEDICINE, 3 rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans etc., eds., Bacterial Infections of Humans; Epidemiology andControl, 2d.Ed., pp 239-254, Plenum Medical Book Co., New York (1991); Mandell etc., Principles and Practice of Infectious Diseases, 3d.Ed., Churchill Livingstone, New York (1990); Berkow etc., eds., The Merck Manual, 16 th edition, Merck and Co., Rahway, N.J., 1992; Wood etc., FEMS Microbiology Immunology, 76:121-134 (1991); Marrack etc., Science, 248:705-711 (1990), the content of above-mentioned list of references is all by complete introducing herein as a reference.

GLP-1 analogue body of the present invention or specific part or variant compositions can further comprise at least a suitable arbitrarily adminicle, for example, but are not limited only to diluent, binding agent, stabilizing agent, buffer agent, salt, lipophilic solvent, antiseptic, adjuvant etc.It is preferred that medicine can be accepted adminicle.The limiting examples of above-mentioned sterile solution and preparation method all are well known in the art, for example, but are not limited only to Gennaro, Ed., Remington ' s Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co. (Easton, PA) 1990.Can select the drug acceptable carrier that matches with the method for application of GLP-1 analogue body compositions, dissolubility and/or stability by well known and/or described herein conventional method.

Drug excipient and additive useful in the present composition comprise, but be not limited only to protein, peptide, aminoacid, lipid and carbohydrate (for example, sugar, comprise monosaccharide, disaccharide, trisaccharide, tetrose and oligosaccharide; Derived carbohydrate is such as sugar alcohol, alduronic acid, esterified saccharides etc.; Polysaccharide or carbohydrate polymer) they can exist separately or with combining form, and comprise separately or in combination, exist with 1-99.99% weight or volume ratio.The example of protein excipient comprises serum albumin, such as human serum albumin (HSA), recombined human albumin (rHA), gelatin, casein etc.Also can bring into play the representative aminoacid/GLP-1 analogue body of cushioning effect or specific part or variant component and comprise alanine, glycine, arginine, betanin, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame etc.A kind of preferred amino acids is a glycine.

Be applicable to that carbohydrate excipient of the present invention comprises, for example, monosaccharide is such as fructose, maltose, galactose, glucose, D-mannose, sorbose etc.; Disaccharide is such as lactose, sucrose, trehalose, cellobiose etc.; Polysaccharide is such as Raffinose, melezitose, maltodextrin, glucosan, starch etc.; And sugar alcohol, such as mannitol, xylitol, maltose alcohol, lactose, xylitol, Sorbitol (glucitol), inositol etc.Be applicable to that preferred saccharide excipient of the present invention is mannitol, trehalose and Raffinose.

GLP-1 analogue body compositions also can comprise buffer agent or pH regulator agent; Typical buffer agent be from organic acid or alkali preparation and salt.Representative buffer agent comprises acylate, such as the salt of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, hydrochloric acid Tris or phosphate buffer.The preferred reducing that is applicable to the present composition is an acylate, such as citrate.

In addition, GLP-1 analogue body of the present invention or specific part or variant compositions can comprise polymeric excipient/additive, such as polyvinylpyrrolidone, water-soluble poly-sucrose (a kind of polymerization sugar), dextrates (cyclodextrin for example, such as 2-hydroxy propyl-Beta cyclodextrin), Polyethylene Glycol, flavoring agent, antibacterial, sweeting agent, antioxidant, antistatic additive, surfactant (for example polysorbate, such as "TWEEN 20 " and " TWEEN 80 "), lipid (for example phospholipid, fatty acid), steroid (for example cholesterol) and chelating agen (for example EDTA).

Be applicable to that the above-mentioned of GLP-1 analogue body compositions of the present invention and other known drug excipient and/or additive all are known in the art, for example at " Remington:TheScience﹠amp; Practice of Pharmacy ", 19th ed., Williams﹠amp; Williams, (1995) and at " Physician ' s Desk Reference ", 52nd ed., MedicalEconomics, Montvale, listed drug excipient and/or additive among the NJ (1998), the disclosure of above-mentioned list of references all by complete introducing herein as a reference.Preferred carrier or excipient materials are carbohydrate (for example saccharide and sugar alcohol) and buffer agent (for example citrate) or polymerizer.

Preparation.As mentioned above, the invention provides stabilization formulations, said preparation can preferably include the suitable buffer agent of brackish water or specific salts and the optional listerine that contains, and the preparation that contains antiseptic is provided and is fit to the preservatives of using of medicinal or veterinary purpose more, these preparations have comprised at least a GLP-1 analogue body or specific part or the variant that medicine can be accepted dosage form.Preservative contains at least a known antiseptic, or be selected from following optional antiseptic, be at least a phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, nitrous acid benzene hydrargyrum, phenoxyethanol, formaldehyde, ammonia butanols, magnesium chloride (for example hexahydrate), alkyl P-hydroxybenzoic acid (methyl, ethyl, propyl group, butyl etc.), alkyldimethylbenzylammonium chloride, benzethonium chloride, dehydro sodium acetate and thimerosal, or the mixture of above-mentioned substance in aqueous diluent.Can be according to the concentration or the mixture of knowledge application of any suitable known in the art, such as 0.001-5%, or any range wherein or numerical value, for example, but be not limited only to 0.001,0.003,0.005,0.009,0.01,0.02,0.03,0.05,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.3,4.5,4.6,4.7,4.8,4.9 or any range wherein or numerical value.Limiting examples comprises, preservative free, 0.1-2%m-cresol (for example 0.2,0.3,0.4,0.5,0.9,1.0%), 0.1-3% benzyl alcohol (for example 0.5,0.9,1.1,1.5,1.9,2.0,2.5%), 0.001-0.5% thimerosal (for example 0.005,0.01%), 0.001-2.0% phenol (for example 0.05,0.25,0.28,0.5,0.9,1.0%), 0.0005-1.0% alkyl P-hydroxybenzoic acid (for example 0.00075,0.0009,0.001,0.002,0.005,0.0075,0.009,0.01,0.02,0.05,0.075,0.09,0.1,0.2,0.3,0.5,0.75,0.9,1.0%) etc.

As mentioned above, the invention provides a kind of goods, these goods comprise that packaging material and at least one contain the bottle of particular solution, this particular solution contains at least a GLP-1 analogue body or specific part or variant and above-mentioned buffer agent and/or antiseptic, and randomly be the aqueous diluents form, wherein above-mentioned packaging material comprise that having indicated above-mentioned solution can preserve and surpassed 1,2,3,4,5,6,9,12,18,20,24,30,36,40,48,54,60,66,72 hour or the label of longer time section.The present invention further comprises a kind of goods, these goods comprise packaging material, contain first bottle of at least a lyophilizing GLP-1 analogue body or specific part or variant, second bottle with the aqueous diluents that contains above-mentioned buffer agent or antiseptic, wherein above-mentioned packaging material comprise a label, can how at least a GLP-1 analogue body or specific part or variant be sneaked in the aqueous diluents again to patient explanation, thereby and form to preserve and surpassed 24 hours or the solution of longer time section.

At least a GLP-1 analogue body of using according to the present invention or specific part or variant can comprise from mammalian cell or transgenic goods preparing that perhaps from then on the place is described or other biological origin purification acquisition known in the art by the reorganization approach.

In product of the present invention, the quantitative range of at least a GLP-1 analogue body or specific part or variant is included under the situation in wet/dry system, restoring the amount that can obtain the about 1000mg/ml concentration of about 1.0ug/ml-on the basis, but low and higher concentration also is feasible, and depend on specific delivery vectors, for example pharmaceutical solutions is different from percutaneous medicine card, lung, strides mucosa, or infiltration or micro pump method.

Preferably, above-mentioned aqueous diluents comprises further that randomly medicine can accept antiseptic.Preferred antiseptic comprises and is selected from following antiseptic, be phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl P-hydroxybenzoic acid (methyl, ethyl, propyl group, butyl etc.), alkyldimethylbenzylammonium chloride, benzethonium chloride, dehydro sodium acetate and thimerosal, or the mixture of above-mentioned substance.The concentration of preservatives that is applied in the preparation of the present invention is the concentration that is enough to produce anti-microbial effect.This concentration depends on selected antiseptic, and the technical staff can easily measure this concentration.

Other excipient, for example isotonic agent, buffer agent, antioxidant, preservative enhancers all can be chosen wantonly and preferably be added in the above-mentioned diluent.Isotonic agent as glycerol, adopts its concentration known usually.Physiology can tolerate buffer agent and preferably be added, to improve pH control.Preparation of the present invention can cover large-scale pH, and such as the about pH10 of about pH4-, preferred range is the about pH9 of about pH5-, and highly preferred scope is the about pH8.0 of about pH6.0-.The preferred pH value of preparation of the present invention is approximately between the 6.8-about 7.8.Preferred reducing agents comprises phosphate buffer, most preferably sodium phosphate, especially phosphate buffered saline(PBS) (PBS).

Other additive, can accept solubilizing agent such as medicine, as Tween 20 (polyethylene glycol oxide (20) Span-20), Tween 40 (polyethylene glycol oxide (20) sorbitan monopalmitate), Tween 80 (polyethylene glycol oxide (20) sorbitan monooleate), Pluronic F68 (polyethylene glycol oxide polyoxypropylene block copolymers) and PEG (Polyethylene Glycol) or non-ionic surface active agent, such as polysorbate20 or 80 or poloxalkol 184 or 188

Polyls, other block copolymer and such as the chelating agen of EDTA and EGTA all can randomly be added in preparation of the present invention or the compositions, to reduce cohesion.When adopting pump or plastic containers to use preparation of the present invention, above-mentioned additive is especially effective.The tendency of protein condenses has been lowered in the existence that medicine can be accepted surfactant.

Preparation of the present invention can prepare by ad hoc approach, this method comprise with at least a GLP-1 analogue body or specific part or variant be selected from one of following antiseptic and mix, be phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl P-hydroxybenzoic acid (methyl, ethyl, propyl group, butyl etc.), alkyldimethylbenzylammonium chloride, benzethonium chloride, dehydro sodium acetate and thimerosal, or the mixture of above-mentioned substance in aqueous diluents.At least a GLP-1 analogue body or specific part or variant and antiseptic are blended in the aqueous diluents, are to adopt conventional dissolving and married operation realization.Be the preparation appropriate formulation, for example, at least a GLP-1 analogue body or the appointment antiseptic in specific part or variant and the buffer accurately measured in the buffer pressed the particular magnitude compatibility, this particular magnitude is enough to provide the prescribed concentration of protein and antiseptic.Any those of ordinary skill in this area all should be approved the change form of this method.For example, add the order of component, whether adopt other additive, the temperature and the pH of preparation said preparation, thereby all be can optimisedly be applicable to the factor of the concentration that adopts and route of administration.

The preparation of institute of the present invention prescription can be used as clear liquid or two doleiform formula is provided for the patient, wherein in two bottles, there is one bottle to contain at least a freeze dried GLP-1 analogue body or specific part or variant, can utilize and contain water, antiseptic and/or excipient, preferably phosphoric acid salt buffer agent and/or saline solution and specific salts, and another bottle of formation aqueous diluents restores it.Single solution bottle or need restorative two bottle all can be repeated to utilize repeatedly, and be enough to satisfy single or many circulation treatments needs to the patient, thereby can provide than existing general treatment scheme Therapeutic Method more easily.

The goods of institute of the present invention prescription are applicable to from using to 24 hours or longer time section span immediately.Correspondingly, the goods of institute of the present invention prescription provide significant benefits for the patient.Preparation of the present invention is safe storage under the about 40 ℃ temperature of about 2-randomly, and keep proteinic biological activity for a long time, thereby can on 6,12,18,24,36,48,72 or 96 hours or longer time period span, be saved and/or use indicating this solution on the packaging label.As adopt anticorrosion diluent, this label can comprise operating period until at least one month in 1-12 the middle of the month number, half a year, a year and a half and/or 2 years.

In the present invention, the solution of at least a GLP-1 analogue body or specific part or variant can prepare by ad hoc approach, and this method comprises at least a GLP-1 analogue body or specific part or variant are blended in the aqueous diluents.This mixing is to adopt conventional dissolving and married operation to realize.For preparing suitable diluent, for example, with in water or buffer, with at least a GLP-1 analogue body or specific part or variant that the particular magnitude compatibility is accurately measured, this value is enough to provide the prescribed concentration of protein and optional antiseptic or buffer agent.Any those of ordinary skill in this area all should be approved the change form of this method.For example, add the order of component, whether adopt other additive, the temperature and the pH of preparation preparation of the present invention, thereby all be can optimisedly be applicable to the factor of the concentration that adopts and route of administration.

The product of institute of the present invention prescription can be used as clear liquid or two doleiform formula is provided for the patient, wherein in two bottles, has one bottle to contain at least a freeze dried GLP-1 analogue body or specific part or variant, can utilize another bottle of property of water-bearing diluent that it is restored.Single solution bottle or need restorative two bottle all can be repeated to utilize repeatedly, and be enough to satisfy single or many circulation treatments needs to the patient, thereby can provide than existing general treatment scheme Therapeutic Method more easily.

By providing the clear liquid of institute of the present invention prescription or the product of two doleiform formulas to pharmacy, clinic or other similar means and facility, it can be offered the patient indirectly, wherein in two bottles, there is one bottle to contain at least a freeze dried GLP-1 analogue body or specific part or variant, can utilizes another bottle of property of water-bearing diluent that it is restored.Clear liquid in this situation can reach one liter or more volume, thereby need provide a kind of big reservoir, by its one or more times will more a spot of at least a GLP-1 analogue body or specific part or variant solution shift in the into less bottle, this less bottle can be provided to its client and/or patient by pharmacy or clinic.

The generally acknowledged device that comprises above-mentioned single bottle system comprises and is used to send pen type-injection device of passing solution, such as Huma

WithThe generally acknowledged device that comprises two bottles system comprises and can be used for freeze-dried drug is restored in cartridge case, and send pen type-injection device of passing this reconstituted solution, such as

The product of institute of the present invention prescription comprises packaging material.Except that the desired information of administrative organization, these packaging material also provide the condition of using this product.Packaging material of the present invention provide description to the patient, told about at product and be two bottles, wet/during dry form, restore at least a GLP-1 analogue body or specific part or variant in the aqueous diluent, form the method for solution, and surpassed 2-24 hour or the span of longer time section on use the method for this solution.For single bottle of solution product, labeled marker this solution can be used above 2-24 hour or longer time section.The product of institute of the present invention prescription is useful aspect people's drug products purposes.

Preparation of the present invention can prepare by ad hoc approach, and this method comprises that with at least a GLP-1 analogue body or specific part or variant and specific buffers the phosphate buffer of preferred brackish water or specific salts is mixed.At least a GLP-1 analogue body or specific part or variant and buffer agent are blended in the aqueous diluents, are to adopt conventional dissolving and married operation realization.Be the preparation appropriate formulation, for example, at least a GLP-1 analogue body or the appointment buffer agent in specific part or variant and the water accurately measured in water or the buffer agent pressed the particular magnitude compatibility, this value is enough to provide the prescribed concentration of protein and buffer agent.Any those of ordinary skill in this area all should be approved the change form of this method.For example, add the order of component, whether adopt other additive, the temperature and the pH of preparation preparation of the present invention, thereby all be can optimisedly be applicable to the factor of the concentration that adopts and route of administration.

Stable or the preservative of institute of the present invention prescription can clear liquid or two doleiform formula be provided for the patient, in this pair bottle, there is one bottle to contain at least a freeze dried GLP-1 analogue body or specific part or variant, can utilizes the antiseptic of property of water-bearing diluent form or another bottle of buffer agent and excipient that it is restored.Single solution bottle or need restorative two bottle all can be repeated to utilize repeatedly, and be enough to satisfy single or many circulation treatments needs to the patient, thereby can provide than existing general treatment scheme Therapeutic Method more easily.

According to the present invention, at least a GLP-analogue body in stable or preservative or the solution described herein or specific part or variant can be applied to the patient by the multiple method of passing of sending, and comprise SC or IM injection; Percutaneous, lung, stride mucosa, transplanting, osmotic pumps, cartridge case, micro pump or well known, and other approach of being understood by those of skill in the art.

Treatment is used.The present invention about analogue body also provides a kind of at cell, tissue, organ, the diabetes of regulating or treating among animal or the patient, the method of I type or type ii diabetes, comprising adult that begin or childhood, insulin-dependent, non-insulin-dependent etc., comprise relevant S﹠S, for example but be not limited only to insulin resistance, hyperglycemia, hypoglycemia, pancreatitis, the Sushing syndrome, acanthosis nigricans, lipoatrophy type diabetes, retinopathy, nephropathy, polyneuropathy, mononeuropathy, idioneurosis, ulcer, ulcer of foot, arthrosis, infect (for example, fungus or antibacterial) etc.

It is a kind of at cell that the present invention also provides, tissue, organ, regulate or treat the method at least a and immune correlated disease that diabetes interrelate among animal or the patient, include but are not limited at least a in I type or the type ii diabetes, comprising adult that begin or childhood, insulin-dependent, non-insulin-dependent etc., comprise relevant S﹠S, for example but be not limited only to insulin resistance, hyperglycemia, hypoglycemia, pancreatitis, the Sushing syndrome, acanthosis nigricans, lipoatrophy type diabetes, retinopathy, nephropathy, polyneuropathy, mononeuropathy, idioneurosis, ulcer, ulcer of foot, arthrosis, infect (for example, fungus or antibacterial) etc.See Merck Manual for example, 12th-17th Editions, Merck﹠amp; Company, Rahway, NJ (1972,1977,1982,1987,1992,1999), Pharmacotherapy Handbook, Wells etc., eds., Second Edition, Appletonand Lange, Stamford, Conn. (1998,2001), each document all is incorporated herein by reference by complete.

This method can randomly comprise cell, tissue, organ, animal or the patient who at least a particular composition of effective dose or pharmaceutical composition is applied to this adjusting of needs, processing or treatment, and said composition or pharmaceutical composition contain at least a GLP-1 analogue body or specific part or variant.

The present invention also provides at cell, tissue, organ, the method of regulating or treating at least a cardiovascular diseases among animal or the patient, this cardiovascular diseases comprises, but be not limited only at least a heart syndrome of fainting, myocardial infarction, congestive heart failure, apoplexy, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, the diabetes arteriosclerotic disease, hypertension, arterial hypertension, renal vascular hypertension, faint, shock, the syphilis of cardiovascular system, heart failure, pulmonary heart disease, primary pulmonary hypertension, arrhythmia, room ectopic beat, atrial flutter, auricular fibrillation (continuing or paroxysmal), irregularity or many focuses atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmia, ventricular fibrillation, His restraints arrhythmia, atrioventricular block, bundle branch block, the myocardial ischaemia sexual maladjustment, coronary heart disease, angina pectoris, myocardial infarction, cardiomyopathy, the dilatation and congestion cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumor, aorta and peripheral arterial aneurysm, aortic dissection, the aorta inflammation, ventral aorta and branched obturation thereof, the peripheral blood vessel imbalance, the imbalance of occlusive tremulous pulse, the peripheral arterial atheromatosis, thromboangiitis obliterans, the imbalance of functional peripheral tremulous pulse, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, phlkebocholosis, thrombophlebitis, cirso-, arteriovenous fistula, lymphedema, lipedema, unstable angina, reperfusion injury, syndrome behind the pump, ischemia-reperfusion injury etc.This method can randomly comprise cell, tissue, organ, animal or the patient who the particular composition of effective dose or pharmaceutical composition is applied to this adjusting of needs, processing or treatment, and said composition or pharmaceutical composition contain at least a GLP-1 analogue body or specific part or variant.

Arbitrary method of the present invention all can comprise cell, tissue, organ, animal or the patient who the particular composition of effective dose or pharmaceutical composition is applied to this adjusting of needs, processing or treatment, and said composition or pharmaceutical composition contain at least a GLP-1 analogue body or specific part or variant.This method can randomly further comprise at treatment the collaborative of above-mentioned immunological disease to be used or therapeutic alliance, wherein above-mentioned at least a GLP-1 analogue body, using of specific part or its variant further is included in before it, simultaneously and/or afterwards, use and be selected from following at least a medicine, be at least a TNF antagonist (for example, but be not limited only to TNF antibody or fragment, soluble TNF acceptor or fragment, its fusion rotein or micromolecule TNF antagonist), antirheumatic, muscle relaxant, anesthetics, on-steroidal anti-inflammatory medicaments (NSAID), analgesic, anesthetis, tranquilizer, local anesthetic, neuromuscular blocking agents, antibacterial (for example, aminoglycoside, antifungal, antiparasitic, antiviral agents, carbamyl, cephalosporin, fluoroquinolone, macrolide, penicillin, sulfanilamide, tetracycline, other antibacterial), antipsoriatic, corticosteroid, anabolic steroid, the diabetes related drugs, mineral, nutrient, the thyroid medicine, vitamin, the calcium associated hormone, diarrhea, cough medicine, antiemetic, antiulcerative, aperient, anticoagulant, erythropoietin (for example according to pool Ai Ting α), Fei Lasiting (G-CSF for example, Neupogen), Sargramostim (GM-CSF, Leukine), immunity, immunoglobulin, immunosuppressant (basiliximab for example, ciclosporin, daclizumab), growth hormone, the hormone replacement medicine, the estrogen receptor instrumentality, mydriatic, cycloplegic, alkylating agent, antimetabolite, mitotic inhibitor, radiopharmaceutical, antidepressants, antimaniacal drugs, psychosis, antianxiety drugs, hypnotic, sympathomimetic, analeptic, donepezil, tacrine, asthma drug, beta-agonists, suck steroid, the leukotriene inhibitor, methylxanthine, cromoglicic acid, epinephrine or analog, streptodornase α (Pulmozyme), cytokine or cytokine antagonist.Proper dosage is well known in the art.See, for example Wells etc., eds., PharmacotherapyHandbook, 2 nd Edition, Appleton and Lange, Starnford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, DeluxeEdition, Tarascon Publishing, Loma Linda, CA (2000), each list of references are all by complete introducing herein as a reference.

Analogue body also can be applied to external, cultivates such as autologous bone marrow.In brief, before chemotherapy, in patient's body, take out bone marrow, and adopt TPO and/or GLP-1 to handle, randomly in conjunction with analogue body, randomly in conjunction with one or more other cytokines.Subsequently, treated bone marrow is returned to through chemotherapeutical patient, to quicken the recovery of bone marrow.In addition, TPO can use separately and with GLP-1 analogue body and/or GLP-1 use in conjunction, also can be used to external expansion bone marrow or peripheral blood CFU-GM (PBPC).Before chemotherapy, can adopt stem cell factor (SCF) or G-CSF to stimulate bone marrow, so that early progenitor cell is discharged in the peripheral circulation.Randomly, from peripheral blood, collect and concentrate these CFU-GM, and adopt TPO and analogue body to handle its culture subsequently, randomly in conjunction with one or more other cytokines, comprise, but be not limited only to SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11, making its differentiation and hypertrophy is high density megalokaryocyte culture, subsequently it is randomly returned in the patient's body after the too high dose chemotherapy.The TPO dosage range that is used for extracorporeal treatment bone marrow should be 100pg/ml-10ng/ml, preferably 500pg/ml-3ng/ml.The dosage of analogue body should be equal to GLP-1 on activity, adoptable dosage range is 0.1 unit/ml-20 unit/ml, 0.5 unit/ml-2 unit/ml preferably, or any range wherein or numerical value.

Be applicable to the present composition, therapeutic alliance, collaboratively use, the TNF antagonist of device and/or method (further comprising at least a antibody of the present invention, specific part or its variant) comprises, but be not limited only to anti-TNF antibodies, its part binding fragment and with the bonded acceptor molecule of TNF specificity; Prevention and/or inhibition TNF are synthetic, TNF discharges or its chemical compound to the effect of target cell, such as thalidomide, tenidap, phosphodiesterase inhibitor (for example pentoxifylline and rolipram), A2b adenosine receptor antagonists and A2b adenosine receptor reinforcing agent; Stop and/or suppress the chemical compound of TNF receptor signalling, such as mitogen-activated protein(MAP) (MAP) inhibitors of kinases; Retardance and/or the cracked chemical compound of inhibition film TNF are such as metalloprotein alcohol inhibitor; Retardance and/or the active chemical compound of inhibition TNF are such as angiotensin converting enzyme (ACE) inhibitor (for example captopril); Generate and/or synthetic chemical compound with retardance and/or inhibition TNF, such as map kinase inhibitor.

Reductions such as " tnf antibody " used herein, " TNF antibody ", " TNF Alpha antibodies " or fragment, block, suppress, eliminate or disturbed external, original position and/or preferred intravital TNF alpha active.For example, suitable TNF people's antibody of the present invention can be in conjunction with TNF α, and comprises anti-TNF antibodies, its Fab and specified mutant or itself and the bonded zone of TNF alpha specific.Suitable TNF antibody or fragment also can reduce, block, eliminate, disturb, stop and/or suppress TNF RNA, DNA or protein synthesis, TNF release, the signalling of TNF receptor, film TNF cracking, TNF activity, TNF generation and/or synthesize.

Chimeric antibody cA2 is by in the high-affinity and mouse anti human TNF alpha IgG1 antibody, and promptly the antigen of A2 is in conjunction with variable region and human IgG1, and promptly the constant region of K immunoglobulin is formed.This human IgG1 Fc district has improved the alloantibody effector function, has prolonged the circulation serum half-life of this antibody, and has reduced its immunogenicity.The affinity of chimeric antibody cA2 and epitope specificity derive from the variable region of murine antibody A 2.In a kind of particular, for the nucleic acid of thesemurine antibody A 2 variable regions of encoding, preferred source is the A2 hybridoma cell line.

Chimeric A2 (cA2) has neutralized natural in dosage dependence mode and the two cellulotoxic effect of reorganization human TNF alpha.With combine of reorganization human TNF alpha tested, the affinity costant of extrapolating chimeric antibody cA2 is 1.04 * 10 from chimeric antibody cA2¹⁰M^-1Method for optimizing that suppress to measure monoclonal antibody specificity and affinity by competition can be with reference to Harlow, etc., Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York, 1988; Colligan etal., eds., Current Protocols inImmunology, Greene Publishing Assoc.and Wiley Interscience, NewYork. (1992-2002); Kozbor etc., Immunol.Today, 4:72-79 (1983); Ausu bel etc., eds.Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2002); And Muller, Meth.Enzymol., 95:589-601 (1983), these lists of references are all by complete introducing herein as a reference.

This area has been described and can be applicable to other monoclonal anti-TNF antibodies example of the present invention and (see that for example U.S. Patent number 5,231,024; M

Ller, A. etc., Cytokine 2 (3): 162-169 (1990); U. S. application number 07/943,852 (JIUYUE was submitted on the 11st in 1992); Rathjen etc., international publication number WO 91/02078 (publication on February 21st, 1991); Rubin etc., GLP-1 patent publication No. 0218868 (publication on April 22nd, 1987); Yone etc., GLP-1 patent publication No. 0288088 (on October 26th, 1988); Liang, etc., Biochem.Biophys.Res.Comm.137:847-854 (1986); Meager, etc., Hybridoma 6:305-311 (1987); Fendly etc., Hybridoma 6:359-369 (1987); Bringman, etc., Hybridoma 6:489-507 (1987); And Hirai, etc., J.Immunol.Meth.96:57-62 (1987), these lists of references are all by complete introducing herein as a reference).

The TNF acceptor molecule.The TNF acceptor molecule that the present invention preferably adopts be those combine with TNF α high-affinity (see, for example Feldmann etc., international publication number WO 92/07076 (publication on April 30th, 1992); Schall etc., Cell 61:361-370 (1990); With Loetscher etc., Cell 61:351-359 (1990), these lists of references are all by complete introducing herein as a reference), and randomly have the TNF acceptor molecule of reduced immunogenicity.55kDa (p55TNF-R) and 75kDa (p75TNF-R) TNF cell surface receptor are particularly useful in the present invention.The clipped form of these receptors comprise the extracellular domain (ECD) of this receptor or its funtion part (see, for example Corcoran etc., Eur.J.Biochem.223:831-840 (1994)), also useful in the present invention.In urine and serum, detected the clipped form that this TNF receptor comprises ECD, i.e. the TNF α of 30kDa and 40kDa inhibition conjugated protein (Engelmann, H. etc., J.Biol.Chem.265:1531-1536 (1990)).TNF receptor multimeric molecule and TNF immunity receptor fusion molecule, and derivant and fragment or part are other TNF acceptor molecule examples that can be applicable to the inventive method and compositions.Can be applicable to TNF acceptor molecule of the present invention and be characterised in that it has the long-term treatment patient, and can well arrive the ability of relief of symptoms admirably, and toxicity is low.Reduced immunogenicity and/or high-affinity, and other not clear and definite feature all may work to the therapeutic effect that is obtained.

Useful in the present invention TNF receptor multimeric molecule comprises via one or more peptide linkers or other non-peptide linker, such as Polyethylene Glycol (PEG), and whole or funtion part of the ECD of the two or more TNF receptors that link together.This multimeric molecule can further comprise the signal peptide of secreted protein, to guide the expression of this multimeric molecule.Described these multimeric molecules and preparation method thereof in U. S. application number 08/437,533 (submission on May 9 nineteen ninety-five), the content of this list of references by complete introducing herein as a reference.

Useful TNF immunity receptor fusion molecule comprises at least one part of one or more immunoglobulin molecules and whole or funtion part of one or more TNF receptors in the inventive method and compositions.These immunity receptor fusion molecule can fitted to be monomer, or heteromultimers or homology polymer.This immunity receptor fusion molecule also can be unit price or multivalence.An example of this TNF immunity receptor fusion molecule is TNF receptor/IgG fusion rotein.This area described TNF immunity receptor fusion molecule and preparation method thereof (Lesslauer etc., Eur.J.Immunol.21:2883-2886 (1991); Ashkenazi etc., Proc.Natl.Acad.Sci.USA 88:10535-10539 (1991); Peppel etc., J.Exp.Med.174:1483-1489 (1991); Kolls etc., Proc.Natl.Acad.Sci.USA91:215-219 (1994); Butler etc., Cytokine 6 (6); 616-623 (1994); Baker etc., Eur.J.Immunol.24:2040-2048 (1994); Beutler etc., U.S. Patent number 5,447,851; With U. S. application number 08/442,133 (submission on May 16 nineteen ninety-five), these lists of references all by complete introducing herein as a reference).The method for preparing the immunity receptor fusion molecule also can be with reference to Capon etc., U.S. Patent number 5,116,964; Capon etc., U.S. Patent number 5,225,538; With Capon etc., Nature 337:525-531 (1989), these lists of references are all by complete introducing herein as a reference.

The function equivalent of TNF acceptor molecule, derivant, fragment or district refer to the specific part of this TNF acceptor molecule, or the part of the TNF acceptor molecule of encoding in this TNF acceptor molecule sequence, its size and sequence be enough to make its on function to can be applicable to TNF acceptor molecule of the present invention similar (for example, with TNF α high-affinity combine, and have reduced immunogenicity).The function equivalent of TNF acceptor molecule be also included within on the function to can be applicable to TNF acceptor molecule of the present invention similar (for example, with TNF α high-affinity combine, and have reduced immunogenicity) modification TNF acceptor molecule.For example, the function equivalent of TNF acceptor molecule can contain " reticent (SILENT) " codon or one or more amino acid replacement, disappearance or interpolation (for example are replaced into another acidic amino acid with an acidic amino acid; Or with one the coding identical or different hydrophobic amino acid codon be replaced into another the coding hydrophobic amino acid codon).See Ausubel etc., Current Protocols in Molecular Biology, GreenePublishing Assoc.and Wiley-Interscience, New York (1987-2003).

Cytokine include but not limited to all known cytokines.See, for example CopewithCytokines.com.Cytokine antagonist include but not limited to any antibody, fragment or analogue body, any solvable receptor, fragment or analogue body, any micromolecule antagonist, or the combination in any of above-mentioned substance.

Any method of the present invention all can comprise the method for the imbalance that is used for the treatment of protein mediation, this method comprises cell, tissue, organ, animal or the patient who the particular composition of effective dose or pharmaceutical composition is applied to this adjusting of needs, processing or treatment, and said composition or pharmaceutical composition contain at least a GLP-1 analogue body or specific part or variant.This method can randomly further comprise at treatment the collaborative of immunological disease to be used or therapeutic alliance, the using of wherein above-mentioned at least a GLP-1 analogue body, specific part or its variant further be included in before it, simultaneously and/or afterwards, use and be selected from least a other cytokine, such as IL-3 ,-6 and-11; Stem cell factor; At least a material among G-CSF and the GM-CSF.

Typically, treatment to pathologic condition is to realize by at least a GLP-1 analogue body compositions of using effective dose or dosage, promptly according to the contained given activity of said composition, the scope of each dosage amounts to average out to, the per kilogram weight in patients is used at least approximately at least a GLP-1 analogue body or the specific part or the variant of 0.0001-500 milligram, and preferably per kilogram weight in patients single or multiple is used at least approximately 0.001-100 milligram GLP-1 analogue body or specific part or variant.Alternatively, effectively serum-concentration can comprise that single or multiple uses the 0.001-5000ug/ml serum-concentration that is obtained.Proper dosage is that the doctor is known, and depends on specified disease situation, the given activity of the compositions of using and the particular patient of just receiving treatment natch.Under some situation,, be necessary to carry out repetitive administration, promptly use separately, and before obtaining appointed date dosage or effect, repeat this and use separately with the repetition of specifically monitored dosage or dosing for obtaining the TA amount.

Preferred dose can randomly comprise uses 0.0001 at every turn, 0.0002,0.0003,0.0004,0.0005,0.0006,0.0007,0.0008,0.0009,0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 and/or 30mg/kg, or any range wherein, numerical value or mark, or use by single or multiple and to obtain 0.0001,0.0002,0.0003,0.0004,0.0005,0.0006,0.0007,0.0008,0.0009,0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.5,0.9,1.0,1.1,1.2,1.5,1.9,2.0,2.5,2.9,3.0,3.5,3.9,4.0,4.5,4.9,5.0,5.5,5.9,6.0,6.5,6.9,7.0,7.5,7.9,8.0,8.5,8.9,9.0,9.5,9.9,10,10.5,10.9,11,11.5,11.9,12,12.5,12.9,13.0,13.5,13.9,14.0,14.5,15,15.5,15.9,16,16.5,16.9,17,17.5,17.9,18,18.5,18.9,19,19.5,19.9,20,20.5,20.9,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,96,100,200,300,400 and/or the 500ug/ml serum-concentration, or any range wherein, numerical value or mark.

Alternatively, can be according to known facts, such as the pharmacokinetic properties of particular agent, and mode of administration and approach; Receiver's age, health condition and body weight; The frequency of the nature and extent of symptom, the kind of combined treatment, treatment and Expected Results change application dosage.Usually, the dosage of active component can be per kilogram of body weight and uses about 0.0001-100 milligram.General dose is 0.001-10, and preferred dose is that per kilogram of body weight is used the 0.001-1 milligram at every turn, or obtains Expected Results effectively by continuous releasing pattern.

A limiting examples is, can be to human or animal's treatment by disposable or periodically use at least a GLP-1 analogue body of the present invention or specific part or the variant dosage of 0.0001-100mg/kg, single such as adopting, infusion or repeated doses, the 1st, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38, at least one sky in 39 or 40 days, or alternatively 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, in at least one week in 19 or 20 weeks, or use 0.0002 the every day in the combination in any cycle of above-mentioned natural law and all numbers, 0.0003,0.0004,0.0005,0.0006,0.0007,0.0008,0.0009,0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.9,1.0,1.1,1.5,2,3,4,5,6,7,8,9 or at least a GLP-1 analogue body of the present invention or or specific part or the variant of 10mg/kg.

The common per unit of the dosage form that use suitable inside (compositions) or each container contain about 0.0001 milligram-about 500 milligrams of active component.In these pharmaceutical compositions, the amount of active component is calculated by weight the about 0.5-99.999% that should be the said composition gross weight usually.

For parenteral administration, GLP-1 analogue body or specific part or variant can be configured to solution, suspension, emulsion or lyophilized powder with the acceptable parenteral excipient of medicine or in the mode that provides respectively.The example of this excipient is water, saline solution, RingerShi solution, glucose solution and 5% human serum albumin.Also can adopt liposome and non-aqueous excipient, such as expressed oi.Excipient or lyophilized powder can contain the additive of keeping isotonicity (for example sodium chloride, mannitol) and chemical stability (for example buffer agent and antiseptic).Said preparation is by known or suitable technique sterilization.

Suitable pharmaceutical carrier canonical reference document in the field, promptly Remington ' sPharmaceutical Sciences all describes in the latest edition of A.Osol to some extent.

Therapeutic administration.According to the present invention, can adopt many known and mode of administration of having researched and developed, be used at least a GLP-1 analogue body of the present invention or the specific part or the variant of administering therapeutic effective dose.GLP-1 analogue body of the present invention can form solution, emulsion, colloid or suspension in carrier, or with powder type, by utilize multiple be applicable to suck or described herein or apparatus and method that other known mode of this area is used in any one, passed and send.

Parenteral administration and using.The preparation that is used for parenteral administration can contain usual excipients, sterilized water or saline, such as the oil of the ployalkylene glycol of Polyethylene Glycol, plant origin, hydrogenated naphthalene etc.According to known method, can prepare the aqueous or the oiliness suspension of injection by adopting suitable emulsifying agent or humidizer and suspending agent.The medicament of injection can be a diluent nontoxic, can not be oral, such as aqueous solution or sterile injectable solution or the suspension that forms in solvent.Effectively excipient or solvent can adopt water, RingerShi solution, isotonic saline solution etc.; Usual vehicle or suspended solvents then can adopt aseptic expressed oi.With regard to these purposes, can adopt the expressed oi and the fatty acid of any kind of, comprise natural synthetic or semisynthetic fatty oil or fatty acid; Natural synthetic or semisynthetic monoglyceride or diester or three esters.Parenteral administration is known in the art, comprise, but be not limited only to conventional injecting pathway, U.S. Patent number 5,851, air-pressure type needleless injection device and the U.S. Patent number 5,839 described in 198, the laser beam perforation apparatus of describing in 446, these two patents all by complete introducing herein as a reference.

Can select and recommend and pass.The invention further relates to by parenteral, subcutaneous, intramuscular, intravenous, pill, vagina, rectum, oral cavity, Sublingual, intranasal or percutaneous approach and use at least a GLP-1 analogue body or specific part or variant.Protein, GLP-1 analogue body or specific part or variant compositions can be produced and be used for parenteral (subcutaneous, intramuscular or intravenous) and use, and are good with liquid solution or suspensions especially; When being used for vagina or rectal administration,, be good such as paste and suppository then especially with semi-solid form; When oral cavity or sublingual administration, be good with tablet or capsule form especially then; During intranasal administration, be good with powder, nasal drop or aerosol or particular agent form especially; It is good that applied dermally then send the delivery system form with gel, ointment, lotion, suspension or plaster especially, this send delivery system to contain can to change skin texture or improves the chemical intensifier of percutaneous medicine card Chinese medicine concentration, such as dimethyl sulfoxine (Junginger, Deng., at " Drug PermeationEnhancement "; Hsieh, D.S., Eds., pp59-90 (Marcel Dekker, Inc.NewYork 1994, complete introducing is herein as a reference)), or contain to make and contain the oxidant (WO 98/53847) that protein and peptide formulations are applied to skin, maybe can apply electric field, and cause the of short duration approach of passing that send, such as electroporation, maybe can improve the mobility that electrically charged medicine passes skin, such as ionotherapy, but or the using ultrasound ripple, import method (U.S. Patent number 4 such as ultrasound wave, 309,989 and 4,767,402) (above-mentioned publication and patent are all by complete introducing herein as a reference).

Lung/nasal administration.For pulmonary administration, preferably, at least a GLP-1 analogue body or specific part or variant compositions are by so that its granular size that arrives at the respiratory tract of lung or hole bottom is effectively sent passs.According to the present invention, can send by any one of multiple suction that is used for suction-type administering therapeutic medicament known in the art or Nasal delivery devices and pass at least a GLP-1 analogue body or specific part or variant.These devices that atomization preparation is deposited in patient's hole chamber or the alveolar comprise metered dose inhaler, nebulizer, dry powder generator, aerosol apparatus etc.Other device that is applicable to guiding GLP-1 analogue body or specific part or variant lung or nasal administration also is known in the art.Above-mentioned all devices all can be used the preparation that is fit to use, so that GLP-1 analogue body or specific part or variant are distributed in the aerosol.This aerosol can be made up of solution (aqueous and non-aqueous) or solid particle.Metered dose inhaler, as

Metered dose inhaler typically adopts propulsive gas, and need start (see that for example WO 94/16970, WO 98/35888) in intake period.Diskus is as Turbuhaler^TM(Astra),

(Glaxo),

(Glaxo), Spiros^TMInhaler (Dura), the device of selling by InhaleTherapeutics and

Powder inhalator (Fisons) all sucks mixed-powder (US 4668218Astra, EP 237507 Astra by respiratory activity, WO97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498Fisons, complete introducing is herein as a reference).Nebulizer such as AERx^TMAradigm,Nebulizer (Mallinckrodt), and Acorn

Nebulizer (MarquestMedical Products) (US 5404871 Aradigm, WO 97/22376), can generate aerosol by solution, inhaler such as metered dose inhaler, Diskus then can produce the granule aerosol, and above-mentioned list of references all by complete introducing herein as a reference.Commerce can provide the above-mentioned particular instance of suction apparatus only category of the present invention not to be construed as limiting the representative of putting into practice specific device of the present invention for being applicable to.Preferably, send by Diskus or aerosol apparatus and pass the compositions that comprises at least a GLP-1 analogue body or specific part or variant.Be applicable to that the suction apparatus of using at least a GLP-1 analogue body of the present invention or specific part or variant has some gratifying features.For example, send the advantage of passing to be reliably, can to reappear by suction apparatus and accurately.This suction apparatus can randomly send passs little dried granule, and for example for good inhalable, this granule is preferably about 1-5 μ m less than about 10 μ m.

Use GLP-1 analogue body or specific part or variant compositions with Sprayable.Comprise that the proteic spraying of GLP-1 analogue body or specific part or variant compositions can be prepared by nozzle by suspension or the solution that forces at least a GLP-1 analogue body or specific part or variant under pressure.The size of this nozzle and configuration, applying pressure and liquid feed rate all can be selected to obtain the output and the particle size of expection.Electron spray can pass through, and for example electric field and capillary tube or nozzle material-feeding obtain.Send the advantage of passing at least a GLP-1 analogue body or specific part or variant compositions protein body to be by aerosol apparatus, particle size is less than about 10 μ m, and preferable range is the about 5 μ m of about 1um-, and highly preferred scope is the about 3 μ m of about 2 μ m-.

Be applicable to that at least a GLP-1 analogue body of aerosol apparatus or specific part or variant compositions protein formulation typically comprise GLP-1 analogue body or the specific part or the variant compositions albumen of aqueous solution form, its concentration is at least a GLP-1 analogue body or specific part or the variant compositions albumen that every ml soln contains the about 20mg of about 1mg-.Said preparation can comprise such as excipient, buffer agent, isotonic agent, antiseptic, surfactant and preferred zinc.Said preparation also can comprise and is used to make protein stabilized excipient of GLP-1 analogue body or specific part or variant compositions or reagent, such as buffer agent, Reducing agent, filling albumen or carbohydrate.Can be used for preparing the proteic filling albumen of GLP-1 analogue body or specific part or variant compositions and comprise albumin, protamine etc.Can be used for preparing the proteic typical carbohydrate of GLP-1 analogue body or specific part or variant compositions and comprise sucrose, mannitol, lactose, trehalose, glucose etc.This GLP-1 analogue body or specific part or variant compositions protein formulation also can comprise surfactant, the GLP-1 analogue body that atomizing caused or specific part or the proteic spatial induction cohesion of variant compositions in the time of can reducing or prevent to form aerosol by solution.Multiple conventional surfactants all can be employed, such as polyoxyethylene fatty acid ester and ethanol and polyoxyethylene sorbitol fatty acid ester.Its application quantity scope should be the 0.001-14% of weight of formulation usually.For the especially preferred surfactant of application of the present invention is Tween-81, polysorbate80, polysorbate20 etc.Proteinic other reagent that is used to prepare such as analogue body or specific part or variant is known in the art, also is included in the preparation.

Use GLP-1 analogue body or specific part or variant compositions by nebulizer.GLP-1 analogue body or specific part or variant compositions albumen can pass through nebulizer, such as blast atomizer or soniclizer and used.Typically, in blast atomizer, adopt compressed air source to form the high-speed air injection stream by nozzle.When gas when nozzle diffuses out, formed low-pressure area, GLP-1 analogue body or specific part or the proteic solution of variant compositions are sucked, and make its capillary tube by linking to each other with liquid reservoir.When being derived from liquid stream capillaceous and leaving this capillary tube, be cut into unsettled filamentous and drop, just formed aerosol.Can use a series of structure, flow velocity and flow deflector type, from the particular spray nebulizer, to obtain the performance characteristic of expection.In soniclizer, adopt high-frequency electrical energy to form the mechanical energy of concussion, typically applying piezoelectric formula transducer.This energy directly or by means of coupling liquid is transferred to GLP-1 analogue body or specific part or variant compositions protein formulation, has just formed to comprise this GLP-1 analogue body or specific part or the proteic aerosol of variant compositions.Send the advantage of passing GLP-1 analogue body or specific part or variant compositions protein body to be by nebulizer, particle size is less than about 10 μ m, and preferable range is the about 5 μ m of about 1 μ m-, and highly preferred scope is the about 3 μ m of about 2 μ m-.

Be applicable to and spray or play GLP-1 analogue body or specific part or the variant compositions albumen that at least a GLP-1 analogue body of acoustic wave type nebulizer or specific part or variant formulations typically comprise the aqueous solution form that its concentration is at least a GLP-1 analogue body or specific part or the misfolded proteins that every ml soln contains the about 20mg of about 1mg-.Said preparation can comprise such as excipient, buffer agent, isotonic agent, antiseptic, surfactant and preferred zinc.Said preparation also can comprise and is used to the excipient or the reagent that make at least a GLP-1 analogue body or specific part or variant compositions protein stabilized, such as buffer agent, Reducing agent, filling albumen or carbohydrate.Can be used for preparing the proteic filling albumen of at least a GLP-1 analogue body or specific part or variant compositions and comprise albumin, protamine etc.The typical carbohydrate that can be used for preparing at least a GLP-1 analogue body or specific part or variant comprises sucrose, mannitol, lactose, trehalose, glucose etc.This at least a GLP-1 analogue body or specific part or variant formulations also can comprise surfactant, at least a GLP-1 analogue body that atomizing caused in the time of can reducing or prevent to form aerosol by solution or the spatial induction cohesion of specific part or variant.Multiple conventional surfactants all can be employed, such as polyoxyethylene fatty acid ester and ethanol and polyoxyethylene sorbitol fatty acid ester.Its application quantity scope should be the 0.001-4% of weight of formulation usually.For the especially preferred surfactant of application of the present invention is Tween-81, polysorbate80, polysorbate20 etc.Other reagent that is used to prepare such as at least a GLP-1 analogue body or specific part or variant proteins is known in the art, also is included in the preparation.

Use GLP-1 analogue body or specific part or variant compositions by metered dose inhaler.In metered dose inhaler (MDI), propellant, at least a GLP-1 analogue body or specific part or variant and any excipient or other additive all are inclusive in the canister, have formed the mixture that comprises liquified compressed gas.Starting of metering valve discharges this mixture with aerosol form, this aerosol preferably contains the granule of size range less than about 10um, and the preferred sizes scope is the about 5um of about 1um-, and highly preferred size range is the about 3um of about 2um-.By using by several different methods well known by persons skilled in the art, comprise GLP-1 analogue body or the specific part or the variant compositions protein formulation of method preparations such as jet grinding, spray drying, critical point condensation, can obtain ideal aerosol particle size.Preferred metered dose inhaler comprises the metered dose inhaler of being produced by 3M or Glaxo, and has used hydrogenation fluorohydrocarbon propellant.

Being applicable to that at least a GLP-1 analogue body of metered dose inhaler device or specific part or variant formulations generally include contains at least a GLP-1 analogue body or specific part or variant, and can in non-aqueous media, form the attritive powder of suspension, for example, be suspended in the propellant by means of surfactant.This propellant can be any conventional material that can be used for this purposes, such as chlorine fluorohydrocarbon, hydrogenated chloride fluorine for hydrocarbon, hydrogenation fluorohydrocarbon or hydrocarbon, comprise Arcton 11, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) etc.Preferred propellant is the hydrogenation fluorohydrocarbon.Can select surfactant, to stablize at least a GLP-1 analogue body or the specific part or the variant of suspended form in this propellant, to protect this activating agent not by chemical degradation etc.Suitable surfactant comprises sorbitan trioleate, soybean lecithin, oleic acid etc.In some situation, the solution aerosol preferably adopts such as alcoholic acid solvent.Known in the artly can be used for preparing protein, also can be included in the preparation of the present invention such as proteinic other reagent of the present invention.

Any those of ordinary skill in this area will be appreciated that all the inventive method can be achieved by at least a GLP-1 analogue body of pulmonary administration or specific part or variant compositions by the device of not describing herein.

Mucosa preparation and using.For absorption via mucomembranous surface, the compositions of at least a GLP-1 analogue body or specific part or variant and application process comprise contain a large amount of submicron particles, mucosa adheres to the peptide of macromole, biologically active and the emulsion of aqueous continuous phase, can adhere to by the mucosa of realizing this emulsion granules and promote its (U.S. Patent number 5 of absorption via mucomembranous surface, 514,670).The mucomembranous surface that is fit to application emulsion of the present invention can comprise the route of administration of cornea, conjunctiva, oral cavity, Sublingual, nose, vagina, lung, stomach, intestinal and rectum.The preparation that is used for vagina or rectal administration, for example suppository can contain excipient, for example ployalkylene glycol, vaseline, cocoa butter etc.The preparation that is used for intranasal administration can be solid, and contains excipient, and for example lactose perhaps can be the aqueous or the oily solution of nasal drop.For oral, excipient comprises (U.S. Patent number 5,849,695) such as sugar, calcium stearate, magnesium stearate, pre-gelatinized starches.

Oral formulations and using.Be used for collaborative the using that oral preparation depends on adjuvant (for example resorcinol and nonionic surfactant, such aspolyoxyl 10 oleyl ether and n-cetyl polyvinylether), permeability with artificial raising intestinal wall, said preparation also depends on collaborative the using (for example trypsin inhibitor, diisopropyl fluorophosphate (DFP) (DFF) and aprotinin) of enzyme inhibitor, degrades with inhibitory enzyme.The activity composition chemical compound that is used for oral solid type dosage form can mix with at least a additive, this additive comprises sucrose, lactose, cellulose, mannitol, trehalose, Raffinose, maltose alcohol, glucosan, starch, agar, arginine, chitin, chitosan, pectin, gum tragacanth, Radix Acaciae senegalis, gelatin, collagen protein, casein, albumin, synthetic or semi synthetic polymer, and glyceride.These dosage forms also can contain the additive of other type, for example torpescence diluent, lubricant, such as magnesium stearate, P-hydroxybenzoic acid, such as the antiseptic of sorbic acid, ascorbic acid, alpha-tocopherol, such as the antioxidant of cysteine, disintegrating agent, binding agent, thickening agent, buffer agent, sweeting agent, flavoring agent, aromatizing agent etc.

Tablet and pill can be further processed in the enteric coating preparation.Be used for the pharmaceutical solutions that oral liquid preparation comprises Emulsion, syrup, elixir, suspensoid and can be used for medical usage.These preparations can contain the torpescence diluent that is usually used in this field, as water.It is the drug delivery system (U.S. Patent number 4,239,754) of insulin and heparin that liposome also once was described to.More recently be that the microsphere of the artificial polymer of kilnitamin (albuminoid) once was used to send drug delivery (U.S. Patent number 4,925,673).In addition, U.S. Patent number .5,879,681 andU.S. Patent number 5,5,871,753 in the carrier compound described be used to orally send that to pass bioactivator be known in the art.

Percutaneous preparation and using.For applied dermally, this at least a GLP-1 analogue body or specific part or variant are sealed in the delivery device, such as liposome or polymerization nanoparticle, microgranule, microcapsule or microsphere (without otherwise indicated, can be called microgranule jointly).It is known that many suitable devices are arranged, comprise by synthetic polymer, as polyhydroxy acid, as polylactic acid, polyglycolic acid and copolymer thereof, poe, poly-anhydride and poly-polyphosphazene, and natural polymer, such as collagen protein, polyamino acid, albumin and other protein, alginate and other polysaccharide, and the microgranule (U.S. Patent No. 5 made of the combination of above-mentioned substance, 814,599).

Chronic administration and preparation.Some the time, making The compounds of this invention cross over the long time period, to be applied to the patient again be ideal, for example, begins a thoughtful year section from single administration.Can utilize multiple slow release, dGLP-1t or transplant dosage form.For example, dosage form can contain the acceptable nontoxic salts of medicine of specific compound, this salt has low solubility in body fluid, for example, (a) has polyprotic acid, such as the acid-addition salts of acid such as phosphoric acid, sulphuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, LOMAR PWA EINECS 246-676-2 or naphthalenedisulfonic acid, Poly Gal A Galacturonan; (b) have multivalent metal cation,, or have, the organic cations salt that N '-diphenyl-ethylenediamine or ethylenediamine form by for example N such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium etc.; (c) (a) with (b) combination, for example tannic acid zinc salt.In addition, The compounds of this invention, or preferred, insoluble salt as above-mentioned salt, can be formulated into gel relatively, for example is applicable to injection, and for example contains, the monostearate alumina gel of Oleum sesami.Especially preferred salt is zinc salt, tannic acid zinc salt, embonate etc.Other type slow release dGLP-1t preparation of injection should contain and be disperseed to be sealed in slow degraded, nontoxic, nonantigenic polymer, such as U.S. Patent number 3,773, and chemical compound or salt in 919 described polylactic acid/polyglycolic acid polymer.This chemical compound or, preferred insoluble salt relatively also can be formulated in the cholesterol substrate silicone ball as above-mentioned salt, especially is applied to animal.Other slow release, dGLP-1t or transplanting preparation, for example gas or the visible this area of liquid fatty body list of references (U.S. Patent number 5,770,222 and " Sustained and Controlled Release Drug DeliverySystems " (continue and controlled release drug send delivery system), J.R.Robinson ed., MarcelDekker, Inc., N.Y., 1978).

Above summarized the present invention, will be more readily understood the present invention, and following example only is used for illustration, the present invention is not construed as limiting by following example.

Cloning and the expression of embodiment 1:GLP-1 analogue body in mammalian cell.Typical case's mammalian expression vector contains the promoter element that at least one can mediate the mRNA transcription initiation, GLP-1 analogue body or specific part or variant coded sequence, and tanscription termination and the needed signal of transcription product polyadenylation.Other element comprises enhancer, Kozak sequence and the both sides intervening sequence with RNA shearing donor and acceptor site.Efficiently transcribe and to realize by early stage and late promoter, retrovirus such as the RSV that adopts the SV40 source, the long terminal repeat (LTRS) in HTLVI, HIVI source and the early promoter in cytomegalovirus (CMV) source.But also can adopt cell element (for example human actin promoter).Can be used for realizing that suitable expression vector of the present invention comprises, for example, pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC67109).The mammalian cell that may be utilized comprises people Hela 293, H9 and Jurkat cell, mouse NIH 3T3 and C127 cell, Cos1, Cos7 and CV1, Carnis Coturnicis japonicae QC1-3 cell, mouse Lcell and Chinese hamster ovary (CHO) cell.

Alternatively, gene can be expressed in the specific stable cell lines, and this cell line contains the said gene that is be integrated in its chromosome.With selectable marker, such as the cotransfection of dhfr, gpt, neomycin or hygromycin can realize to the evaluation of transfectional cell with separate.

Rotaring redyeing gene also can be amplified, with GLP-1 analogue body or the specific part or the variant of expressing a large amount of codings.DHFR (dihydrofolate reductase) be marked with help develop carried be concerned about the hundreds of of gene or even the cell line of thousands of copies.Another useful selected marker be glutamine synthase (GS) (Murphy, etc., Biochem.J.227:277-279 (1991); Bebbington, etc., Bio/Technology 10:169-175 (1992)).Utilize these labellings, can in selective medium, cultivate mammalian cell, and select to have the cell of high resistance.These cell lines contain the amplification gene that is be integrated in its chromosome.Chinese hamster ovary (CHO) and NSO cell are normally used for generating GLP-1 analogue body or specific part or variant.

Expression vector pC1 and pC4 contain the sarcoma viral strong promoter in Louth (Rous) (LTR) (Cullen, Deng., Molec.Cell.Biol.5:438-447 (1985)), add a fragment (Boshart of CMV-enhancer, Deng., Cell 41:521-530 (1985)).Multiple clone site for example has restriction endonuclease cracking site BamHI, XbaI and Asp718, helps the cloning of genes of interest.This carrier also contains the polyadenylation and the termination signal of rat proinsulin protogene except that containing 3 ' intron.

Cloning in Chinese hamster ovary celI and expression.Carrier pC4 is used to the expression of GLP analogue body or specific part or variant.Plasmid pC4 is the derivant (ATCC is numbered No.37146) of plasmid pSV2-dhfr.This plasmid contains the mice DHFR gene that is subjected to the control of SV40 early promoter.By at the selective medium that is supplemented with the chemotherapeutant methotrexate (α minus MEM for example, Life Technologies, Gaithersburg, MD) middle cultured cell, can filter out by these plasmid transfections, and lack the active Chinese hamster ovary cell of dihydrofoilic acid or other cell.Many lists of references have all write down the amplification of DHFR gene in the cell that methotrexate (MTX) is had resistance and (have seen that F.W.Alt for example is etc., J.Biol.Chem.253:1357-1370 (1978); J.L.Hamlin and C.Ma, Biochem.et Biophys.Acta 1097:107-143 (1990); With M.J.Pa ge and M.A.Sydenham, Biotechnology 9:64-68 (1991)).The cell of growing in progressive concentration MTX causes the excessive generation of target enzyme DHFR by gene-amplification DHFR gene, thereby has produced the resistance to medicine.If second gene is connected to this DHFR gene, then this second gene is usually by common amplification and overexpression.This method known in the art can be used to exploitation and carry the cell line that surpasses 1,000 copy of this amplification gene.Subsequently, when extracting methotrexate, can obtain to contain the cell line of amplification gene, this amplification gene has been integrated in one or more chromosomes of host cell.

Plasmid pC4 contains the strong promoter (Cullen that is useful on the rous sarcoma virus long terminal repeat (LTR) of expressing genes of interest, Deng., Molec.Cell.Biol.5:438-447 (1985)), add the fragment (Boshart of separation from human cytomegalic inclusion disease virus (CMV) immediate early gene enhancer, Deng., Cell 41:521-530 (1985)).The downstream of this promoter is BamHI, XbaI and the Asp718 restriction endonuclease cracking site that can realize that said gene is integrated.Behind these cloning sites, this plasmid contains the polyadenylation site of 3 ' intron and rat proinsulin protogene.Other efficient promoter also can be used to express, for example people b-actin promoter, SV40 is early stage or the long terminal repeat in late promoter or other retrovirus such as HIV and HTLVI source.The Tet-Off of Clontech and Tet-On gene expression system and similar system can be used to express GLP-1 (M.Gossen with the scalable approach in mammalian cell, and H.Bujard, Proc.Natl.Acad.Sci.USA 89:5547-5551 (1992)).For the polyadenylation of mRNA, also can adopt other signal, for example from the signal of human growth hormone or globulin gene.Adopting selectable marker, on the basis such as gpt, G418 or the common transfection of hygromycin, also can select carried be concerned about the stable cell lines of gene, this gene has been integrated in its chromosome.At first adopt more than one selectable markers to have superiority, for example G418 adds methotrexate.

By operational approach known in the art, adopt restriction endonuclease digested plasmid pC4, and adopt calf enteral phosphatase dephosphorylation subsequently.From 1% agarose gel, isolate this carrier subsequently.

According to the known method step, adopt the DNA sequence of the complete GLP-1 analogue body of coding or specific part or variant, this DNA sequence is corresponding with the HC and the LC variable region of GLP-1 analogue body of the present invention.Also can adopt the isolating nucleic acid of the suitable human constant region of coding (being HC and LC district) in this construct.

Make subsequently that the separation of coding DNA is variable to be connected with the T4DNA ligase with the dephosphorylation carrier with constant region.Subsequently, transfection Escherichia coli HB101 or XL-1Blue cell, and adopt, for example the restriction enzyme analysis method is identified among the plasmid pC4 and is contained the segmental antibacterial of above-mentioned insertion.

Adopt Chinese hamster ovary (CHO) cell that lacks active DHFR gene to be used for transfection.Adopt cationic-liposome lipofectin, by 0.5ug plasmid pSV20-neo cotransfection 5 μ g expression plasmid pC4.Plasmid pSV2neo contains dominant selectable marker, and the neo gene code that derives from Tn5 can make it to one group of antibiotic, comprises that G418 has the enzyme of resistance.This cell inoculation is nourished among the α minus MEM that is filled with 1ug/ml G418.After 2 days, this cell of trypsinization, and inoculation advance hybridoma cloning flat board (Greiner, Germany) in, this flat board added be supplemented with 10,25 or the 50ng/ml methotrexate add the α minus MEM of 1ug/ml G418.About after 10-14 days, the single clone of trypsinization, and inoculate the 6 hole culture dishs or the 10ml that advance to contain variable concentrations methotrexate (50nM, 100nM, 200nM, 400nM, 800nM) subsequently and shake in the bottle.Subsequently, the clone who grows under the maximum concentration methotrexate condition is transferred in the new 6 hole flat boards that contain taller concentration methotrexate (1mM, 2mM, 5mM, 10mM, 20mM).Repeating same steps as can be till the clone who grows under the 100-200mM concentration conditions until acquisition.Analyze the expression of ideal gene outcome, for example, by SDS-PAGE and Western blotting (Western blot), or by the reversed-phase HPLC analysis.

Embodiment 2: the limiting examples of GLP-1 analogue body of the present invention.GLP-1 is by the peptide of 37 secreted amino acid longs of intestinal L-cell after oral glucose stimulates.The analogue body construct that comprises biological activity GLP-1 (7-37) peptide, variant or derivant is considered to the half-life of peptide in the energy extension body and for thetype 2 diabetes mellitus patient, provides a kind of new Therapeutic Method that is used for blood sugar lowering.The Toplink of natural GLP-1 (7-37) peptide or DPP-IV resistance analog of encoding is inserted in the analogue body supporting structure.Prepared some such molecules, and proved that the analogue body that produces has activity in external test based on cell.Should be pointed out that and can adopt different experiment in vitro and body inner model in the above-mentioned research, and report tire each other or therewith the place introduce the result and can not compare.

In order to produce GLP-1 analogue body variant, the GLP-1 peptide in the analogue body, joint sequence, hinge sequence or CH2 and CH3 sequence can be by disappearances, add, displacement, variation or modify improve its expression, tire, stability or effector functions.

The anti-GLP-1 variant of wild type GLP-1 sequence and DPP-IV, for example GLP-1 (A2S) or GLP-1 (A2G) can be inserted in the analogue body supporting structure.The sudden change that can carry out peptide improves the characteristic of GLP-1 analogue body.For example the sudden change at n terminal residue may improve the signal effect, and may stablize spiral in the sudden change in helical structure territory, thereby improves and the combining and/or the stability of analogue body of receptor.

The length and form of joint sequence of can suddenling change changes the flexibility that is connected between GLP-1 peptide and the Fc district and stable.Different isotypes can be inserted in the hinge region of molecule.In addition, can suddenly change at the hinge region of analogue body and stablize this molecule.For example, but mutant human IgG4 hinge region prepares Ser²²⁸The mutant of-Pro, thereby the interchain disulfide bond in the stable simulation body.The variation of analogue body Fc part can cause improving the stability of molecule, and changes effector functions, connects as FcR.For example, can the end user or the isotype (or variant of these molecules) of muroid, as the IgG4 of Ala/Ala sudden change.

GLP-1 analogue body of the present invention.A specific limiting examples of the present invention is the GLP-1 analogue body construct (SEQ ID NO:2) according to molecular formula (I):

(P (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is single copy of the GLP-1 peptide (7-36) of biologically active, L is that the series connection of Gly-Ser or Gly-Gly-Gly-Ser flexible joint repeats, V is V_HThe C-end of sequence, the promptly natural J district that has IgG, H is complete IgG1 hinge region, and CH2﹠amp; CH3 belongs to IgG1 isotype subclass.It should be GLP-1 peptide itself or its variant or the manyfold of derivant half-life that the half-life of this construct is considered to, and approximate with the half-life of IgG.

Except that above-mentioned base structure, this paper has also described the variant with potential favourable biological property.The construct that these variants comprise that tendency may reduce from connecting, immune effector miopragia or immunogenicity reduce.The present invention has also looked forward to other can give special characteristic, such as the modification that improves conformation and can cross over blood brain barrier of biologically active peptide.The variant and the modification of suggestion can be combined in any way, to obtain construct with given activity.

Utilize the recombinant DNA method, the GLP-1 peptide is inserted in the intermediate carrier, between immunoglobulin signal peptide and people J sequence.Complementary synthetic oligonucleotide has been adopted in this operation, and its end is compatible with the restriction site in this carrier.These oligonucleotide contain the coded sequence of GLP-1 peptide and the flexible joint coded sequence that is repeated to form by two GGGS.The restricted fragment that contains function element is transferred in the expression vector subsequently.This carrier contains coded sequence, HC constant region 2 (CH2) and the constant region 3 (CH3) of anti-CD4 immunoglobulin promoter and enhancer, human IgG1's hinge region, also contain plasmid replication in the antibacterial and screening, and for the necessary element of screening of stably express in the mammalian cell.

This carrier is directed in the HEK293E cell, and has obtained the expression of wild type GLP-1 MMB in the transient transfection cell.Finish the purification of GLP-1 MMB with standard protein A andSuperose 12 affinity chromatographies, approximately produce the 1.5mg/L transfectional cell.This albumen is the parent material of following described experiment.

The aminoacid sequence of GLP-1 as shown in Figure 1.Functional domain is the coded sequence of above-mentioned peptide.The J sequence is considered to the flexibility that can provide bigger, makes the GLP-1 dimer present correct conformation, and makes this dimer outstanding from the spherical structure of immunoglobulin, and deeply to the breach between two GLP-1 receptors.Contain three cysteine in the IgG1 hinge region.First cysteine usually matches with light chain immunoglobulin (LC), and two other cysteine has then participated in the interchain key between two HC.CH2﹠amp; The CH3 district has constituted this proteic filling part.It is the bonded ability of itself and FcRn that immunoglobulin is considered to have one of reason of long serum half-life, and this FeRn can be by returning to extracellular space with the pinocytosis immunoglobulin, thereby prolong serum half-life.The junction in the binding site of FeRn and CH2 and CH3 district overlapping (Sheilds etc., 2O01, J.Bio.Chem., vol.276 (9), 6591-6604).

As everyone knows, two IgG heavy chains during cell processing by hinge region in disulfide bond between two cysteine be assembled in together, formed homodimer.It is believed that this phenomenon also can occur between the modified peptides, thereby form the GLP-1 analogue body construct of assembling.In addition, it is believed that between two cysteine in the GLP-1 peptide and also will form intrachain disulfide bond.The expected structure of GLP-1 analogue body contains two GLP-1 peptides.This peptide is in the spatial arrangements of N-terminal, and the flexibility of contiguous sequence makes this peptide form the dimer of biologically active.

Embodiment 3:FACS is in conjunction with test.With external FACS in conjunction with testing the activity that detects the GLP-1 analogue body.In order to determine whether GLP-1MMB combines with GLP-1R, the HEK293 cell (1x10 that expresses GLP-1R will be crossed⁶Cell) cultivated 2 hours down at 4 ℃ with GLP-1 MMB (20nM).Washed cell adds fluorescently-labeled secondary detection antibody (1ug/mL, goat anti-human igg, Fc γ specificity) down at 4 ℃, keeps 30 minutes.The fluorescence intensity of these cells detects by flow cytophotometer.Fig. 2 A shows GLP-1 MMB and crosses the HEK293 cell of expressing GLP-1R (Lycoperdon polymorphum Vitt, GLP-1 MMB but do not have two anti-; Black only has two to resist; Redness, negative contrast MMB and two resists; Blueness, GLP-1 MMB and two resists) combination.Fig. 2 B show GLP-1 MMB not with the HEK293 cell of contrast (Lycoperdon polymorphum Vitt, GLP-1 MMB but do not have two anti-; Black only has two to resist; Blueness, GLP-1 MMB and two resists) combination.Fig. 2 C show GLP-1 peptide analogues (A2S) can with the competitive HEK293 cell of expressing GLP-1R (Lycoperdon polymorphum Vitt, the GLP-1 MMB but do not have two and resist of combine of GLP-1 MMB; Black, negative contrast MMB and two resists; Orange, GLP-1 MMB, 0.2nM competitor, two is anti-; Blueness, GLP-1 MMB, 20nM competitor, two is anti-; Redness, GLP-1 MMB, 100nM competitor, two is anti-).

Embodiment 4:cAMP test.GLP-1 combines with its receptor G-G-protein linked receptor, causes the metering dependency of signaling molecule 3 ', 5 '-ring AMP (cAMP) to increase.Can in the cell (Applied Biosystems) of expressing GLP-1R, measure cAMP by analyzed in vitro.In brief, Rinm cell (100,000 cell) is cultivated with GLP-1 peptide (0-30nM) or the GLP-1 MMB (0-100nM) that concentration increases progressively.Cell is dissolved, uses competitive assay to determine the amount of cAMP, and this competitive assay is used the cAMP conjugate and the chemical luminous substrate of alkali phosphatase enzyme mark

The concentration dependent cAMP activity (Fig. 3 A) of wild type GLP-1 MMB can be comparable to GLP-1 peptide (Fig. 3 B) (respectively naturally: EC₅₀=11nM is to 0.4nM).In similar test, GLP-1 in the IgG4 supporting structure (A2G) (Fig. 3 C) and the GLP-1 in the IgG4 supporting structure (A2S) MMB (Fig. 3 D), compare with the wild type GLP-1 MMB in the IgG4 supporting structure, all make the cAMP level in the Rinm cell be increased to a remarkable higher level.

Embodiment 5:DPP-IV breaking test.Because GLP-1 is by the rapid inactivation of DPP-IV, it is quantitative to complete (that is: uncracked) GLP-1 MMB to carry out in vitro tests.In brief, with GLP-1 MMB or peptide (1.2nM) and DPP-IV (1 μ g/mL, R﹠amp; D system) at room temperature cultivates.At different time (0,5,10,15,20,30,40 minutes) afterwards, (100uM Linco) stops this reaction to add the DPP-IV inhibitor.Measure the complete GLP-1 MMB or the amount of peptide with the active ELISA of GLP-1, and draw GLP-1 MMB or peptide standard curve separately.Fig. 4 shows that GLP-1 MMB obviously has stronger resistance to the cracking of DPP-IV with respect to GLP-1.

Embodiment 6: the human serum stability test.Also measured in the serum stability of GLP-1 MMB and guaranteed that other serum albumin enzyme can not cracking or inactivation GLP-1 MMB.In brief, GLP-1 peptide or GLP-1 MMB (30nM) are cultivated down at 37 ℃ in human serum.After different time, (100uM Linco) stops this reaction, uses the active elisa assay sample from the GLP-1 of Linco with the DPP-IV inhibitor.Fig. 5 shows that GLP-1MMB is stable in 24 hours in human serum, and peptide has been decomposed rapidly.

Embodiment 7:GLP-1 MMB causes the insulin secretion in the RINm cell.In order to measure the effect of GLP-1 MMB in insulin secretion, the GLP-1 that working concentration increases progressively (7-36) peptide (0-5nM), exendin-4 peptide (0-5nM) or various GLP-1 analogue body (5 or 50nM) are handled the RINm cell, and measure the amount of insulin secretion with ELISA.All GLP-1 MMB in the RINm cell of test have the activity (Fig. 6) that strengthens insulin secretion.When 50nM, MMB has the activity that is comparable to wild type GLP-1 (7-36) peptide.

Embodiment 8:GLP-1MMB reduces the glucose level of db/db Mus.Carrier vein calibrated shot GLP-1 peptide or GLP-1 (A2S) MMB are used in the db/db Mus fasting in six ages in week two hours then.0.5,1,2,3 and 4 hour monitoring blood glucose after injection.The GLP-1 peptide is blood sugar lowering in 30 minutes, but blood glucose may begin to increase once more owing to the short-half-life of GLP-1 peptide in 60 minutes.Comparatively speaking, dosage causes the reduction (Fig. 7 A) of blood glucose in the whole 4 hours cycle than GLP-1 (A2S) MMB of low 100 times of GLP-1 peptide dosage.In addition, the reduction of blood glucose is dose dependent (Fig. 7 B).

Embodiment 9: the pharmacokinetics of GLP-1 MMB in Mus and machin.In order to measure the pharmacokinetics of four kinds of GLP-1 analogue bodies (A2G, A2S, exedin-cap and wild type), to the MMB of C57/Bl6 Mus intravenous injection 1mg/kg.Carry out cardiac puncture at different time points by the execution Mus and obtain blood plasma.Use Fc, total MMB, active MMB and active fragment that different ELISA is determined at the animal body intracellular metabolite.The complete N-end of active MMB performance peptide still links to each other with the Fc district of analogue body.Second aminoacid (alanine) of peptide is replaced into serine or glycine, has prolonged the half-life of active MMB in the circulation.

To four kinds of different GLP-1MMB constructs of machin Mus intravenous injection, after injection, get blood serum sample with 1.0mg/kg from 10 minutes to 5 days different time points.Identify that with ELISA blood serum sample carries out the quantitative of active MMB.As shown in Figure 8, all four kinds of MMB have shown the phase that distributes fast, are thereafter slower interval phase.The pharmacokinetics constant that is fit to each construct shows that T1/2 is about 3 days, and it is close with the exposure that AUC determined from T=0 to T=120 hour.

Embodiment 10: the effect of GLP-1 MMB in the diabetic mice oral glucose tolerance test.The db/db Mus in eight ages in week was subcutaneous injection GLP-1 MMB (0.02 to 2mg/kg) fasting before 6 hours.After injection, Mus is again by fasting 6 hours, and records the fasting glucose baseline.When t=0, Mus is raised the glucose of 1.0mg/g by force from mouth, and measure blood glucose at different time.Result's hint GLP-1MMB in the oral glucose tolerance test of all dosage shown in Figure 9 is effective for reducing the glucose excursion.

Embodiment 11:GLP-1 MMB is to the effect of fasting glucose in the chronic medication of diabetic mice.To the diabetes db/db Mus in ten ages in week, continuous six every day in week subcutaneous injection carrier or GLP-1MMB (1mg/kg).In research process, measure fasting glucose twice weekly.In the research, the fasting glucose of the animal of processing comparison is according to low (Figure 10), and through six weeks, gap has surpassed 200mg/dL, and (contrast and the animal of handling are respectively: 466 vs 221mg/dL).

Embodiment 12:GLP-1 MMB effect in the oral glucose tolerance test after chronic medication to diabetic mice.As embodiment 11, to the diabetes db/db Mus in ten ages in week, continuous six every day in week subcutaneous injection carrier or GLP-1 MMB (1mg/kg).After 40 days the administration, Mus is carried out oral glucose tolerance test.In brief, when t=0, Mus is raised the glucose of 1.0mg/g by force from mouth, and measure blood glucose at different time.Result's hint shown in Figure 11 GLP-1 MMB in oral glucose tolerance test is effectively for reducing the glucose excursion, and the Mus that this oral glucose tolerance test explanation is handled with GLP-1 MMB is for a long time compared with control animal and can more effectively solve the glucose load.

Embodiment 13:GLP-1 MMB reduces the effect of HbA1c after to the chronic medication of diabeticmice.As embodiment 11 and 12, to the diabetes db/db Mus in ten ages in week, continuous six every day in week subcutaneous injection carrier or GLP-1 MMB (1mg/kg).Before or after 40 days the administration, get whole blood sample, and analyze the percent of HbA1c.As shown in figure 12, in six time-of-weeks, the HbA1C of the animal of handling with GLP-1 increases by 109%, and the animal of contrast increases by 142%.The treated animal of this data declaration can be regulated their long-term blood glucose compared with the control better.

Embodiment 14:GLP-1 MMB is to the effect in the oral glucose tolerance test of normal machin.Normal machin single-dose GLP-1 MMB (1mg/kg) before or after six days, is being carried out oral glucose tolerance test (OGTT).In brief, when t=0, monkey is raised the glucose of 2.0mg/g by force from mouth, and measure blood glucose at different time.Blood sugar level obviously reduces (Figure 13 A) in the OGTT that administration was done after six days, insulin level obviously raise (Figure 13 B).This explanation GLP-1 MMB causes pancreas excreting insulin when high concentration of glucose.

Embodiment 15:GLP-1 MMB after the single dose medication to the effect in the dyeing of the islets of langerhans of insulin Mus (db/db).The diabetic mice (db/db) in 12 ages in week, the GLP-1 MMB (1.5mg/kg) of subcutaneous injection single dose gets pancreas after 4 weeks.In order to determine the existence of insulin, with pancreas section, dyeing.As shown in figure 14, treated animal is compared with control animal, and more insulin dyeing is obviously arranged.

Embodiment 16:GLP-1 MMB postpones gastric emptying to normal Canis familiaris L..By surgical operation stomach tube is inserted in female sleuth (10-15kg) body of general anesthesia, and allows it to recover at least two weeks.Canis familiaris L. was by fasting 24 hours, and water can freely obtain afterwards.Open stomach tube, remove gastric juice and remaining food with 40-50ml warm water.At the 60 minutes before the meal group subcutaneous injection lidamidines to six Canis familiaris L.s, α 2 agonist (0.63mg/kg) were over against the delay according to gastric emptying.15 minutes before the meal cephalic veins of Canis familiaris L. are injected vehicle Control or GLP-1 MMB (0.1mg/kg).5 minutes before the meal, open stomach tube and determine fluidic under one's belt at that time amount, as baseline value, fluid is imported in the stomach rapidly once more.Comprise the test meal of 250ml glucose solution (5g/l) then by intubate, and allow it to stop under one's belt 30 minutes.After 30 minutes, gastric content derived from stomach measure total amount.The gastric content that stays 1ml is analyzed, and surplus is imported in the stomach once more by intubate.In the time of 60,90 and 120 minutes, repeat the mensuration of gastric content amount and the extraction of sample.To the sample determination concentration of glucose of collecting, and be used for being determined at the absolute quantity of the remaining glucose of each time point place stomach.By initial value, in the concentration of each time point place glucose and can determine the percent of remaining glucose in the stomach as the function described point mapping of time.The time of gastric content residue 50% is determined the match of first power by curve.As shown in figure 15, the Canis familiaris L. that gives carrier after in 12.35 ± 3.69 minutes 50% gastric content be drained, and lidamidine over against according to and the Canis familiaris L. that gives GLP-1 MMB shown that the delay of tangible gastric emptying (is respectively: 30.60 ± 6.47 and 59.23 ± 14.46).

Embodiment 17:GLP-1 MMB is blood sugar lowering in to the oral glucose tolerance test (IPGTT) of the Mus of diet induced obesity.In order to set up a kind of muroid model of diet induced obesity, made Mus keep providing high fat diet at least in 27 weeks.It is fat that Mus becomes, and when its fasting blood sugar surpasses 120mg/dl, determine its trouble diabetes.In order to estimate the effect of GLP-1 MMB treatment to level of postprandial blood sugar, the Mus of diet induced obesity is by overnight fasting, and subcutaneous injection 0.02,0.2, or 2mg/kg GLP-1 MMB or vehicle Control.Injected back six hours, the Mus stomach tube is raised the 1.5mg/g glucose.Before injection MMB, t=0, measure blood sugar level withtail vein 15,30,60,90,120,150 and 180 minutes the time.As shown in figure 16, at t=0 and all time point places afterwards, fasting blood sugar has tangible GLP-1 MMB dose dependent to reduce.Calculated the area under curve between t=0 and the t=180, shown that glucose is handled all significantly reduction under all dosage.

Embodiment 18:GLP-1 MMB is (IPGTT) blood sugar lowering in to the intraperitoneal glucose tolerance test of db/db Mus.The male db/db Mus in about 13-15 age in week according to fasting blood glucose level, is divided into 6 every group treatment group randomly.At glucose tolerance test before 6 hours, GLP-1 MMB is pressed 0.02mg/kg or 0.1mg/kg to the Mus administration, maybe will bear contrast MMB by 0.1mg/kg to the Mus administration.Before 5 minutes, from tail vein blood, measure glucose at glucose tolerance test with portable glucometer.Then to Mus peritoneal injection 1mg/g D-glucose, 10 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes monitoring blood sugar levels.Shown in Figure 17 A, blood sugar level obviously reduces in All Time in two groups that handle with GLP-1 MMB.Other several treated animals that execution is handled with the same manner were measured insulin level in the time of t=10 minute.The amount of the insulin that discharged in 10 minutes has the dose dependent of following GLP-1 MMB dosage to increase.

Advantage: this recruit provides some advantages that surpass other GLP-1 analog as the application of the therapeutic agent oftreatment type 2 diabetes mellitus.For example, it may prolong the half-life of GLP-1 peptide.And, wild type GLP-1 peptide resistant protease degraded, especially DPP-IV in the analogue body structure stand.This may make wild type GLP-1 Toplink be used for the treatment of, rather than the mutant peptide.Because GLP-1 is a kind of natural peptide, so may be littler than the immunoreation among the patient who is using mutant GLP-1 analogue treatment with the immunoreation among the patient of GLP-1 analogue body treatment.In addition, the large scale of GLP-1 MMB may prevent that it from passing through blood brain barrier.This may provide an advantage, because feel sick and anxiety is associated with the bonded GLP-1R of GLP-1 in the brain.In addition, the analogue body platform causes the expression of two peptides on an analogue body molecule.This may make the GLP-1 peptide interact, and forms a dimer part that can increase pair cell surface GLP-1 receptor affinity.

Should be understood that and to adopt other method different to put into practice the present invention with specific descriptions method in above-mentioned explanation and the example.

According to foregoing, many improvement of the present invention and variation all are possible, thereby also within the scope of the invention.

Table 1

Sequence table

SEQ?ID?NO：1

SEQ?ID?NO：2

SEQ?ID?NO：3

SEQ?ID?NO：5

SEQ?ID?NO：6

SEQ?ID?NO：7

SEQ?ID?NO：8

SEQ?ID?NO：9

SEQ?ID?NO：10

SEQ?ID?NO：11

SEQ?ID?NO：12

SEQ?ID?NO：13

SEQ?ID?NO：14

SEQ?ID?NO：15

SEQ?ID?NO：16

SEQ?ID?NO：17

SEQ?ID?NO：18

SEQ?ID?NO：19

SEQ?ID?NO：20

SEQ?ID?NO：21

SEQ?ID?NO：22

SEQ?ID?NO：23

SEQ?ID?NO：24

SEQ?ID?NO：25

SEQ?ID?NO：26

SEQ?ID?NO：27

SEQ?ID?NO：28

SEQ?ID?NO：29

SEQ?ID?NO：30

SEQ?ID?NO：31

SEQ?ID?NO：32

SEQ?ID?NO：33

SEQ?ID?NO：34

SEQ?ID?NO：35

SEQ?ID?NO：36

SEQ?ID?NO：37

SEQ?ID?NO：38

SEQ?ID?NO：39

SEQ?ID?NO：40

SEQ?ID?NO：41

SEQ?ID?NO：42

SEQ?ID?NO：43

SEQ?ID?NO：44

SEQ?ID?NO：45

SEQ?ID?NO：46

SEQ?ID?NO：47

SEQ?ID?NO：48

SEQ?ID?NO：49

SEQ?ID?NO：50

SEQ?ID?NO：51

SEQ?ID?NO：52

SEQ?ID?NO：53

SEQ?ID?NO：54

SEQ?ID?NO：55

SEQ?ID?NO：56

SEQ?ID?NO：57

SEQ?ID?NO：58

SEQ?ID?NO：59

SEQ?ID?NO：60

SEQ?ID?NO：61

SEQ?ID?NO：62

SEQ?ID?NO：63

SEQ?ID?NO：64

SEQ?ID?NO：65

SEQ?ID?NO：66

SEQ?ID?NO：67

SEQ?ID?NO：68

Claims (37)

1. at least a GLP-1CH1 lacks analogue body nucleic acid, comprises at least a polynucleotide of the aminoacid sequence of coding SEQ ID NO:2 or 4, or polynucleotide complementary with it.

2. at least a GLP-1 CH1 lacks analogue body nucleic acid, comprises at least a polynucleotide of at least a aminoacid sequence among the coding SEQ ID NO:7-14, or polynucleotide complementary with it.

3. at least a GLP-1 CH1 lacks analogue body nucleic acid, comprises at least a polynucleotide of the polypeptide of coding molecule formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby for it provides the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

4. at least a GLP-1CH1 disappearance analogue body polypeptide comprises all continuous amino acids in SEQ ID NO:2 or 4.

5. at least a GLP-1 CH1 disappearance analogue body polypeptide comprises all at least a among SEQ ID NO:7-14 continuous amino acids.

6. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide that is selected from the biologically active of at least a aminoacid sequence in SEQ ID NO:1 and 6, and L is selected from GS, GGS, GGGS (SEQ ID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ ID NO:20); V is selected from GTLVTVSS (SEQ ID NO:21), GTLVAVSS (SEQ IDNO:22), GTAVTVSS (SEQ ID NO:23), TVSS (SEQ ID NO:24) and AVSS (SEQ ID NO:25); H is EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO:26), CH2 is SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:43), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:44), n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

7. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is selected from the GLP-1 polypeptide of the biologically active of SEQ ID NO:6 at least one, and L is selected from GS, GGS, GGGS (SEQ ID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ ID NO:20); V is selected from GTLVTVSS (SEQID NO:21), GTLVAVSS (SEQ ID NO:22), GTAVTVSS (SEQID NO:23), TVSS (SEQ ID NO:24) and AVSS (SEQ ID NO:25); H is ESKYGPPCPSCPAPEFLGGP (SEQ ID NO:27), CH2 is SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:45), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:46), n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

8. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is selected from the GLP-1 polypeptide of the biologically active of SEQ ID NO:6 at least one, and L is selected from GS, GGS, GGGS (SEQ ID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ ID NO:20); V is selected from GTLVTVSS (SEQIDNO:21), GTLVAVSS (SEQ ID NO:22), GTAVTVSS (SEQ ID NO:23), TVSS (SEQ ID NO:24) and AVSS (SEQ ID NO:25); H is ESKYGPPCPPCPAPEAAGGP (SEQ ID NO:28), CH2 is SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:45), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:46), n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

9. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby for it provides the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO:43), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:44), n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

10. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby for it provides the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO:45), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:46), n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

11. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is selected from the GLP-1 polypeptide of the biologically active of SEQ ID NO:6 at least one, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby for it provides the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

12. at least a GLP-1 CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is selected from GS, GGS, GGGS (SEQ ID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ ID NO:20); V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, and o, p, q, r, s and t can be the integer among the 0-10 independently.

13. at least a GLP-1 CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant; L is at least one joint sequence, and it can have optional orientation and binding characteristic by making analogue body, thereby provides the polypeptide of structural flexibility for it; V is selected from GTLVTVSS (SEQ ID NO:21), GTLVAVSS (SEQ ID NO:22), GTAVTVSS (SEQ ID NO:23), TVSS (SEQ ID NO:24) and AVSS (SEQ ID NO:25); H is at least one part of immunoglobulin variable hinge region; CH2 is at least one part of immunoglobulin CH2 constant region; CH3 is at least one part of immunoglobulin CH3 constant region; N is the integer among the 1-10, and o, p, q, r, s and t can be the integer among the 0-10 independently.

14. at least a GLP-1CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby provides the polypeptide of structural flexibility for it; V is at least one part of immune globulin variable region C-end, H is selected from EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO:26), ESKYGPPCPSCPAPEFLGGP (SEQ ID NO:27) and ESKYGPPCPPCPAPEAAGGP (SEQ ID NO:28), CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, and o, p, q, r, s and t can be the integer among the 0-10 independently.

15. at least a GLP-1 CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby for it provides the polypeptide of structural flexibility, V is at least one part of immune globulin variable region C-end, H is selected from EPKSADKTHTCPPCPAPEAAGGP (SEQ ID NO:29), EPKSADKTHTCPPCPAPELAGGP (SEQ ID NO:30), EPKSADKTHTCPPCPAPEALGGP (SEQ ID NO:31), EPKSADKTHTCPPCPAPELEGGP (SEQ ID NO:32), EPKSSDKTHTCPPCPAPEFLGGP (SEQ ID NO:33), EPKSADKTHACPPCPAPELLGGP (SEQ ID NO:34), EPKSADKAHTCPPCPAPELLGGP (SEQ ID NO:35) and EPKSADKTHTCPPCPAPELLGGP (SEQ ID NO:36), ADKTHTCPPCPAPELLGGP (SEQ ID NO:37), THTCPPCPAPELLGGP (SEQ ID NO:38), ESKYGPPCPSCPAPEAAGGP (SEQ ID NO:39), ESKYGPPCPPCPAPELLGGP (SEQ ID NO:40), CPPCPAPELLGGP (SEQ ID NO:41) and CPPCPAPEAAGGP (SEQ ID NO:42), CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, o, p, q, r, s and t can be the integer among the 0-10 independently.

16. at least a GLP-1 CH1 lacks the analogue body polypeptide, comprises the polypeptide of molecular formula (I):

(Pep (n)-L (o)-V (p)-H (q)-CH2 (r)-CH3 (s)) (t), wherein P is the GLP-1 polypeptide of at least one biologically active, variant or derivant, L is at least one joint sequence, it can have optional orientation and binding characteristic by making analogue body, thereby provides the polypeptide of structural flexibility for it; V is at least one part of immune globulin variable region C-end, H is at least one part of immunoglobulin variable hinge region, CH2 is at least one part of immunoglobulin CH2 constant region, CH3 is at least one part of immunoglobulin CH3 constant region, n is the integer among the 1-10, and o, p, q, r, s and t can be the integer among the 0-10 independently.

17. according to the GLP-1 CH1 disappearance analogue body nucleic acid of claim 1-16 described in each or GLP-1 CH1 disappearance analogue body polypeptide, wherein said polypeptide has at least a activity of at least a P polypeptide.

18. anti-hereditary sexual type monoclonal or polyclonal antibody, fusion rotein, or its fragment can combine with at least a GLP-1 CH1 disappearance analogue body polypeptid specificity of claim 4-16 in each.

19. according to the GLP-1 CH1 disappearance analogue body nucleic acid of claim 1-3 described in each or coding claim 4-18 in each at least a GLP-1 CH1 disappearance analogue body polypeptide or the GLP-1 CH1 disappearance analogue body nucleic acid of GLP-1 CH1 disappearance analogue body antibody, or polynucleotide complementary with it.

20. comprise the GLP-1 CH1 disappearance analogue body carrier of at least a isolating nucleic acid of claim 19.

21. comprise the GLP-1 CH1 disappearance analogue body host cell of the isolating nucleic acid of claim 19.

22. according to the disappearance of the GLP-1 CH1 described in the claim 21 analogue body host cell, wherein said host cell is for being selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,653, SP2/0,293, NSO, DG44CHO, CHO K1, HeLa, myeloma or lymphoma cell, or at least a cell in the deriving arbitrarily of above-mentioned cell, immortalization or the transformant.

23. method that generates at least a GLP-1 CH1 disappearance analogue body polypeptide or GLP-1 CH1 disappearance analogue body antibody, be included in external, the body or under the original position condition, the nucleic acid of translation claim 19 makes GLP-1 CH1 disappearance analogue body or antibody can detect or callable quantity is expressed.

24. a compositions comprises at least a GLP-1 CH1 disappearance analogue body nucleic acid, GLP-1 CH1 disappearance analogue body polypeptide, or the GLP-1 CH1 disappearance analogue body antibody of claim 1-19 in each.

25. according to the compositions described in the claim 24, wherein said composition further comprises at least a medicine acceptable carrier or diluent.

26. according to the compositions described in the claim 24, further comprise at least a compositions, said composition contains at least a chemical compound for the treatment of effective dose, compositions or polypeptide, this chemical compound, compositions or polypeptide are selected from diabetes or insulin metabolism related drugs, detectable label or reporter molecule, the TNF antagonist, anti-infectives, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomic nervous system (ANS) medicine, respiratory drugs, gastrointestinal (GI) tract drug, hormonal medicaments, the medicine that is used for balance body fluid or electrolyte, blood substance, antitumor drug, immunoregulation medicament, eye, ear or nose medication, local application, nutritional drugs, at least a in cytokine or the cytokine antagonist.

27. according to the compositions described in the claim 24, its form is to be selected from least a in liquid, gas or dry thing, solution, mixture, suspension, emulsion or colloid, lyophilized formulations or the powder.

28. diagnose or treat the conditions associated method of GLP-1 in cell, tissue, organ or the animal, comprising for one kind:

(a) make cell, tissue, organ or animal contact or it is used a kind of compositions, said composition contains at least a GLP-1 CH1 disappearance analogue body nucleic acid, polypeptide or the antibody of claim 1-19 in each of effective dose.

29. according to the method described in the claim 28, wherein GLP-1 is conditions associated is diabetes or congestive heart failure.

30. according to the method described in the claim 28, wherein said effective dose is the GLP-1 CH1 disappearance analogue body antibody that every kilogram of above-mentioned cell, tissue, organ or animal are used 0.0001-50mg; 0.1-500mg above-mentioned GLP-1 CH1 disappearance analogue body; Or the above-mentioned GLP-1 CH1 disappearance analogue body nucleic acid of 0.0001-100 μ g.

31. according to the method described in the claim 28, wherein above-mentioned contact or to use be by being selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical; in the pathological changes; medicine group; vagina; rectum; cheek; Sublingual; at least a pattern in intranasal or the percutaneous approach realizes.

32. according to the method described in the claim 28, further be included in before above-mentioned (a) contact or use, simultaneously or afterwards, use at least a compositions, said composition contains at least a chemical compound or the polypeptide for the treatment of effective dose, and this chemical compound or polypeptide are selected from diabetes or insulin metabolism related drugs, detectable label or receptor, the TNF antagonist, anti-infectives, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomic nervous system (ANS) medicine, respiratory drugs, gastrointestinal (GI) tract drug, hormonal medicaments, the medicine that is used for balance body fluid or electrolyte, blood substance, antitumor drug, immunoregulation medicament, eye, ear or nose medication, local application, nutritional drugs, at least a in cytokine or the cytokine antagonist.

33. device, contain at least a isolating GLP-1 CH1 disappearance analogue body polypeptide of claim 1-19 in each, antibody or nucleic acid, wherein this device is applicable to by being selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical; in the pathological changes; medicine group; vagina; rectum; cheek; Sublingual; at least a pattern in intranasal or the percutaneous approach contacts or uses described at least a GLP-1 CH1 disappearance analogue body polypeptide; antibody or nucleic acid.

34. goods that are used for people's medicine or diagnostic uses comprise packaging material and container, this container contains at least a isolating GLP-1 CH1 disappearance analogue body polypeptide, antibody or the nucleic acid of claim 1-19 in each.

35. the goods of claim 34, wherein said vesse is a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, in the bronchus, in the abdomen, in the capsule, in the cartilage, intracavity, in the body cavity, in the cerebellum, Intraventricular, colonic, in the cervix uteri, gastric, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate, in the lung, internal rectum, in the kidney, in the retina, in the spinal column, in the synovial membrane, intrathoracic, intrauterine, intravesical; in the pathological changes; medicine group; vagina; rectum; cheek; Sublingual; an ingredient of intranasal or percutaneous delivery device or system.

36. one kind is used for generating each the method for at least a isolating GLP-1 CH1 disappearance analogue body polypeptide, antibody or nucleic acid of claim 1-19, this method comprise provide at least a can be can detect or callable scale reaches host cell, transgenic animal, transgenic plant, the plant cell of aforementioned polypeptides, antibody or nucleic acid.

37. at least a GLP-1 CH1 disappearance analogue body polypeptide, antibody or nucleic acid that method generated by claim 36.

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