CN109762059A - A kind of novel glucagon analogue and its application - Google Patents

Abstract

The present invention relates to a kind of novel glucagon analogue and its applications.Novel glucagon analogue of the invention has longer half-life period and better insulin secretion accelerating activity compared with Wild type human GLP1, has very strong long-acting blood sugar reducing function.

Description

A kind of novel glucagon analogue and its application

Technical field

The invention belongs to polypeptide drugs technical fields.Promoting pancreas islet more particularly to -1 receptor stimulating agent drug of novel glp-1 Element secretion and it is hypoglycemic on application, and the application for the treatment of type-2 diabetes mellitus and fat aspect.

Background technique

Type-2 diabetes mellitus is a kind of long-term metabolic disorder, it is characterised in that hyperglycemia, insulin resistance and opposite shortage pancreas Island element.Long-term complications caused by patient's hyperglycemia include heart disease, apoplexy, and diabetic retinopathy even results in blindness, Renal failure, four limbs thrombosis even result in amputation etc..Type-2 diabetes mellitus accounts for about the 90% of diabetes cases.From 1960 Since year, the significant increase of the disease incidence of type-2 diabetes mellitus.By 2017, about 4.5 hundred million people were diagnosed with II type glycosuria Disease, in contrast, there are about 30,000,000 people within 1985.The World Health Organization, which predicts the whole world in 2035 and there will be over 600,000,000 people, to be suffered from Type-2 diabetes mellitus.

158 amino acid compositions of glucagon reason.Glucagon-like-peptide-1 is cut at different positions (glucagon-like peptide-1,GLP-1).Glucagon-like-peptide-1 (glucagon-like peptide-1, GLP- 1) the single peptides mainly secreted by food stimulus intestinal epithelial cells.GLP-1 passes through its exciting receptor, promotes pancreas islet Element secretion, protects beta Cell of islet, and glucagon suppression secretion inhibits gastric emptying, reduces appetite.Thus, it can be used for two types sugar The treatment of urine disease and obesity.Biologically active GLP-1 is mainly GLP-1 (7-36) amide and GLP-1 (7- in human body 37), but all by dipeptidyl peptidase IV (DPP-IV), (half-life period less than 5min), does not have clinical use valence to hydrolytic inactivation rapidly Value.Current GLP-1 receptor stimulating agent drug multi-pass carries out structural modification to GLP-1 excessively, eliminates or cover DPP-IV digestion position Point, while retaining its pharmacological activity and obtaining, such as Liraglutide (Liraglutide) He Dula glycopeptide (Dulaglutide). In addition, the natural polypeptides similar with GLP-1 structure may also pharmacological activity having the same.Exendin-4 is exactly from lizard saliva The glucagon analogue isolated in liquid is made of 39 amino acid, there is about 53% homology with GLP-1, not by DPP-IV degradation, and there is longer half-life period and stronger bioactivity.Exenatide based on Exendin-4 sequent synthesis (Exenatide) it has listed.

This field needs a kind of novel glucagon analogue, has longer half compared with Wild type human GLP1 Decline phase and better insulin secretion accelerating activity.

Summary of the invention

By screening and verifying on a large scale, the inventors discovered that a kind of novel glucagon analogue.

Specifically, the present invention provides the following contents:

In one embodiment, the present invention provides a kind of glucagon analogues, wherein the glucagon Analog has longer half-life period and better insulin secretion accelerating activity compared with Wild type human GLP1.

In further embodiment, the present invention provides a kind of glucagon analogues, wherein the pancreas hyperglycemia The sequence of plain analog are as follows:

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly- Xaa17-Ala-Thr-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Leu-Glu(SEQ.ID NO.3),

Xaa17 is Ser, His, Gln, and any one of Ala or Lys amino acid, Xaa20 is Ser, His, Gln, Ala Or any one of Lys amino acid, Xaa28 are Ser, His, Asp, any one of Ala or Lys amino acid.

In further embodiment, the present invention provides a kind of glucagon analogues, wherein the pancreas hyperglycemia The sequence of plain analog are as follows:

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly- Xaa17-Ala-Ala-Xaa20-Glu-Phe-Val-Ala-Trp-Leu-Val-Xaa28-Ser-Leu-Glu(SEQ.ID NO.4),

Xaa17 is Ser, His, Gln, and any one of Ala or Lys amino acid, Xaa20 is Ser, His, Gln, Ala Or any one of Lys amino acid, Xaa28 are Ser, His, Asp, any one of Ala or Lys amino acid.

In further embodiment, the present invention provides a kind of glucagon analogues, wherein the pancreas hyperglycemia The sequence of plain analog is selected from SEQ.ID NO.1-16.

In further embodiment, the present invention provides the pharmaceutically acceptable of above-mentioned glucagon analogue Salt, solvate, prodrug or their any combination.

It is demonstrated experimentally that glucagon analogue of the present invention is readily synthesized, and can effectively prolonged excitement GLP-1 receptor promotes the secretion of insulin, enhances insulin sensitivity, reduces blood glucose, and have better stability.Into One step, the glucagon analogue have the vivo biodistribution stability of enhancing and extend half-life period, obtain more long-acting work Property.

In another embodiment, the present invention provides a kind of dimer or polymer, it includes two or more Glucagon analogue of the invention.

In another embodiment, the present invention provides a kind of conjugates, and it includes glucagons of the invention Analog and conjugate fraction.

In further embodiment, the present invention provides a kind of conjugate, wherein the glucagon analogue with Heterologous peptide analogue fusion.

In further embodiment, the present invention provides a kind of pharmaceutical compositions, and it includes pancreas hyperglycemia of the invention Plain analog, dimer of the invention or polymer, conjugate of the invention, or combinations thereof and pharmaceutically acceptable carrier, Diluent or excipient.

In another embodiment, the present invention provides one kind loses weight increase or is lured in subject in need The method for leading weight loss comprising weight gain is effectively reduced or the amount of weight loss is induced to apply to patient in need Pharmaceutical composition of the invention.In further embodiment, the amount effectively treats the obesity of subject in need.

In another embodiment, the present invention provides a kind of methods for treating diabetes comprising in need Patient applies pharmaceutical composition of the invention.

In another embodiment, the present invention provides a kind of glucagon analogues of the invention, of the invention Dimer or polymer or conjugate of the invention increase or induce for losing weight in subject in need in preparation Purposes in the drug of weight loss.

In another embodiment, the present invention provides a kind of glucagon analogues of the invention, of the invention Dimer or polymer or conjugate of the invention are preparing the purposes in the drug for treating diabetes.

The method for preparing peptide

Glucagon analogue of the invention can be obtained by method as known in the art.Recombine the suitable of peptide Method is described in such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press,Oxford,United Kingdom,2005；Peptide and Protein Drug Analysis,ed.Reid, R.,Marcel Dekker,Inc.,2000；Epitope Mapping, ed.Westwood et al., Oxford University Press,Oxford,United Kingdom,2000；In U.S. Patent number 5,449,752.

In some embodiments, glucagon analogue or peptide as described herein can be synthesized by company trade.At this Aspect, the peptide can be synthesis, recombination, separation and/or purifying.

In addition, mark can be used in the case where analog of the invention does not include any non-coding or unnatural amino acid Quasi- recombination method generates glucagon analogue using the nucleic acid recombination of the amino acid sequence of encoding analogs.See, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual. the 3rd edition, Cold Spring Harbor Press,Cold Spring Harbor,NY 2001；With Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, NY, 1994.

In some embodiments, glucagon analogue of the invention is separation.Term as used herein " divides From " mean to remove from its natural surroundings.In an exemplary embodiment, analog is via recombination method preparation and analog It is separated from host cell.

In some embodiments, glucagon analogue of the invention is purifying.Term as used herein is " pure Change " referring to molecule or compound, (it is in some respects usually with molecule or compound natural to be substantially free of pollutant Or in natural environment in conjunction with) form separation and mean to cause purity to increase since the other components with original composition separate Add.Purified peptide or compound includes for example being substantially free of nucleic acid molecules, lipid and carbohydrate or other starting materials The peptide of matter or the intermediate for using or being formed during the chemical synthesis of peptide.Generally acknowledge that " purity " is relative terms, and need not understand For absolute purity or absolutely enrichment or absolutely selection.In some respects, purity be at least or about 50%, at least or about 60%, extremely Less or about 70%, at least or about 80% at least or about 90% (for example, at least or about 91%, at least or about 92%, at least or About 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least Or about 99% or about 100%).

Conjugate

The present invention further provides be conjugated to heterologous portion comprising one or more glucagon analogues as described herein The conjugate divided.As used herein term " heterologous moiety " and term " conjugate fraction " are synonymous, and refer to it is described herein The different any molecule of glucagon analogue (chemistry or biochemistry, naturally occurring or non-coding).It can be connected to The exemplary conjugate fraction of any analog as described herein includes but is not limited to heterologous peptides or polypeptide (including such as blood plasma egg It is white), targeting agent, immunoglobulin or part thereof (for example, variable region, the area CDR or Fc), diagnostic flag (the same position of such as radioactivity Element, fluorogen or enzyme label), polymer (including water-soluble polymer) or other treatment or diagnosticum.In some embodiments In, the conjugate comprising analog and plasma protein of the invention is provided, wherein the plasma protein is selected from albumin, turns iron egg White, fibrinogen and globulin.In some embodiments, the plasma protein fraction of conjugate for albumin or turns iron egg It is white.In some embodiments, during conjugate includes one or more glucagon analogues as described herein and is following One or more: (it is different from glucagon as described herein to peptide and/or GLP-1 receptor active glucagon is similar Object), polypeptide, nucleic acid molecules, antibody or its segment, polymer, quantum dot, small molecule, toxin, diagnosticum, carbohydrate, ammonia Base acid.

In some embodiments, heterologous moiety is the peptide and conjugation different from glucagon analogue as described herein Object is fusogenic peptide or chimeric peptide.In some embodiments, heterologous moiety is that the peptide of 1-21 amino acid extends.In particular implementation It is described to extend the C-terminal for being connected to glucagon analogue in scheme.

It is described to extend to single amino acid or dipeptides in some particular aspects.In specific embodiments, the extension packet Containing amino acid selected from the following: charge residue (for example, negatively charged amino acid (such as Glu), positively charged amino acid), Amino acid comprising hydrophilic parts.In some respects, described to extend to Gly, Glu, Cys, Gly-Gly, Gly-Glu.

In some embodiments, described to extend comprising GPSSGAPPPS, GGPSSGAPPPS, KRNRNNIA or KRNR Amino acid sequence.In a particular aspect, amino acid sequence is connected via the C-terminal amino acid of glucagon analogue.Some In embodiment, GPSSGAPPPS, GGPSSGAPPPS, KRNRNNIA or KRNR any amino acid sequence is bonded via peptide It is bonded to the C-terminal of glucagon analogue.

In some embodiments, heterologous moiety is polymer.In some embodiments, polymer is selected from: polyamide； Polycarbonate；Polyalkylene class (polyalkylenes) and its derivative, including polyalkylene glycol, polyalkylene oxide, poly- pair Alkylene；The polymer of acrylate and methacrylate, including poly- (methyl methacrylate), poly- (first Base ethyl acrylate), poly- (butyl methacrylate), poly- (Isobutyl methacrylate), poly- (hexyl methacrylate), poly- (isodecyl methacrylate), poly- (lauryl methacrylate), poly- (methyl acrylate), gather poly- (phenyl methacrylate) (isopropyl acrylate), poly- (isobutyl acrylate) and poly- (octadecyl ester)；Polyvinyl, including polyethylene Alcohol, polyvinylether, polyvinyl ester, polyvinylhalide, poly- (vinyl acetate) and polyvinylpyrrolidone；Polyglycolide；Poly- silicon oxygen Alkane；Polyurethane and its copolymer；Cellulose, including alkylcellulose, hydroxy alkyl cellulose, cellulose ether, cellulose esters, nitre Base cellulose, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxyl-propyl methylcellulose, hydroxybutyl methyl Cellulose, cellulose acetate, cellulose propionate, cellulose acetate-butyrate, Cellacefate, carboxyethylcellulose Element, cellulose triacetate and sulfate cellulose sodium salt；Polypropylene；Polyethylene, including it is poly(ethylene glycol), poly- (ethylene oxide) and poly- (ethylene terephthalate)；And polystyrene.

In some respects, polymer is biodegradable polymer, including synthesising biological degradable polymer (for example, cream Polymer, polyanhydride, poly- (original) acid esters, the polyurethane, poly- (butyric acid), poly- (valeric acid) and poly- (lactide-co-of acid and glycolic Caprolactone)) and natural biodegradable polymer (for example, alginate and other polysaccharide (including glucan and cellulose), Collagen, its chemical derivative (replace, add chemical group such as alkyl, alkylidene, hydroxylating, oxidation and this field skill Other modifications that art personnel usually carry out), albumin and other hydrophilic proteins (for example, zeins (zein) and its His prolamin (prolamine) and hydrophobic proteins)) and its any copolymer or mixture.In general, these materials It by enzymatic hydrolysis or is exposed to water in vivo, degraded by surface or bulk erosion (bulk erosion).

In some respects, polymer is bioadhesive polymer, such as bioerodible hydrogel (by H.S.Sawhney, C.P.Pathak and J.A.Hubbell are described in Macromolecules, and 1993,26,581-587, religion Lead and be incorporated herein), poly- hyaluronic acid, casein, gelatin, glutin (glutin), polyanhydride, polyacrylic acid, alginic acid Salt, chitosan, poly- (methyl methacrylate), poly- (ethyl methacrylate), poly- (butyl methacrylate), poly- (methyl-prop Olefin(e) acid isobutyl ester), poly- (hexyl methacrylate), poly- (isodecyl methacrylate), poly- (lauryl methacrylate), poly- (phenyl methacrylate), poly- (methyl acrylate), poly- (isopropyl acrylate), poly- (isobutyl acrylate) and poly- (acrylic acid Octadecane ester).

In some embodiments, polymer is water-soluble polymer or hydrophilic polymer.Herein in " hydrophily portion Point " under further describe hydrophilic polymer.Suitable water-soluble polymers are known in the art and including such as polyethylene pyrroles Pyrrolidone, hydroxypropyl cellulose (HPC；Klucel), hydroxypropyl methyl cellulose (HPMC；Methocel), NC Nitroncellulose, hydroxyl Ethyl cellulose, hydroxypropyl butylcellulose, droxypropylpentylcellulose, methylcellulose, ethyl cellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxy alkyl cellulose, various cellulose ethers, cellulose acetate, Carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, vinyl acetate/crotonic acid copolymers, polymethyl Sour hydroxyl alkyl ester, methacrylic acid hydroxyl methyl esters, methacrylic acid copolymer, polymethylacrylic acid, polymethyl methacrylate, Maleic anhydride/ethylene methacrylic ether copolymer, polyvinyl alcohol, Sodium Polyacrylate and calcium polyacrylate (CPA), polyacrylic acid, acidic carboxypolymer are poly- Close object, carbomer, carboxyl vinyl polymer, poloxalkol, poly- ethylene methacrylic ether -co- horse Come acid anhydrides, carboxymethylamide, methacrylic acid potassium divinyl benzene copolymer, polyoxy ethyl glycol, polyethylene oxide, He Qiyan Biology, salt and combination.

In specific embodiments, polymer is polyalkylene glycol, including such as polyethylene glycol (PEG).

In some embodiments, heterologous moiety is carbohydrate.In some embodiments, carbohydrate is single Sugared (for example, glucose, galactolipin, fructose), disaccharides (for example, sucrose, lactose, maltose), oligosaccharides are (for example, gossypose, wood Sugar) or polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose (callose), laminarin (laminarin), xylan, mannosan, fucoidin (fucoidan) or galactomannans).

In some embodiments, heterologous moiety is lipid.In some embodiments, lipid is fatty acid, class 20 Alkanoic acid (eicosanoid), prostaglandin, leukotriene, thromboxane, N- acyl ethanol amine, glycerol lipid are (for example, monosubstituted sweet Oily, disubstituted glycerol, tri-substituted glycerol), glycerophosphatide is (for example, phosphatidyl choline, phosphatidylinositols, phosphatidyl-ethanolamine, phosphorus Acyl serine), sphingolipid (for example, sphingol, ceramide), sterol lipid (for example, steroids, cholesterol), prenol Lipid (prenol lipid), glycolipid matter or polyketide (polyketide), oil, wax, cholesterol, sterol, fat-soluble dimension Raw element, monoglyceride, Diglyceride, triglycerides, phosphatide.

In some embodiments, heterologous moiety is connected to analog of the invention via non-covalent or covalent bonding.? Illustrative aspect, heterologous moiety are connected to analog of the invention via connector.It can be by covalent chemical bond, physical force (such as Electrostatic interaction, interaction of hydrogen bond, ionic interaction, Van der Waals interaction or hydrophobicity or hydrophily phase interaction With) come realize connection.It can be used a variety of non-covalent associations systems, including biotin-avidin, ligand/receptor, enzyme/ Substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cell adhesion molecule companion；Or there is affinity each other Any binding partners or its segment.

In some embodiments, by make the Target amino acid Residue of analog with can be with these target amino acids The organic derivatizing agents reaction of selected side chain or N-terminal or the reaction of C-terminal residue and via direct covalent bonds by glucagon class Conjugate fraction is connected to like object.Reactive group on analog or conjugate fraction include for example aldehyde, amino, ester, sulfydryl, α-halogen acetyl group, Maleimido or diazanyl.Derivating agent includes that such as maleimidobenzoyl sulfosuccinic acyl is sub- Amine ester (being conjugated via cysteine residues), n-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride or Other reagents as known in the art.Alternatively, conjugate fraction can via between intermediate vector such as polysaccharide or peptide carrier in succession It is connected to analog.The example of polysaccharide carrier includes aminoglucan.The example of appropriate polypeptides carrier includes polylysine, polyglutamic The mixed polymer of acid, poly-aspartate, its copolymer and these amino acid and other amino acid (such as serine), to assign Dissolution properties needed for giving gained load carriers.

Most commonly make Cysteinyl residues anti-to alpha-halogenate acetic acid esters (and corresponding amine) (such as monoxone, chloroacetamide) It should be to obtain carboxymethyl or carboxamide groups methyl-derivatives.Cysteinyl residues also by with bromine trifluoroacetone, the bromo- β-of α- (5- imidazole radicals (imidozoyl)) propionic acid, chloroacetyl phosphate, N- alkyl maleimide, 3- nitro -2- pyridyl group curing Object, methyl 2- pyridyl disulfide, pCMBA ester, 2- chloromercuri -4- nitrophenol or chloro- 7- nitro benzo - 2- oxa- -1,3- diazole reacts and derivatization.

Histidyl residues at pH 5.5-7.0 with pyrocarbonic acid diethyl ester by reacting and derivatization, because of the reagent pair Histidyl side chain is relative specificity.P-bromophenacyl bromide is also useful；Reaction is preferably in the 0.1M of pH 6.0 It is carried out in sodium cacodylate.

Lysyl- and n terminal residue and succinic anhydride or other carboxylic acid anhydride reactants.It is performed the derivatization with these reagents Have the function of reversing the charge of lysinyl residues.Other suitable agents for derivatization containing alpha-amino residue include Asia Propylhomoserin ester (such as pyridinecarboxylic methyl ester imidate), phosphopyridoxal pyridoxal phosphate, pyridoxal, chlorine boron hydride, trinitrobenzene sulfonic acid, O- first Base isourea, 2,4- pentanedione, and reacted with the transaminase-catalyzed of glyoxylic ester.

Arginyl- residue is by (wherein having phenyl glyoxal, 2,3- diacetyl, 1,2- with one or more of conventional reagents Cyclohexanedione and ninhydrin) it reacts and is modified.Because guanidine functional group has high pKa, the derivatization of arginine residues It needs to react and carry out under alkaline condition.In addition, these reagents can be reacted with the group of lysine and arginine epsilon-amino group.

Can carry out the specific modification of tyrosyl- residue, wherein especially concern by with aromatic diazonium compounds or tetranitro Methane reaction and spectral marker is introduced into tyrosyl- residue.Most commonly, N- acetyl imidazole and tetranitro first are used respectively Alkane forms O- acetyl tyrosyl substratess matter and 3- nitro-derivative.

Carboxyl side group (aspartyl or glutamyl) is selected and reacting with carbodiimides (R-N=C=N-R') It modifies to selecting property, wherein R and R' is different alkyl, such as 1- cyclohexyl -3- (2- morpholinyl -4- ethyl) carbodiimides or 1- Ethyl -3- (4- nitrogen (azonia) -4,4- dimethyl amyl group) carbodiimides.In addition, aspartyl and glutamyl are residual Base is converted to asparaginyl- and glutaminyl residues and reacting with ammonium ion.

Other modifications include: the hydroxylating of proline and lysine；The phosphorus of the hydroxyl of seryl- or threonyl residues Acidification；Lysine, arginine and histidine side chains alpha-amino methylation (T.E.Creighton, Proteins: Structure and Molecular Properties,W.H.Freeman&Co.,San Francisco,pp.79-86 (1983))；Asparagine or glutamine go amidation；The acetylation of N-terminal amine；And/or the amide of C-terminal carboxylic acid group Change or is esterified.

Another type of covalent modification is related to making glucosides chemistry or enzymatic of glucosides to analog.Sugar can be connected to: (a) smart ammonia Acid and histidine；(b) free carboxy；(c) free sulfhydryl groups, the free sulfhydryl groups of such as cysteine；(d) free hydroxyl group, such as silk The free hydroxyl group of propylhomoserin, threonine or hydroxy-proline；(e) aromatic moieties of aromatic moieties, such as tyrosine or tryptophan；Or (f) amide groups of glutamine.These methods are described in WO87/05330 disclosed on September 11st, 1987；With Aplin and Wriston,CRC Crit.Rev.Biochem.,pp.259-306(1981)。

In some embodiments, glucagon analogue via glucagon analogue amino acid side chain with it is different Covalent bond between the part of source is conjugated to heterologous moiety.In some embodiments, glucagon analogue is via internal ammonia The side chain of base acid, C-terminal extend in position or the combinations of C-terminal amino acid or these positions be conjugated to heterologous moiety.

In some embodiments, conjugate includes the connector that glucagon analogue is bonded to heterologous moiety.? Some aspects, connector include that length is 1 to about 60 atom or 1 to 30 atom or longer, 2 to 5 atoms, 2 to 10 originals The chain of son, 5 to 10 atoms or 10 to 20 atoms.In some embodiments, chain atom is carbon atom.In some realities It applies in scheme, the chain atom in the skeleton of connector is selected from C, O, N and S.Chain atom can be selected according to expected dissolubility (hydrophily) With connector to provide more soluble conjugate.In some embodiments, connector is provided due to enzyme or other catalyst or target Hydrolysising condition present in tissue or organ or cell and the functional group that is cracked.In some embodiments, the length of connector Long enough is spent to reduce the possibility of steric hindrance.If connector is covalent bond or peptide bond and conjugate is polypeptide, entire conjugate can To be fusion protein.Such peptidyl linkers can be any length.Exemplary adapter length is about 1 to 50 amino acid, length For 5 to 50,3 to 5,5 to 10,5 to 15 or 10 to 30 amino acid.Or it can be by known to persons of ordinary skill in the art heavy Group genetic engineering remodeling method prepares such fusion protein.

Conjugate: Fc fusion

As described above, in some embodiments, analog conjugation (such as fusion) is to immunoglobulin or part thereof (for example, variable region, the area CDR or Fc).The immunoglobulin (Ig) of known type includes IgG, IgA, IgE, IgD or IgM.The area Fc For the C-terminal area of Ig heavy chain, it is responsible for such as recycling (it leads to Increased Plasma Half-life), antibody dependent cellular with operative activities Mediating cytotoxicity (ADCC) and the Fc receptor of complement-dependent cytotoxicity (CDC) combine.

For example, the human IgG area heavy chain Fc extends to the C-terminal of heavy chain from Cys226 according to some definition." the hinge of human IgG1 Sequence " usually extends to Pro230 from Glu216 (can be by other by comparing cysteine involved in cysteine bonding The hinge area of IgG isotype is compared with IgG1 sequence).The area Fc of IgG includes two constant domains CH2 and CH3.People The CH2 structural domain in the area IgG Fc usually extends to amino acid 341 from amino acid 231.The CH3 structural domain in the area human IgG Fc usually from Amino acid 342 extends to amino acid 447.Refer to the equal base of the amino acid number of immunoglobulin or immunoglobulin fragment or region In Kabat et al., Sequences of Proteins of Immunological Interest, U.S.Department of Public Health,Bethesda,Md.In a related embodiment, the area Fc may include one or more from immune ball Ferritin heavy chain natural or modification constant region (in addition to CH1), for example, the CH3 of the area CH2 and CH3 of IgG and IgA or IgE and The area CH4.

Suitable conjugate fraction includes the part of the immunoglobulin sequences comprising FcRn binding site.FcRn (rescue Receptor) it is responsible for recycling immunoglobulin and be back to it in blood to recycle.It has been based on X-ray crystallography (Burmeister Et al., 1994, Nature 372:379) IgG of the description in conjunction with FcRn receptor the part Fc region.Fc's and FcRn is main Contact surface is close to the joint of CH2 and CH3 structural domain.Fc-FcRn is contacted in single Ig heavy chain.Dominant touch site includes The amino acid residue 248 of CH2 structural domain, 250-257,272,285,288,290-291,308-311 and 314 and CH3 structural domain The amino acid residue 385-387,428 and 433-436 of structure.

Some conjugate fractions may include or may not include Fc γ R binding site.Fc γ R is responsible for ADCC and CDC.With The example of position is amino acid 234-239 (lower end hinge area), amino acid 265-269 (B/C in the area Fc that Fc γ R is directly contacted Ring), amino acid 297-299 (C'/E ring) and amino acid 327-332 (F/G) ring (Sondermann et al., Nature 406: 267-273,2000).The lower end hinge area of IgE also has been directed to FcRI and combines (Henry et al., Biochemistry36,15568- 15578,1997).The residue for being related to the combination of IgA receptor is described in Lewis et al. (J Immunol.175:6694-701,2005) In.Be related to IgE receptor combination amino acid residue be described in Sayers et al. (J Biol Chem.279 (34): 35320-5, 2004) in.

Can the area Fc to immunoglobulin carry out amino acid modification.Such variable area Fc is included in the CH3 structural domain in the area Fc At least one amino acid modification in (residue 342-447) and/or in the CH2 structural domain (residue 231-341) in the area Fc extremely A few amino acid modification.It is believed that the mutation for assigning the increased affinity of FcRn includes T256A, T307A, E380A and N434A (Shields et al., 2001, J.Biol.Chem.276:6591).Other mutation can reduce the area Fc and Fc γ RI, Fc γ RIIA, The combination of Fc γ RIIB and/or Fc γ RIIIA is without significantly reducing the affinity to FcRn.For example, the position 297 in the area Fc Asn by Ala or the removal of another amino acid substitution high conservative N- glycosylation site and can lead to immunogenicity reduce it is adjoint simultaneously The Increased Plasma Half-life in the area Fc, and and Fc γ R combination reduce (Routledge et al., 1995, Transplantation 60:847；Friend et al., 1999, Transplantation68:1632；Shields et al., 1995, J.Biol.Chem.276:6591).The amino acid modification of the position 233-236 of IgG1, the combination of reduction and Fc γ R are carried out (Ward and Ghetie, 1995, Therapeutic Immunology 2:77 and Armour et al., 1999, Eur.J.Immunol.29:2613).Some exemplary amino acid substitutions are described in United States Patent (USP) 7,355,008 and 7,381,408 In, respectively it is incorporated herein by reference with it.

Conjugate: hydrophilic parts

Glucagon analogue as described herein can further be modified to improve it in aqueous solution at physiological ph Dissolubility and stability, while keeping the high bioactivity relative to natural glucagon.It can be for making albumen and activation Hydrophilic parts such as PEG group is connected to analog under any appropraite condition of reacted polymer molecule.Ability can be used Known any mode in domain, including via acylation, standard reductive alkylation, Michael's addition (Michael addition), mercapto Base alkylation or other chemo-selective conjugation/connection methods via on peg moiety reactive group (for example, aldehyde, amino, Ester, sulfydryl, α-halogen acetyl group, Maleimido or diazanyl) to the reactive group on target compound (for example, aldehyde, ammonia Base, ester, sulfydryl, α-halogen acetyl group, Maleimido or diazanyl).It can be used for for water-soluble polymer being connected to a kind of or more The activated group of kind albumen includes but is not limited to sulfone, maleimide, sulfydryl (sulfhydryl), mercaptan (thiol), fluoroform Sulphonic acid ester, trifluoro esilate, aziridine, ethylene oxide, 5- pyridyl group and α-acid halide are (for example, alpha-iodine acetic acid, α-bromine second Acid, α-monoxone).If being connected to analog by standard reductive alkylation, selected polymer should have single reaction aldehyde To control the degree of polymerization.See, for example, Kinstler et al., Adv.Drug.Delivery Rev.54:477-485 (2002)； Roberts et al., Adv.Drug Delivery Rev.54:459-476 (2002)；With Zalipsky et al., Adv.Drug Delivery Rev.16:157-182(1995)。

In a particular aspect, the amino acid residue of the analog with sulfydryl is modified by hydrophilic parts such as PEG.

In some embodiments, sulfydryl is modified in nucleophilic substitution by the PEG that halogen acetyl group activates to cause to produce Raw includes the pegylated analogs of thioether bond.

Suitable hydrophilic parts include polyethylene glycol (PEG), polypropylene glycol, oxyethylated polyols (such as POG), Polyoxyethylated sorbitol, oxyethylated glucose, oxyethylated glycerol (POG), polyalkylene oxide, polyethylene glycol third Aldehyde, the copolymer of ethylene glycol/propylene glycol, mono methoxy-polyethylene glycol, mono- (C1-C10) alkoxy-polyethylene glycol or mono- (C1-C10) aryloxy group-polyethylene glycol, carboxymethyl cellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly- 1,3- dioxolane, poly- 1,3,6- trioxane, ethylene/copolymer-maleic anhydride, poly- (beta-amino acids) (homopolymer or nothing Advise copolymer), poly- (n-VP) polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyalkylene oxides, polycyclic oxygen third Alkane/ethylene oxide copolymer, colon acid (colonic acid) or other polysaccharide polymers, Ficoll or glucan and its mixing Object.Glucan is mainly by the polysaccharide polymer of the α 1-6 glucose subunit being keyed.Glucan can be with various molecular weights model Acquisition is enclosed, for example, about 1kD to about 100kD, or about 5,10,15 or 20kD are to about 20,30,40,50,60,70,80 or 90kD.Contain Cover linear or branched polymer.Gained conjugate formulations substantially can be monodisperse or polydispersion, and each analog can With about 0.5,0.7,1,1.2,1.5 or 2 polymer moieties.

In some or any embodiment, glucagon analogue via glucagon analogue amino acid side Covalent linkage between chain and hydrophilic parts is conjugated to the hydrophilic parts.In some or any embodiment, the high blood of pancreas Sugared element analog via internal amino acid side chain, C-terminal extend in position or C-terminal amino acid or these positions combination It is conjugated to hydrophilic parts.

Conjugate: rPEG

In some or any embodiment, conjugate of the invention include with auxiliary analog merge with GIP by The glucagon analogue of body agonist activity is capable of forming the extension configuration similar with chemistry PEG (for example, recombination PEG (rPEG) molecule), such as International Patent Application Publication No. WO2009/023270 and U.S. Patent Application Publication No. Those of described in US20080286808.In some respects, rPEG molecule is to include glycine, serine, glutamic acid, asparagus fern One of propylhomoserin, alanine or proline or a variety of polypeptides.In some respects, rPEG is homopolymer, for example, gathering sweet ammonia Acid, polyserine, polyglutamic acid, poly-aspartate, polyalanine or polyproline.In other embodiments, rPEG includes It is two kinds of to repeat amino acid, for example, poly- (Gly-Ser), poly- (Gly-Glu), poly- (Gly-Ala), poly- (Gly-Asp), poly- (Gly-Pro), poly- (Ser-Glu) etc..In some respects, rPEG includes three kinds of different types of amino acid, for example, poly- (Gly- Ser-Glu).In a particular aspect, rPEG increases the half-life period of glucagon and/or GLP-1 agonist analog.In some sides Face, rPEG include net positive charge or net negative charge.In some respects, rPEG lacks secondary structure.In some embodiments, The length of rPEG is greater than or equal to 10 amino acid and length is about 40 to about 50 amino acid in some embodiments.One A little aspects, auxiliary peptide is merged via peptide bond or protease cracking site with the N-terminal of analog of the present invention or C-terminal, or insertion In the ring of analog of the present invention.In some respects, rPEG includes affinity label or is connected to PEG greater than 5kDa.Some In embodiment, rPEG assigns the increased hydrodynamic radius of analog of the present invention, serum half-life, protease resistant or molten Solution property and the immunogenicity for assigning analog reduction in some respects.

Conjugate: polymer

The present invention further provides the polymer of analog disclosed herein or dimers, including-or miscellaneous-polymer or - or miscellaneous-dimer.Standard connection agent well known by persons skilled in the art and program can be used to connect for two or more analogs It is connected together.For example, can be via using bifunctional thiol crosslinkers and bifunctional amine's crosslinking agent to form dimerization between two peptides Body, particularly with the class replaced by cysteine, lysine, ornithine, homocysteine or acetyl phenyl alanine residue Like object.Dimer can be equal dimer or optionally can be heterodimer.In an exemplary embodiment, two are connected (or more) connector of analog is PEG, for example, 5kDa PEG, 20kDa PEG.In some embodiments, connector two Sulfide linkage.For example, each monomer of dimer may include Cys residue (for example, Cys of end or interior location) and each Cys residue Sulphur atom participates in forming disulfide bond.In illustrative aspect, each monomer of dimer is keyed via thioether.In illustrative aspect, The ε amine key of the Lys residue of one monomer is bonded to Cys residue, which is connected to another monomer via chemical part in turn The ε amine of Lys residue.The method of the dimer described further herein for preparing such thioether bonding.In some respects, monomer via End amino acid (for example, N-terminal or C-terminal), via internal amino acid, or via at least one monomer end amino acid and The internal amino acid of at least another monomer connects.In a particular aspect, monomer is not connected via N-terminal amino acid.In some sides Face, " tail-tail " orientation that the monomer of polymer is linked together with the C-terminal amino acid of each monomer link together.

Prodrug

The present invention further provides the prodrugs of peptide as described herein and analog.Term " prodrug " as used herein is determined Justice is that any compound of chemical modification is undergone before showing its complete pharmacotoxicological effect.

In an exemplary embodiment, prodrug is the peptide prodrug based on amide, with the world for being disclosed on June 24th, 2010 It is similar those of described in patent application publication number WO/2010/071807.Such prodrug is intended to delayed-action starting and extends medicine The half-life period of object.Delayed-action starting is advantageous, because it allows systemic distribution of the prodrug before its activation.Therefore, it applies The complication as caused by peak activity and the therapeutic index of increase parent drug after applying can be eliminated with prodrug.

In illustrative aspect, prodrug includes structure: A-B-Q；Wherein Q is peptide or analogue as described herein；A is amino acid Or carboxylic acid；B is the N- Alkylation of Amino Acids that Q is connected to via the amido bond between A-B and the amine of Q；Wherein A, B or and A-B The amino acid of the Q of connection is undoded amino acid, and furthermore wherein A-B and Q chemical cracking partly declines in PBS in physiological conditions Phase (t1/2) is at least about 1 hour to about 1 week.Term " carboxylic acid " as used herein, which refers to, have been modified to be set with hydroxyl Change the amino acid of α carbon amino.

Pharmaceutical composition, purposes and kit

Salt

In some embodiments, glucagon analogue is in salt form, for example, pharmaceutically acceptable salt.Such as this Term used in text " pharmaceutically acceptable salt " refer to the bioactivity for keeping parent compound and be not biologically or The salt of other undesirable compounds of aspect.Such salt can be by making in situ during the final separation and purifying of analog It is standby, or reacted with suitable acid by making free alkali functional group and it is independently prepared.Many compounds as disclosed herein are due to depositing Ackd salt and/or basic salt are capable of forming in amino and/or carboxyl or similar group.

Pharmaceutically acceptable acid-addition salts can be prepared by inorganic acid and organic acid.Representative acid-addition salts include but unlimited In acetate, adipate, alginates, citrate, aspartate, benzoate, benzene sulfonate, disulfate, butyric acid Salt, camphor hydrochlorate, camsilate, digluconate, glycerophosphate, Hemisulphate, enanthate, caproate, fumaric acid Salt, hydrochloride, hydrobromate, hydriodate, 2- isethionate (isothionate), lactate, maleate, methylsulphur Hydrochlorate, nicotinate, 2- naphthalene sulfonate, oxalates, palmitate, pectate, persulfate, 3- phenylpropionic acid salt, picric acid It is salt, pivalate, propionate, succinate, tartrate, rhodanate, phosphate, glutamate, bicarbonate, right Toluene fulfonate and undecanoate.Salt derived from inorganic acid includes the salt of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.. Salt derived from organic acid include acetic acid, propionic acid, glycolic, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, Fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-methyl benzenesulfonic acid, salicylic acid etc. Salt.Can be used to form pharmaceutically acceptable acid-addition salts acid example include such as inorganic acid, for example, hydrochloric acid, hydrobromic acid, Sulfuric acid and phosphoric acid；And organic acid, for example, oxalic acid, maleic acid, succinic acid and citric acid.

It can also be prepared in situ during the final separation and purifying of bigcatkin willow acid source, or by making containing carboxylic moiety and suitably Alkali (hydroxide, carbonate or the bicarbonate of such as pharmaceutically acceptable metal cation) or with ammonia or organic primary amine, Secondary amine or reactive tertiary amine prepare base addition salts.Pharmaceutically acceptable salt includes but is not limited to be based on alkali or alkaline earth metal Cation, lithium salts, sodium salt, sylvite, calcium salt, magnesium salts and aluminium salt etc.；With nontoxic quaternary amine and amine cation, especially include Ammonium, tetramethyl-ammonium, tetraethyl ammonium, methyl ammonium, dimethyl ammonium, trimethyl ammonium, triethyl ammonium, diethyl ammonium and ethyl ammonium.It can use In other the representative organic amines for forming base addition salts include such as ethylenediamine, ethanol amine, diethanol amine, piperidines, piperazine.Spread out The salt for being born from organic base includes but is not limited to the salt of primary amine, secondary amine and tertiary amine.

In addition, Basic nitrogen-containing groups can be quaternized through analog of the invention, such as low-carbon alkyl halide, such as methyl, Ethyl, propyl and butyl chloride compound, bromide and iodide；Long chain halide, such as decyl, lauryl, myristyl and ten Eight alkanoyl chlorides, bromide and iodide；Arylalkyl halide, such as benzyl and phenylethyl bromide.Thus to obtain Water-soluble or oil-soluble or dispersible products.

Preparation

According to some embodiments, pharmaceutical composition is provided, wherein the composition includes glucagon class of the invention Like object or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.Term as used herein is " pharmaceutically acceptable Carrier " includes any standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsion (such as oil/water or water/oil cream Agent) and various types of wetting agents.The term is also covered by U.S. Federal Government management organization (regulatory agency Of the US Federal government) approval or United States Pharmacopeia (US Pharmacopeia) in list animal (including People) used in any reagent.

Pharmaceutical composition may include any pharmaceutically acceptable ingredient, including for example acidulant, additive, adsorbent, Aerosol propellants, basifier, anti-caking agent, anti-coagulants, resist micro- life at air displacement agent (air displacement agent) Object preservative, antioxidant, fungicide, base-material, adhesive, buffer, chelating agent, coating agent (coating agent), coloring Agent, desiccant, detergent, diluent, disinfectant, disintegrating agent, dispersing agent, solubilizer, dyestuff, emollient, emulsifier, emulsion are steady Determine agent, filler, film forming agent, odor enhancers, corrigent, flow enhancing agent, gelling agent, granulating agent, moisturizer, lubrication Agent, ointment base, ointment, oiliness solvent, organic base, pastille base-material, pigment, plasticizer, polishing agent, is prevented mucoadhesive Rotten agent, sequestering agent, skin penetrant, solubilizer, solvent, stabilizer, suppository base, surfactant, surfactant, The miscible cosolvent of suspending agent, sweetener, therapeutic agent, thickener, tonicity agent, toxic agents, viscosity increasing agent, water absorbing agent, water, water Softening agent or wetting agent.

In some embodiments, pharmaceutical composition includes any of following components or combination: Arabic gum, acetyl ammonia Base potassium sulfonate, tributyl 2-acetylcitrate, acetyl triethyl citrate, agar, albumin, ethyl alcohol, dehydrated alcohol, denaturation wine Essence, dilute alcohol, aleuritic acid, alginic acid, aliphatic polyester, aluminium oxide, aluminium hydroxide, aluminum stearate, amylopectin, α-straight chain Starch, ascorbic acid, ascorbyl palmitate, aspartame, injection bacteriostatic water, bentonite, bentonite magma, benzalkonium chloride, benzyl Rope oronain, benzoic acid, benzyl alcohol, Ergol, bronopol, butylated hydroxy anisole, Butylated Hydroxytoluene, para hydroxybenzene first Acid butyl ester, butyl P-hydroxybenzoic acid sodium, calcium alginate, Calcium Ascorbate, calcium carbonate, calcium cyclamater, anhydrous phosphoric acid hydrogen Calcium, dehydration calcium monohydrogen phosphate, tricalcium phosphate, calcium propionate, calcium silicates, calcium sorbate, calcium stearate, calcium sulfate, calcium sulfate half are hydrated Object, Canola oil (canola oil), carbomer (carbomer), carbon dioxide, calcium carboxymethylcellulose, carboxymethyl cellulose Sodium, beta carotene, carrageenan, castor oil, rilanit special, cationic emulsified wax, cellulose acetate, acetic acid O-phthalic Acid cellulose, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, 16 Alcohol octadecyl alcolol mixture (cetostearyl alcohol), cetrimonium bromide, cetanol, Chlorhexidine, methaform, chloreresol, gallbladder are solid Alcohol, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), F-22, contains chlorine at chlorhexidine acetate Fluorohydrocarbon (CFC), mycotetracid, chloroxylenol, corn-syrup solids, anhydrous citric acid, citric acid monohydrate close object, cocoa Rouge, colorant, corn oil, cottonseed oil, cresols, metacresol, o-cresol, paracresol, cross-linked croscarmellose sodium, Crospovidone, Cyclamic acid, cyclodextrin, dextrates, dextrin, dextrose, anhydrous dextrose, diazonium alkyl imidazole urea (diazolidinyl Urea), dibutyl phthalate, dibutyl sebacate, diethanol amine, diethyl phthalate, Difluoroethane (HFC), Dimethyl-β-cyclodextrin, cyclodextrin type compound are for exampleDimethyl ether, repefral, edetic acid(EDTA) Dipotassium, natrium adetate, disodium hydrogen phosphate, docusa, docusate potassium, docusate sodium, lauryl gallate, dodecyl Trimethylammonium bromide, CaEDTA, ethylenediamine tetra-acetic acid, eglumine, ethyl alcohol, ethyl cellulose, progallin A, the moon Ethyl cinnamate, ethylmaltol, ethyl oleate, to hydroxymethyl-benzoic acid ethyl ester, to hydroxymethyl-benzoic acid ethyl ester potassium, to methylol Ethyl benzoate sodium, ethyl vanillin, fructose, fructose liquid, the fructose ground, apyrogeneity fructose, powdery fructose, fumaric acid, Gelatin, glucose, liquid glucose, the glyceride mixture of saturated vegetable fatty acid, glycerol, Compritol 888 ATO, single oleic acid Glyceride, glycerin monostearate, self-emulsifying glycerin monostearate, glyceryl palmitostearate, glycine, ethylene glycol, four Hydrogen furans polyglycol ether (glycofurol), guar gum, heptafluoro-propane (HFC), cetyltrimethylammonium bromide, high fructose sugar Slurry, human serum albumins, hydrocarbon (HC), dilute hydrochloric acid, II type hydrogenated vegetable oil, hydroxyethyl cellulose, 2- hydroxyethyl-β-cyclodextrin, Hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, 2-HP-BETA-CD, hydroxypropyl methyl cellulose, phthalic acid Hydroxypropyl methyl cellulose, miaow urea, indigo, ion-exchanger, iron oxide, isopropanol, isopropyl myristate, palmitinic acid isopropyl Ester, isotonic saline solution, kaolin, lactic acid, Lactitol, lactose, lanolin, lanolin alcohol, wool grease, lecithin, silicic acid Magnalium, magnesium carbonate, normal magnesium carbonate, anhydrous magnesium carbonate, basic magnesium carbonate, magnesium hydroxide, lauryl magnesium sulfate, magnesia, silicon Sour magnesium, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, malt, maltitol, maltitol solution, malt paste Essence, maltol, maltose, mannitol, medium chain triglyceride, meglumine, menthol, methylcellulose, methyl methacrylate, Methyl oleate, to methyl hydroxy-benzoate, to methyl hydroxy-benzoate potassium, fine to methyl hydroxy-benzoate sodium, crystallite Dimension element and sodium carboxymethylcellulose, mineral oil, light mineral oil, mineral oil and lanolin alcohol, oil, olive oil, monoethanolamine, illiteracy De- stone, octyl gallate, oleic acid, palmitinic acid, paraffin, peanut oil, vaseline, vaseline and lanolin alcohol, pharmaceutical glaze, phenol, Liquefied phenol, phenoxetol, phenoxypropanol, phenylethanol, phenylmercuric acetate, Phenylmercuric Borate, phenylmercuric nitrate, polacrilin (polacrilin), polacrilin potassium, poloxamer (poloxamer), dextrosan, polyethylene glycol, polyethylene oxide, poly- third Olefin(e) acid ester, polyethylene-polyoxypropylene-block polymer, polymethacrylates, polyoxyethylene alkyl ether, polyoxyethylene caster Oily derivative, Polyoxyethylene Sorbitol Fatty Acid Esters, Myrj 45, polyvinyl alcohol, polyvinylpyrrolidone, alginic acid Potassium, Potassium Benzoate, saleratus, potassium acid sulfate, potassium chloride, potassium citrate, anhydrous citric acid potassium, potassium hydrogen phosphate, inclined sulfurous acid Hydrogen potassium, potassium dihydrogen phosphate, potassium propionate, potassium sorbate, povidone, propyl alcohol, propionic acid, propylene carbonate, propylene glycol, propylene glycol alginic acid Ester, propylgallate, to hydroxymethyl-benzoic acid propyl ester, to hydroxymethyl-benzoic acid propyl ester potassium, to hydroxymethyl-benzoic acid propyl ester sodium, Protamine sulfate, rapeseed oil, RingerShi solution, saccharin, ammonium saccharin, calcium benzosulphimide, saccharin sodium, safflower oil, saponite, serum egg White, sesame oil, silica gel, colloidal silicon dioxide, mosanom, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfate, chlorination Sodium, anhydrous citric acid sodium, Sodium citrate dehydrate, sodium chloride, Sodium Cyclamate, edetate sodium, lauryl sodium sulfate, the moon Osmanthus base sodium sulphate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, tertiary sodium phosphate, anhydrous sodium propionate, sodium propionate, sorb Sour sodium, Explotab, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan ester (sorbitan fatty ester), mountain Pears alcohol, sorbitol solution 70%, soybean oil, spermaceti, starch, cornstarch, potato starch, pregelatinized starch, sterilizing corn Starch, stearic acid, the stearic acid of purifying, stearyl alcohol, sucrose, sugar, sompressible sugar, candy sugar, sugar ball, inverted sugar, sucrose-turn Change glycopolymers (Sugartab), sunset yellow FCF, synthesis paraffin, talcum powder, tartaric acid, tartrazines, tetrafluoroethane (HFC), can It can oil (theobroma oil), thimerosal, titanium dioxide, alpha tocopherol, tocopherol acetate, alpha tocopherol succinate, β-fertility Phenol, Delta-Tocopherol, Gamma-Tocopherol, tragacanth, glyceryl triacetate, tributyl citrate, triethanolamine, triethyl citrate, front three Group-beta-cyclodextrin, trimethyl tetradecyl base ammonium bromide, tris buffer, edetate trisodium, vanillic aldehyde, I type hydrogenated vegetable oil, Water, hard water, carbon dioxide-free water, apirogen water, water for injection, sucking sterile water, Injectable sterile water, is rinsed and is used soft water Sterile water, wax, anionic emulsifying wax, Brazil wax, cationic emulsified wax, spermaceti ester type waxes, microwax, non-ionic emulsifying wax, Suppository wax, Chinese wax, yellow wax, white petrolatum, lanolin, xanthan gum, xylitol, zein, zinc propionate, zinc salt, zinc stearate or Handbook of Pharmaceutical Excipients, the 3rd edition, A.H.Kibbe (Pharmaceutical Press, London, UK, 2000) any excipient in (its with its entirely through be incorporated by).Remington's Pharmaceutical Sciences, the 16th edition, E.W.Martin (Mack Publishing Co., Easton, Pa., 1980) (its with its entirely through be incorporated by) disclose prepare various components used in pharmaceutically acceptable composition and Its known technology of preparing.Other than any conventional dose is incompatible with pharmaceutical composition, cover it makes in pharmaceutical composition With.Supplement active constituent also may be incorporated into composition.

In some embodiments, one or more aforementioned components can be present in pharmaceutical composition with any concentration, all Such as, for example, at least A, wherein A be 0.0001%w/v, 0.001%w/v, 0.01%w/v, 0.1%w/v, 1%w/v, 2%w/v, 5%w/v, 10%w/v, 20%w/v, 30%w/v, 40%w/v, 50%w/v, 60%w/v, 70%w/v, 80%w/v or 90% w/v.In some embodiments, one or more aforementioned components can be present in pharmaceutical composition with any concentration, such as, example Such as, at most B, wherein B is 90%w/v, 80%w/v, 70%w/v, 60%w/v, 50%w/v, 40%w/v, 30%w/v, 20% W/v, 10%w/v, 5%w/v, 2%w/v, 1%w/v, 0.1%w/v, 0.001%w/v or 0.0001%.In other embodiments In, one or more aforementioned components can be present in pharmaceutical composition with any concentration range, such as, for example, about A to about B.? In some embodiments, A is 0.0001% and B is 90%.

In some embodiments, pharmaceutically acceptable ingredient is selected from: sugar is (for example, glucose, sucrose, trehalose, cream Sugar, fructose, maltose, glucan, glycerol, glucan, melibiose (mellibiose), melezitose (melezitose), cotton seed Sugar, manninotriose, stachyose (stachyose), maltose, milk ketose (lactulose), maltulose (maltulose) or Isomaltoketose or these sugar combinations), sugar alcohol (for example, ethylene glycol, glycerol, erythritol, threitol, arabite, Xylitol, ribitol, mannitol, D-sorbite, dulcitol, iditol, isomaltose, maltitol, lactitol or Portugal The combination of grape sugar alcohol or these sugar alcohols), salt (for example, sodium chloride), emulsifier or surfactant (for example, polysorbate, Such as other block copolymers of 20 dehydrated sorbitol mono-fatty acid ester of polyoxyethylene or ethylene oxide and propylene oxide), freeze-drying Protective agent and its mixture.For example, excipient such as sugar or sugar alcohol are for example with about 20mg/mL to about 40mg/mL or 25mg/ The concentration of mL to 45mg/mL, such as 35mg/mL exist.

Pharmaceutical composition can be prepared to reach the pH of physical compatibility.In some embodiments, according to preparation and application Approach, the pH of pharmaceutical composition can be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10 or at least 10.5 until and including pH 11, such as between 4 and 7, or in 4.5 and 5.5 Between.In an exemplary embodiment, pharmaceutical composition may include buffer to reach the pH of physical compatibility.Buffer can wrap Any compound that can be buffered at required pH is included, such as, phosphate buffer (such as PBS), triethanolamine, Tris, two Hydroxyethyl glycine (bicine), TAPS, trihydroxy methyl glycine (tricine), HEPES, TES, MOPS, PIPES, dimethyl Arsonate (cacodylate), MES, acetate, citrate, succinate, histidine or other are pharmaceutically acceptable slow Electuary.In an exemplary embodiment, the intensity of buffer is at least 0.5mM, at least 1mM, at least 5mM, at least 10mM, at least 20mM, at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 120mM, at least 150mM or at least 200mM.In some embodiments, the intensity of buffer be no more than 300mM (for example, at most 200mM, at most 100mM, at most 90mM, at most 80mM, at most 70mM, at most 60mM, at most 50mM, At most 40mM, at most 30mM, at most 20mM, at most 10mM, at most 5mM, at most 1mM).For example, buffer concentration can be about 2mM to about 100mM, or about 10mM to about 50mM.

Administration method

Discussion below in relation to administration method is only provided to illustrate exemplary implementation scheme and should not be construed as with any side Formula limits range.

Preparation suitable for oral administration can be made up of: (a) liquid solution, such as a effective amount of to be dissolved in diluent such as The analog of the present invention of water, salt water or orange juice；(b) capsule, sachet, tablet, pastille and dragee, respectively contain predetermined amount Active constituent, as solid or particle；(c) powder；(d) suspension in appropriate liquid；(e) suitable emulsion.Liquid Body preparation may include diluent, such as water and alcohols (for example, ethyl alcohol, benzylalcohol and polyethylene glycol), wherein adding or being not added with medicine Acceptable surfactant on.Capsule form can be (all containing such as surfactant, lubricant and inert filler Such as lactose, sucrose, calcium phosphate and cornstarch) common hard shell or soft-shelled gelatin type.Tablet form may include below one Kind or it is a variety of: it is lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, Arabic gum, bright Glue, guar gum, colloidal silicon dioxide, croscarmellose sodium, talcum powder, magnesium stearate, calcium stearate, zinc stearate, Stearic acid and other excipient, colorant, diluent, buffer, disintegrating agent, wetting agent, preservative, corrigent and other pharmacology Learn compatible excipient.Lozenge form may include of the invention in corrigent (usually sucrose and Arabic gum or tragacanth) Analog, and include analog of the invention in inert base (such as gelatin and glycerol or sucrose and Arabic gum) Pastille, emulsion, the gelling agent etc. of this kind of excipient known in the art are contained other than analog of the invention.

Can by analog of the invention, individually or with other suitable group subassemblys, delivered via pulmonary administration and The aerosol preparation via sucking application can be made into.These aerosol preparations can be placed in the acceptable propellant of pressurization In dicholorodifluoromethane, propane, nitrogen etc..They can also be configured to be used for the drug of non-pressured preparations, such as sprayed In day with fog or atomizer.Such spray formulation can also be used for spray mucosa.In some embodiments, analog is configured to powder Last blend is configured to particle or nanoparticle.Suitable pulmonary formulations are known in the art.See, for example, Qian et al., Int J Pharm 366:218-220(2009)；Adjei and Garren, Pharmaceutical Research, 7 (6): 565- 569(1990)；62 (1-2): 279-287 (1999) of Kawashima et al., J Controlled Release；Liu et al. people, Pharm Res 10(2):228-232(1993)；International application published WO 2007/133747 and WO 2007/ 141411。

Preparation suitable for parenteral administration includes aqueous and non-aqueous isotonic aseptic injectable solution, can be contained anti-oxidant Agent, buffer, bacteriostatic agent and the solute for keeping the blood of preparation and intended recipient isotonic；With aqueous and non-aqueous sterile suspensions, It may include suspending agent, solubilizer, thickener, stabilizer and preservative.Term " parenteral " means not lead to via alimentary canal Cross that some other approach are such as subcutaneous, in intramuscular, vertebra or intravenous.Analog of the invention can in pharmaceutical carrier with life Acceptable diluent is applied together in Neo-Confucianism, the pharmaceutical carrier such as sterile liquid or liquid mixture, including water, physiology salt Water, aqueous dextrose and related sugar solutions, alcohol (such as ethyl alcohol or hexadecanol), glycol (such as propylene glycol or polyethylene glycol), Dimethyl sulfoxide, glycerol, ketal (such as 2,2- dimethyl -1,3- dioxolane -4- methanol), ether, poly(ethylene glycol) 400, Oil, fatty acid, aliphatic ester or glyceride or acetylated fatty acid glyceride, wherein adding or being not added with and is pharmaceutically acceptable Surfactant (such as soap or detergent), suspending agent (such as pectin), carbomer, methylcellulose, hydroxypropyl methyl it is fine Dimension element or carboxymethyl cellulose or emulsifier and other drugs adjuvant.

The oil that can be used in parenteral administration includes petroleum, animal oil, vegetable oil or synthetic oil.Oil particular instance include Peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, paraffin oil and mineral oil.Suitable for parenteral administration Fatty acid includes oleic acid, stearic acid and isostearic acid.Ethyl oleate and isopropyl myristate are the realities of suitable aliphatic ester Example.

Soap suitable for parenteral administration includes fatty alkali metal, ammonium and tetraethoxide amine salt, and suitable detergent packet Include: (a) cationic detergent, such as, for example, dimethyl dialkyl ammonium halide and alkyl pyridinium, (b) anion is gone Dirty agent, such as, for example, the sulfonate of alkyl, aryl and alkene, alkyl, alkene, ether and monoglyceride sulfate and sulfonation amber Amber hydrochlorate, (c) non-ionic octoxynol detergent, such as, for example, fatty amine oxide, Marlamid and polyoxyethylene poly- third Alkene copolymer, (d) both sexes detergent, for example, alkyl-Beta-alanine ester and 2- alkyl-imidazole hyamine, and (e) it is mixed Close object.

Parenteral administration usually will contain about the analog of the present invention in the solution of 0.5 weight % to about 25 weight %.It can Use preservative and buffer.In order to minimize or eliminate the stimulation of injection site, such composition can contain one or more Nonionic surface active agent, the hydrophilic-lipophilic balance (HLB) with about 12 to about 17.Surface-active in such preparation The amount of agent usually will be in the range of about 5 weight % to about 15 weight %.Suitable surfactant includes polyethylene glycol dehydration Sorbitan fatty acid ester (such as dehydrated sorbitol mono-fatty acid ester) and ethylene oxide contract with by propylene oxide and propylene glycol Close the high molecular weight adducts of the hydrophobic base formed.Parenteral administration can be presented in unit dose or multi-dose sealing container It in (such as ampoule and bottle), and can be stored under the conditions of freeze-drying (freeze-drying), it is only necessary to injection is added before facing use With sterile liquid excipient, such as water.Extemporaneous injection solutions can be prepared from the sterile powders, particle and tablet of previously described type And suspension.

Injectable formulation is according to the present invention.Those of ordinary skill in the art are it is known that for the effective of Injectable composition The requirement of pharmaceutical carrier is (see, for example, Pharmaceutics and Pharmacy Practice, J.B.Lippincott Company, Philadelphia, PA, Banker and Chalmers are compiled, and the 238-250 pages (1982)；With ASHP Handbook On Injectable Drugs, Toissel, the 4th edition, the 622-630 pages (1986)).

In addition, analog of the invention can pass through the mixing and system with a variety of matrix such as emulsified bases or water-soluble base At the suppository for rectal administration.Preparation suitable for vaginal application can be used as vaginal suppository, tampon, cream, gelling agent, paste Agent, foaming agent or spray formulation are presented, other than containing active constituent, also containing such load appropriate as known in the art Body.

It will be understood by those skilled in the art that analog of the invention can also be configured to other than aforementioned pharmaceutical compositions Inclusion complex, such as cyclodextrin inclusion complexes or liposome.

Dosage

It is believed that analog of the invention can be used for treating GIP receptor agonism, GIP/GLP-1 receptor is total to agonism, GIP/ glucagon receptor is total to agonism or the triple agonisms of GIP/GLP-1/ glucagon receptor play a role In the method for disease or medical condition.For purposes of the present invention, the amount or dosage for applying analog of the present invention should be enough Such as therapeutic or preventative response is realized within the scope of reasonable time in subject or animal.For example, the dosage of analog of the present invention Should from application at about 1 minute to 4 minutes, 1 hour to 4 hours or 1 week to 4 weeks or longer (for example, 5 weeks to 20 weeks or more More weeks) period in be enough that cAMP is stimulated to secrete from cell as described herein or be enough to reduce the blood glucose water of mammal Flat, fat level, food intake level or weight.In an exemplary embodiment, the period is possible or even longer.Spy should be passed through The weight of the situation and animal to be treated (such as people) of the effect of fixed analog of the present invention and animal (such as people) determines agent Amount.

As is generally known in the art for determining many measuring methods of administration dosage.For the purposes herein, a kind of survey can be used Method is determined to determine the initial dose to be administered to mammal, which is included in mammal (belonging to the mammal One group of mammal be respectively given the analog of various dose) compare blood after the analog of the present invention of application given dose The degree that sugar level reduces.The journey that blood glucose level reduces after applying given dose can be measured by method as known in the art Degree.

Also by the presence of any adverse side effect that may be adjoint by applying specific analog of the present invention, property and degree To determine the dosage of analog of the present invention.In general, attending physician will allow for many factors such as the age, weight, general health, Diet, gender, analog of the present invention to be administered, administration method and the patient's condition to be treated seriousness, determine it is each for treating The dosage of the analog of the present invention of other patient.By way of example and it is not intended to limit the present invention, analog of the present invention Dosage can be about 0.0001 to the treated subject of about 1g/kg body weight/day, about 0.0001 to about 0.001g/kg weight/ It or about 0.01mg are to about 1g/kg body weight/day.In an exemplary embodiment, dosage can be about 1mg to about 40mg or about Total weekly dosage of 4mg to about 30mg or about 4 to about 20mg or about 10 to about 20mg or about 12mg to about 30mg.

In some embodiments, any being disclosed herein of purity level of the pharmaceutical composition comprising being suitable for application to patient Analog.In some embodiments, analog have at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% purity level and pharmaceutically acceptable diluent, carrier or figuration Agent.In some respects, pharmaceutical composition includes the analog of the present invention that concentration is at least A, and wherein A is about 0.001mg/ml, about 0.01mg/ml, about 0.1mg/ml, about 0.5mg/ml, about 1mg/ml, about 2mg/ml, about 3mg/ml, about 4mg/ml, about 5mg/ml, About 6mg/ml, about 7mg/ml, about 8mg/ml, about 9mg/ml, about 10mg/ml, about 11mg/ml, about 12mg/ml, about 13mg/ml, About 14mg/ml, about 15mg/ml, about 16mg/ml, about 17mg/ml, about 18mg/ml, about 19mg/ml, about 20mg/ml, about 21mg/ Ml, about 22mg/ml, about 23mg/ml, about 24mg/ml, about 25mg/ml or 25mg/ml or more.In some embodiments, drug Composition include concentration be at most B analog, wherein B be about 30mg/ml, about 25mg/ml, about 24mg/ml, about 23mg/ml, About 22mg/ml, about 21mg/ml, about 20mg/ml, about 19mg/ml, about 18mg/ml, about 17mg/ml, about 16mg/ml, about 15mg/ Ml, about 14mg/ml, about 13mg/ml, about 12mg/ml, about 11mg/ml, about 10mg/ml, about 9mg/ml, about 8mg/ml, about 7mg/ Ml, about 6mg/ml, about 5mg/ml, about 4mg/ml, about 3mg/ml, about 2mg/ml, about 1mg/ml or about 0.1mg/ml.In some realities It applies in scheme, composition is A to B mg/ml (for example, class of the about 0.001mg/ml to about 30.0mg/ml) containing concentration range Like object.

Targeting form

Will be readily appreciated by those of ordinary skill in the art that analog of the invention can with any various ways through modifying, So that treatment or prevention effect of analog of the present invention increases via modification.For example, analog of the present invention can be directly or indirectly Targeting moiety is conjugated to via connector.The compound of glucagon analogue for example as described herein is sewed as is generally known in the art It is bonded to the operation of targeting moiety.See, for example, Wadhwa et al., J Drug Targeting, 3,111-127 (1995) and the U.S. The patent No. 5,087,616.Term " targeting moiety " as used herein refers to specific recognition and is bound to cell surface receptor So that targeting moiety guides analog of the present invention to be delivered to expressed receptor on surface (glucagon receptor, GLP-1 receptor) Cell colony any molecule or reagent.Targeting moiety includes but is not limited to be bound to cell surface receptor (for example, epithelium is raw Growth factor receptor body (EGFR), T cell receptor (TCR), B-cell receptor (BCR), CD28, platelet derived growth factor receptor (PDGF), nicotinic acetylcholine receptor (nAChR) etc.) antibody or its segment, peptide, hormone, growth factor, cell factor, and Any other natural or non-natural ligand." connector " combines togather Liang Ge independent community as used herein Key, molecule or molecular group.Connector, which can provide the optimal interval of two entities or can further supply, allows two entities to divide each other From unstable connection.Unstable connection includes light cleavable moiety, acid labile moiety, alkali labile moiety and enzyme cleavable Group.In some embodiments, term " connector " refer to by analog of the present invention bridge to targeting moiety any reagent or Molecule.Those skilled in the art will appreciate that on analog of the present invention site (its for analog of the present invention function simultaneously It is nonessential) be jointing and/or targeting moiety ideal site, condition is connector and/or targeting moiety, once be connected to Analog of the present invention would not interfere the function of analog of the present invention, that is, stimulation cAMP from cell secrete with treat diabetes or The ability of obesity.

Control delivery formulations

Alternatively, depot form can be changed into glucagon analogue as described herein, so that analog of the present invention The mode for being discharged into applied body be controlled on time and internal position (see, for example, U.S. Patent number 4,450, 150).The depot form of analog of the present invention can be for example, comprising analog of the present invention and porous or non-porous materials The implantable composition of such as polymer, wherein by being packed in material or making it to be diffused into entire material analog of the present invention In and/or non-porous materials degradation.Depot formulation is implanted required position again, and analog of the present invention is with pre- constant speed Rate is discharged from the implantation material.

In illustrative aspect, pharmaceutical composition is altered to have any kind of internal release overview.In some respects, Pharmaceutical composition is to release immediately, control release, sustained release, extended release, sustained release or two-phase delivery formulations.This field In it is known prepare peptide with carry out control release method.See, for example, Qian et al., Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publication No. WO 2008/130158；WO2004/033036；WO2000/032218；And WO 1999/040942。

This composition can further include for example, micella or liposome or certain other encapsulated form, or can be to extend Releasing pattern application stores and/or delivers effect to provide to extend.Disclosed pharmaceutical preparation, packet can be applied according to any scheme Include for example daily (one time a day, 2 times a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day), three-times-weekly, weekly two Secondary, every two days, it is three days every, four days every, five days every, six days every, weekly, every two weeks, it is three weeks every, monthly or two months every.

Combination

Glucagon analogue as described herein other therapeutic agents can be applied alone or in combination, be intended to treat or prevent Any disease or medical condition as described herein.For example, glucagon analogue as described herein can be with anti-diabetic or anti- Obesity medicament (simultaneously or sequentially) is co-administered.As is generally known in the art or the anti-diabetic medicament studied includes pancreas islet Element, leptin (leptin), Peptide YY (PYY), pancreas peptide (PP), fibroblast growth factor 21 (FGF21), Y2Y4 receptor agonism Agent, sulfonylureas such as orinase (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorine sulphur third Urea (Diabinese), Glipizide (Glucotrol), glibenclamide (Diabeta, Micronase, Glynase), Ge Liemei Urea (Amaryl) or gliclazide (Diamicron)；Meglitinide, such as Repaglinide (Prandin) or Nateglinide (Starlix)；Biguanides such as melbine (Glucophage) or insoral；Thiazolidinedione such as Rosiglitazone (Avandia), Pioglitazone (Actos) or troglitazone (Rezulin) or other PPAR gamma inhibitors；Inhibit carbon hydrate The α glucosidase inhibitor of object digestion, such as Miglitol (Glyset), acarbose (Precose/Glucobay)；Chinese mugwort Fill in that peptide (exenatide) (Byetta) or pramlintide；Dipeptidyl peptidase-4 (DPP-4) inhibitor, such as vildagliptin (vildagliptin) or sitagliptin (sitagliptin)；SGLT (sodium dependent glucose transport protein 1) inhibitor；Portugal Sugared kinase activation agent (GKA)；Glucagon receptor antagonist (GRA)；Or FBPase (fructose 1,6-bisphosphatase) inhibitor.

As is generally known in the art or the anti-obesity medicament studied includes appetite inhibitor, including the stimulation of phenyl ethylamine type Agent, Phentermine (optionally with fenfluramine or Dexfenfluramine), diethylpropionPhendimetrazine), benzphetamine (benzphetamine)Sibutramine (sibutramine) Rimonabant (rimonabant)Other are big Numb hormone receptor antagonists；Oxyntomodulin；Fluoxetine hydrochloride (fluoxetine hydrochloride) (Prozac)； Qnexa (Topiramate (topiramate) and Phentermine), Excalia (diethylpropion and Zonisamide) or Contrave (An Fei Draw ketone and naltrexone (naltrexone))；Or lipase inhibitor, it is similar to Xenical (XENICAL) (orlistat Or celeste (Cetilistat) (also referred to as ATL-962) or GT389-255 (Orlistat)).

In some embodiments, peptide as described herein with for treating non-alcoholic fatty liver disease or the medicament one of NASH It rises and is co-administered.Medicament for treating non-alcoholic fatty liver disease include ursodesoxycholic acid (also known as Actigall, URSO and Ursodiol), melbine (Glucophage), Rosiglitazone (Avandia), Clofibrate, Gemfibrozil, polymyxin B And glycine betaine.

In some embodiments, peptide as described herein with for treating neurodegenerative disorders (for example, Parkinson's disease (Parkinson's Disease)) medicament cooperatively apply.In addition, Mirapexin agent is known in the art And including but not limited to levodopa (levodopa), carbidopa (carbidopa), cholilytic drug (anticholinergics), bromocriptine (bromocriptine), Pramipexole (pramipexole) and ropinirole (ropinirole), amantadine (amantadine) and Rasagiline (rasagiline).

In view of aforementioned, the present invention further provides the pharmaceutical compositions and reagent that additionally comprise one of these other therapeutic agents Box.The additional therapeutic agent can simultaneously or sequentially be applied with analog of the present invention.In some respects, analog is in additional therapeutic agent It applies before, and in other respects, analog is applied after additional therapeutic agent.

Purposes

Based on the information provided for the first time herein, covering composition of the invention (for example, relevant pharmaceutical composition) can be used for Disease or medical condition are treated, wherein for example lacking for GIP receptor, GLP-1 receptor or the activity for two kinds of receptors is this The breaking-out of disease or medical condition and/or the factor of progress.Therefore, the disclosure provides the disease or medicine for treating or preventing patient The method of the patient's condition, wherein the disease or medical condition are wherein to lack GIP receptor activation and/or GLP-1 receptor activation and disease Or the breaking-out and/or the relevant disease of progress or medical condition of medical condition.This method includes to provide effectively treatment to the patient Or prevent the composition or conjugate according to any those described herein of the amount of the disease or medical condition.

In some embodiments, disease or medical condition are metabolic syndrome.Metabolic syndrome (also referred to as Metabolic syndrome Levy X, insulin resistance syndrome or thunder Wen syndrome (Reaven's syndrome)) it is a kind of influence more than 5,000 Wan Meiguo The illness of people.Metabolic syndrome is usually characterized by that at least three or more following risk factors: (1) abdomen occurs in cluster Portion is fat (abdomen neutralizes the excessive adipose tissue of surrounding)；(2) (dyslipidemia, including height are sweet for atharosclerosis dyslipidemia Oily three esters, low HDL cholesterol and high LDL cholesterol enhance accumulation of the patch in arterial wall)；(3) hypertension；(4) pancreas islet Plain resistance or glucose intolerance；(5) before embolism state (for example, high fibrinogen or activator of plasminogen in blood Inhibitor -1)；(6) proinflammatory state (for example, C- proteins C reactive increases in blood).Other risk factors may include old Change, hormone imbalances and genetic predisposition.

Metabolic syndrome relevant other illnesss (such as apoplexy and week to coronary heart disease and with vascular plaque accumulation Side vascular diseases (referred to as Atherosclerotic cardiovascular disease (ASCVD))) risk increase it is related.With metabolic syndrome Patient can be from the insulin resistant state of early stage to full grown type-2 diabetes mellitus, and the risk of ASCVD is further Increase.It is not intended to be bound to any particular theory, the relationship between insulin resistance, metabolic syndrome and vascular diseases can be related to one A or multiple concurrent pathogenic mechanisms, impaired vasodilation including insulin stimulating, due to pancreas caused by oxidative stress enhancing Hormone (such as adiponectin (adiponectin)) derived from the relevant NO availability reduction of island element resistance and fat cell is abnormal (Lteif and Mather, (2004) Can.J.Cardiol.20 (supplementary issue B): 66B-76B).

According to U.S. national cholesterol education program adult treatment experimental subjects group (2001National in 2001 Cholesterol Education Program Adult Treatment Panel (ATP III)), it is following in same individual Any three kinds in the characteristic standards for meeting metabolic syndrome: (a) (male's waistline is greater than 102cm and women waistline to abdominal obesity Greater than 88cm)；(b) serum triglyceride (150mg/dl or more)；(c) HDL cholesterol (male be 40mg/dl or following and Women is 50mg/dl or following)；(d) blood pressure (130/85 or more)；(e) fasting blood-glucose (110mg/dl or more).According to The World Health Organization (World Health Organization, WHO) has height at least two in following standard The individual of insulin level (individual high fasting blood glucose or high postprandial glucose) meets the standard of metabolic syndrome: (a) abdomen Fat (waist-to-hipratio is greater than 0.9, and body-mass index is at least 30kg/m2 or waist measurement is greater than 37 inches)；(b) it shows At least cholesterol experimental subjects group of the triglyceride levels of 150mg/dl or the HDL cholesterol lower than 35mg/dl；(c) blood pressure It is 140/90 or more, or is treating hypertension.(Mathur,Ruchi,"Metabolic Syndrome,",Shiel, Jr., William C. is compiled, MedicineNet.com, on May 11st, 2009).

For the purposes herein, if individual meets U.S. national cholesterol education program adult treatment experiment pair in 2001 As either one or two of group or WHO standard proposed, then the individual is considered as with metabolic syndrome.

Without being bound by any particular theory, composition and conjugate as described herein can be used for treating metabolic syndrome.Cause This, the present invention provides the metabolic syndrome of prevention or treatment subject or reduces the one, two, three or more of subject The method of metabolic syndrome risk factors comprising provide effectively prevention or treatment metabolic syndrome or its risk to the subject The composition as described herein of the amount of factor.

In some embodiments, this method treats hyperglycemia medical condition.In illustrative aspect, the hyperglycemia medicine disease Condition is diabetes, type-1 diabetes mellitus, type-2 diabetes mellitus or gestational diabetes mellitus (insulin-dependent or non-insulin-dependent).? Some aspects, this method is by reducing one or more complication (including nephropathy, retinopathy and the blood vessel disease of diabetes Disease) treat hyperglycemia medical condition.

In some respects, disease or medical condition are obesity.In some respects, obesity is that drug-induced property is fat Disease.In some respects, this method treats fertilizer by preventing or reducing the weight gain of patient or increase the body weight loss of patient Fat disease.In some respects, this method is by reducing appetite, reducing food intake, reduce the fat level of patient or reduce food Obesity is treated via the rate travel of gastronintestinal system.

Because obesity is related with the breaking-out of other diseases or progress, the method for the treatment of obesity is further useful for Reduction complication related with obesity (including vascular diseases (coronary artery disease, apoplexy, peripheral vascular disorder, Ischemia Reperfusion Note etc.), hypertension, type-2 diabetes mellitus breaking-out, hyperlipidemia and musculoskeletal disease) method in.Therefore the disclosure provides The method for treating or preventing complication related with these obesity.

In some embodiments, disease or medical condition are non-alcoholic fatty liver disease (NAFLD).NAFLD refers to more Kind of liver diseases, range be from simple fatty liver (steatosis) to nonalcoholic fatty liver disease (NASH) to cirrhosis (no Reversible advanced stage liver scarring).The NAFLD in all stages has fatty (fatty infiltration) in liver cell (liver cell) jointly Accumulation.Simple fatty liver is certain types of fatty (triglycerides) abnormal accumulation and no inflammation or scar shape in liver cell At.In NASH, fat generation is related with the different level of the inflammation (hepatitis) of liver and cicatrization (fibrosis).Inflammatory is thin Born of the same parents can destroy liver cell (necrosis of liver cells).In term " steatohepatitis " and " adiponecrosis ", fatty (steato) is Refer to fatty infiltration, hepatitis refers to liver inflammation, and necrosis refers to that liver cell is damaged.NASH finally can lead to liver cicatrization (fibrosis) and with then irreversible advanced stage cicatrization (cirrhosis).The cirrhosis as caused by NASH is in NAFLD range Final and most serious stage.(Mendler, Michel, " Fatty Liver:Nonalcoholic Fatty Liver Disease(NAFLD)and Nonalcoholic Steatohepatitis(NASH),"Schoenfield,Leslie J. It compiles, MedicineNet.com, on August 29th, 2005).

Alcoholic Jiver disease or alcohol inductivity liver diseases cover excessively consumed to alcohol it is related or excessive by alcohol The different liver diseases of three kinds of pathology caused by consuming: fatty liver (steatosis), chronic or acute hepatitis and cirrhosis. The range of alcoholic hepatitis can arrive serious liver function for mild hepatitis (unique indication that abnormal laboratory is detected as disease) Obstacle simultaneously has complication such as jaundice (yellow skin as caused by bilirubin retention (bilirubin retention)), liver property Encephalopathy (neurological dysfunction as caused by liver failure), ascites (fluid accumulation of abdomen), hemorrhagic esophageal varication be (oesophagus Varication), abnormal blood coagulation and stupor.Alcoholic hepatitis histologically has Hepatocellular ballooning, neutrophilia The characteristic appearance of granulocyte inflammation and sometimes Mallory body (cellular intermediate filament protein abnormal aggregation).The dissection of cirrhosis is special Sign is on liver extensive nodule and fibrosis.(Worman,Howard J.,"Alcoholic Liver Disease", Columbia University Medical Center website)。

Without being bound by any particular theory, composition and conjugate as described herein can be used for treating alcoholic liver disease Disease, NAFLD or its any stage, including for example steatosis, steatohepatitis, hepatitis, liver inflammation, NASH, cirrhosis or And it sends out disease.Therefore, the disclosure provides the alcoholic Jiver disease for preventing or treating subject, NAFLD or its any stage Method comprising provide the sheet of effectively prevention or treatment alcoholic Jiver disease, NAFLD or the amount in its any stage to subject Composition described in text.Such treatment method include reduce it is one of following, two kinds, it is three or more: liver fat contains Amount, the generation of cirrhosis or progress, the incidence of hepatocellular carcinoma, inflammatory condition such as exception liver enzyme level (such as aspartic acid Aminopherase AST and/or alanine aminotransferase ALT or LDH), raised serum ferritin, raised serum bilirubin And/or for example raised TGF-β of fibrosis symptom is horizontal.In an exemplary embodiment, the composition for treat into Exhibition to be more than simple fatty liver (steatosis) and show inflammation or hepatitis symptom patient.Such method can lead to for example AST and/or ALT level reduces.

GLP-1 and exendin-4 have been displayed with some neuroprotections.The disclosure also provides as described herein Purposes of the composition in treatment neurodegenerative disorders, the disease includes but is not limited to: Alzheimer's disease (Alzheimer ' s disease), Parkinson's disease (Parkinson ' s disease), multiple sclerosis, amyotrophic lateral sclerosis (Amylotrophic Lateral Sclerosis), other demyelinate associated disease, senile dementia, subcortical dementias, arteries Hardenability is dull-witted, relevant dull-witted or other dementias of AIDS-, central nervous system cancer, traumatic brain injury, spinal cord injury, Apoplexy or cerebral ischemia, cerebral vasculitis, epilepsy, Huntington's chorea (Huntington ' s disease), tourette's syndrome (Tourette's syndrome), Guillain Barre syndrome (Guillain Barre syndrome), hepatolenticular degeneration (Wilson Disease), Pick disease (Pick's disease), neuro-inflammatory disorders, encephalitis, encephalomyelitis or viral, fungoid or thin The meningitis of bacterium property origin or other central nervous system infections, prion disease, cerebellar ataxia, cerebellar degeneration, spinal cord are small Cerebral degeneration's syndrome, Friedreich ataxia (Friedreichs ataxia), ataxia-telangiectasia, backbone Muscular dystrophy (spinal dysmyotrophy), dystonia, muscle cramp, is trembled, view at stein-leventhal syndrome Membranochromic pigments denaturation, striatum substantia nigra degeneration, mitochondria brain-myopathy, neuronal waxy lipophilic disease, hepatic encephalopathy, nephro-encephalopathy, The encephalopathy and radiation-induced cerebral injury of metabolic encephalopathy, toxin-induced.

In some embodiments, the parenteral administration of composition combination nutrient is for the non-diabetic in hospital environment Patient, for example, the patient for receiving parenteral absorption or total parenteral absorption.Non-limiting example includes patient with operation；Dusk Patient in fan；Patient with following disease: disease of digestive tract or nonfunctional gastrointestinal tract (such as because operation excision, occlusion Or damaged absorbing ability), Crohn disease (Crohn ' s disease), ulcerative colitis, gastrointestinal obstruction, gastrointestinal fistula, Acute pancreatitis, intestine ischemia, gastrointestinal major surgery, certain congenital gastrointestinal abnormalities, extended diarrhea or because operation caused by Short bowel syndrome；Patient in shock, and healing process is just undergone, often receive parenteral administration carbohydrate and rouge Matter, electrolyte, minerals, vitamin and amino acid multiple combinations patient.Comprising GIP agonist peptide as described herein and The composition and parenteral nutrition composition of glucagon antagonist peptide can simultaneously, when different, each other successively application, condition It is biological effect needed for the composition is played when parenteral nutrition composition is digested.For example, parenteral absorption can daily 1 It is secondary, 2 times or 3 times application, and the composition every other day once, three-times-weekly, twice a week, once a week, once every 2 weeks, It is primary every 3 weeks or monthly apply.

Term " treatment " as used herein includes prevention particular condition or the patient's condition, or is mitigated and particular condition or patient's condition phase The symptom of pass, and/or prevent or eliminate the symptom.For example, term " treatment diabetes " as used herein will generally refer to Change blood glucose level with normal horizontal orientation and may include that blood glucose level is increased or decreased according to given situation.

" effective " amount of glucagon peptide as used herein or " therapeutically effective amount ", which refer to, provides the required nothing acted on The peptide of poison but sufficient amount.For example, effect needed for a kind of will be prevention or treatment hypoglycemia, such as example increased by blood glucose level It is measured.Effect needed for the substitution of disclosure glucagon peptide will include treatment hyperglycemia, such as such as pass through blood glucose level Measured by change closer to normal level；Or induction weight loss/prevention weight gain, such as surveyed by weight loss Amount；Or prevent or reduce weight gain；Or body fat is made to be distributed normalization.Age and overall state, application depending on individual Mode etc., " effective " amount will be different between subject.Therefore, definite " effective quantity " not may always be specified.However, at any In other situation it is appropriate it is " effective " amount can be determined by those of ordinary skill in the art using routine experiment method.

Subject

About above-mentioned treatment method, patient is any host.In some embodiments, host is mammal.Such as this Term used in text " mammal " refers to mammiferous any vertebrate, including but not limited to any monotreme, marsupial And placental.In some embodiments, mammal is one of Rodentia (Rodentia) mammal (such as mouse And hamster) and Lagomorpha (Logomorpha) mammal (such as rabbit).In an exemplary embodiment, mammal comes from Carnivora (Carnivora), including felid (cat) and canid (dog).In an exemplary embodiment, mammal From Artiodactyla (Artiodactyla), including bovid (ox) and porcine (pig), or come from Perissodactyla Including equid (horse) (Perssodactyla),.In some cases, mammal be Primates (Primates), Ceboids or Simoids (monkey) or anthropoids (Anthropoids) (human and ape).In certain embodiments, lactation is dynamic Object is people.

Kit

The a part of glucagon analogue of the invention as kit can be provided according to an embodiment.Cause This provides the kit for applying glucagon analogue to patient in need in some embodiments, wherein should Kit includes glucagon analogue as described herein.

In one embodiment, the reagent with the device for applying glucagon analogue to subject is provided Box.The device is syringe needle, pen device, jet injector or other needleless injectors in some respects.Kit can With optionally or additionally include one or more containers, for example, bottle, pipe, bottle, single-chamber or multi-chamber precharging injection syringe, cylindrantherae, Infusion pump (External or implanted), jet injector, pre-filled pen device etc. optionally contain lyophilized form or aqueous solution shape The glucagon analogue of formula.In some embodiments, kit includes operation instructions.According to an embodiment, The device of kit is aerosol-dispensing device, wherein composition is prepackaged in aerosol device.In another embodiment party In case, kit includes syringe and syringe needle, and is in one embodiment prepackaged in aseptic glucagon composition In syringe.

In some embodiments, kit includes operation instructions.In some respects, the specification includes according to this The operation instructions of any one of literary the method.The specification can be also comprised about maintenance healthy diet and/or body Educate the specification of degree of physical exercise.The specification can be papery booklet form or electronic form, for example, the meter comprising specification The readable storage device of calculation machine.

Detailed description of the invention

Fig. 1 shows that SEQ.ID NO.1 and SEQ.ID NO.2 effectively excitement GLP-1 receptor and can resist human body The hydrolysis of interior DPP4.

Fig. 2 shows influence of SEQ.ID NO.1 and the SEQ.ID NO.2 to pancreas islet BETA cells secrete insulin.SEQ.ID NO.1, SEQ.ID NO.2 do not promote insulin secretion in low sugar, avoid causing hypoglycemia, have good safety；In Portugal Grape sugar can promote BETA cells secrete insulin when increasing, and when concentration of glucose is higher can act (11uM grape Sugar), it is more sensitive to change of blood sugar relative to hGLP1, there is better hypoglycemic effect.

Fig. 3 shows that diabetic obese mice (BKS db) blood glucose level is adjusted in SEQ.ID NO.1 and SEQ.ID NO.2, Reduce blood glucose (monitoring within 0-60 minutes).

Fig. 4 shows that diabetic obese mice (BKS db) insulin secretion is adjusted in SEQ.ID NO.1 and SEQ.ID NO.2 (monitoring within 0-60 minutes).

Fig. 5 shows that SEQ.ID NO.1 and SEQ.ID NO.2 can long-acting adjusting diabetic obese mice (BKS db) blood glucose water It is flat, reduce blood glucose (monitoring within 0-240 minutes).

Fig. 6 shows that SEQ.ID NO.1 and SEQ.ID NO.2 can long-acting adjusting diabetic obese mice (BKS db) insulin Secretion (monitors) for 0-240 minutes.

Specific embodiment

Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this Invention is not for limiting the scope of the invention.

Embodiment 1, new discovery peptide fragment SEQ.ID NO.1 (His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Lys-Ala-Thr-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys- ) and SEQ.ID NO.2 (His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser- Gly-Leu-Glu Tyr-Leu-Asp-Gly-Lys-Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Lys-Ser-Leu-Glu) Effectively excitement GLP-1 receptor and the hydrolysis of the intracorporal DPP4 of people can be resisted

In order to verify sequence SEQ.ID NO.1 and SEQ.ID NO.2 whether can be as GLP-1 receptor stimulating agent, we Chemical synthesis purity is greater than 95% polypeptide.Polypeptide is diluted to containing 20mM HEPES and 2.5mM benemid In Hank ' s balanced salt solution (HBSS, pH 7.4), concentration 100nM.In order to which can the GLP-1 polypeptide that detect synthesized activate GLP-1 receptor has purchased instant GLP-1 detection cell (article No. HTS163RTA) and corresponding complete by Millipore company Full culture medium.The cell is derived by Chem-9 cell line, and further expresses GLP-1 receptor and G- α albumen.It is bought Cell saves in liquid nitrogen.Used time melts in 37 DEG C of water-baths.Cell is transferred to the centrifugation of the 15ml containing 6ml complete medium Guan Zhong, 1000rpm are centrifuged 5 minutes, remove supernatant.Cell is resuspended in 10ml complete medium, is seeded to by the hole 200ul/ 96- orifice plate.It is cultivated 24 hours in 5%CO2,37 DEG C of incubators.96 orifice plates are taken out by incubator before analysis.To contain 20mM HEPES Every hole cell is washed with Hank ' the s balanced salt solution (HBSS, pH 7.4) of 2.5mM benemid.Then, every hole adds 100ul to contain There are 5mM Fluo-8NW (ABD Bioquest 21080) calcon-carboxylic acid, the Hank ' of 20mM HEPES and 2.5mM benemid S balanced salt solution.It is incubated for 20 minutes in 5%CO2,37 DEG C of incubators.Contain 20mM HEPES and 2.5mM oxybenzene sulphur with 100ul Hank ' the s balanced salt solution (HBSS, pH 7.4) of propylamine washes every hole cell.Before fluorescence microscopy microscopic observation, corresponding aperture is removed 50ul GLP-1 polypeptide to be detected is added in interior Hank ' s balanced salt solution.Using GFP optical filter, with cameras record cell fluorescence Dynamic change.Since the cell line is overexpressed GLP-1 receptor and G- α albumen, so can be opened after GLP-1 receptor is activated Dynamic Ca2+ influx.So can reflect whether GLP-1 receptor is activated by the variation of fluorescence.As shown in table one and Fig. 1: peptide fragment SEQ.ID NO.1 and SEQ.ID NO.2 can be with acute activation GLP-1 receptors, and DPP4 is added and does not have an impact peptide fragment activity, Illustrate that peptide fragment SEQ.ID NO.1 and SEQ.ID NO.2 can resist the hydrolysis of the intracorporal DPP4 of people.Peptide fragment as the result is shown simultaneously The GLP1 activity that SEQ.ID NO.1 and SEQ.ID NO.2 activity is compared to people is more lasting.

The variation of cell Ca signal caused by one .SEQ.ID NO.1 and SEQ.ID NO.2 of table

Embodiment 2, the adjustable insulin secretion of SEQ.ID NO.1 and SEQ.ID NO.2

832/13 cell line of INS-1 derives from the rat pancreatic β cell converted through human proinsulin gene.GLP1 receptor is expressed, After by active GLP1 or the stimulation of its analog, and under the conditions of high concentration glucose, insulin secretion increases.I Passed through Ca2+ influx experiments have shown that GLP1 analog to be measured can activate GLP-1R.The experiment is with 832/13 cell of INS-1 Can GLP1 analog to be measured detects in system promote insulin releasing by activation GLP-1R.

The complete medium of 832/13 cell line of INS-1 is RPMI 1640 and is added to 10% fetal calf serum, 50IU/ml Penicillin, 50mg/L streptomysin, 10mM HEPES, 2mM L-Glutamine, 1mM Sodium Pyruvate and 50uM beta -mercaptoethanol. 832/13 cell of INS-1 passes on weekly twice, cultivates in 5%CO2,37 DEG C of incubators.

832/13 cell of 1x105INS-1 is inoculated in every hole of 24- orifice plate.It is cultivated in 5%CO2,37 DEG C of incubators, every other day A culture solution is changed, is co-cultured 6 days.Remove culture solution, with 400ul sugar-free KRB solution (116mM NaCl, 1.8mM CaCl2, 0.8mM MgSO4,5.4mM KCl, 1mM NaH2PO4,26mM NaHCO3,0.5%BSA, pH 7.4) wash cell twice.Every hole 400ul sugar-free KRB solution is added, is placed 1 hour in 5%CO2,37 DEG C of incubators.Remove KRB, with 400ul sugar-free KRB solution Wash cell twice.The KRB solution that 400ul contains 2.2mM or 16.8mM glucose or 100nM GLP1 polypeptide is added.5% CO2 is placed 2 hours in 37 DEG C of incubators.Cell culture supernatant is taken, with the content of ELISA kit analysis insulin.

Rat insulin ELISA kit is purchased from Thermo Fisher.To every hole INS-1832/13 cell, 100ul is taken Cell culture supernatant is added in elisa plate corresponding aperture.Blank control is added simultaneously and the rat insulin of various concentration is done For reference.96 orifice plates are sealed, are incubated at room temperature 2.5 hours.Board-washing 4 times.Then the anti-rat of 100ul biotin labeling is added in every hole Insulin antibody seals 96 orifice plates.Incubation at room temperature 1 hour.Board-washing 4 times.The horseradish mistake of 100ul streptavidin label is added in every hole Oxide enzyme.Incubation at room temperature 45 minutes.Board-washing 4 times.50ul tmb substrate is added in every hole.It is incubated for 30 minutes in dark at room temperature.Often 50ul reaction terminating liquid is added in hole.The absorbance of 450nm and 550nm is read with plate reading machine, and is corrected and read with blank control wells Numerical value.Again by subtracting the reading of 550nm in the reading of 450nm.According to the reference insulin hole of rat (700uIU/ml, 300uIU/ml, 150uIU/ml, 75uIU/ml, 37.5uIU/ml, 18.75uIU/ml, 9.38uIU/ml, and 0uIU/ml) production Standard curve.Calculate the corresponding sample insulin concentration in each hole.As the result is shown under the conditions of low sugar (2.8uM glucose), HGLP1, SEQ.ID NO.1, SEQ.ID NO.2 do not promote insulin secretion, under the conditions of high sugar (16.7uM glucose), HGLP1, SEQ.ID NO.1, SEQ.ID NO.2 promote insulin secretion (P < 0.01), (11uM grape under the conditions of higher sugar Sugar) SEQ.ID NO.1, SEQ.ID NO.2 promotes insulin secretion (P < 0.01).This illustrates SEQ.ID NO.1, SEQ.ID NO.2 does not work in low sugar, avoids causing hypoglycemia, has good safety.Meanwhile SEQ.ID NO.1, SEQ.ID NO.2 can promote BETA cells secrete insulin when glucose increases, and when concentration of glucose is higher can act (11uM glucose) has better hypoglycemic effect relative to hGLP1 (table 2, Fig. 2) more sensitive to change of blood sugar.

Influence of table 2.SEQ.ID NO.1 and the SEQ.ID NO.2 to insulin secretion.

Obesity mice (BKS db) insulin level is adjusted in embodiment 3, SEQ.ID NO.1 and SEQ.ID NO.2, reduces Blood glucose.

Whether can promote to verify polypeptide SEQ.ID NO.1 and SEQ.ID NO.2 in the model animal of diabetes B Into insulin secretion, blood sugar concentration is reduced, we select BKS.Cg-Dock7<m>+/+Lepr<db>/J (db/db) obese male Mouse is as experimental subjects (The Jackson Laboratory).The starvation of 18 hours is carried out to experimental animal first, so Afterwards with 25nmol/kg peptide concentration (SEQ.ID NO.1, SEQ.ID NO.2, Exendin-4) and 18mmol/kg concentration of glucose With progress intraperitoneal injection.Control group injecting normal saline.Blood sugar concentration is measured before the injection, is then measured at interval of 15 minutes Once (0 minute, 15 minutes, 30 minutes, 45 minutes, 60 minutes).Rat-tail blood is collected using enzyme linked immunosorbent assay (ELISA) point Analysis measurement insulin concentration, blood glucose monitoring system measure concentration of glucose.It can be found that polypeptide SEQ.ID NO.1 and SEQ.ID NO.2 can be effectively reduced blood glucose level (table 3, Fig. 3), insulin level (table 4, Fig. 4) in elevating blood.

Whether can to further verify SEQ.ID NO.1 and SEQ.ID NO.2 in the model animal of diabetes B The long-term sensibility for increasing insulin, adjusts insulin secretion, reduces blood sugar concentration, we continue to select BKS.Cg-Dock7 < m >+/+Lepr<db>/J (db/db) obese male mice as experimental subjects (The Jackson Laboratory), with 25nmol/kg peptide concentration (SEQ.ID NO.1, SEQ.ID NO.2, Exendin-4) and 18mmol/kg concentration of glucose and into Row intraperitoneal injection, control group injecting normal saline.The experimental animal normally raised is injected intraperitoneally, is measured before the injection Then blood sugar concentration measured primary (0 minute, 60 minutes, 120 minutes, 180 minutes, 240 minutes) collection mouse at interval of 60 minutes Tail blood measures concentration of glucose using enzyme linked immunosorbent assay (ELISA) analysis measurement insulin concentration, blood glucose monitoring system.It can be with It was found that blood glucose level (table 5, Fig. 5) can be effectively reduced in polypeptide SEQ.ID NO.1 and SEQ.ID NO.2, insulin is increased Sensibility (table 6, Fig. 6).

Embodiment 4, SEQ.ID NO.1 and SEQ.ID NO.2 modification sequence SEQ.ID NO.3 and SEQ.ID NO.4 can be grown Effect adjusts obesity mice (BKS db) insulin level, reduces blood glucose.

Further peptide C terminal amino acid is modified, is partly declined with increasing the vivo biodistribution stability of the polypeptide and extending Phase prepares more long-acting -1 derivative of novel glp-1, and sequence is as follows:

SEQ.ID NO.3:

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly- Xaa17-Ala-Thr-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Leu-Glu。

SEQ.ID NO.4:His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu- Asp-Gly-Xaa17-Ala-Ala-Xaa20-Glu-Phe-Val-Ala-Trp-Leu-Val-Xaa28-Ser-Leu-Glu。

Wherein Xaa17 is Ser, His, Gln, any one of Ala or Lys amino acid, and Xaa20 is Ser, His, Gln, Any one of Ala or Lys amino acid, Xaa28 are Ser, His, Asp, any one of Ala or Lys amino acid.

Whether can be grown to verify polypeptide SEQ.ID NO.3 and SEQ.ID NO.4 in the model animal of diabetes B Effect promotes insulin secretion, reduces blood sugar concentration, we continue to select BKS.Cg-Dock7<m>+/+Lepr<db>/J (db/db) Obese male mice is as experimental subjects (The Jackson Laboratory).Negative control group physiological saline, positive control Group Liraglutide (Liraglutide) and that peptide (Exenatide) (25nmol/kg) of Ethiopia, compound group (25nmol/kg):

SEQ.ID NO.5, His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu- Glu-Gly-Lys-Ala-Thr-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Ala-Gly-Leu-Glu

SEQ.ID NO.6, His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu- Glu-Gly-Lys-Ala-Thr-Ala-Glu-Phe-Ile-Ala-Trp-Leu-Val-Ala-Gly-Leu-Glu

SEQ.ID NO.7, His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu- Glu-Gly-Ala-Ala-Thr-Ala-Glu-Phe-Ile-Ala-Trp-Leu-Val-Ala-Gly-Leu-Glu

SEQ.ID NO.8,

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

SEQ.ID NO.9, His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu- Asp-Gly-Lys-Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.10,

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Ala- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.11,

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

SEQ.ID NO.12,

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys- Ala-Ala-Asp-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.13,

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Asp- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.14,

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

SEQ.ID NO.15,

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

SEQ.ID NO.16,

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

Mouse normal water, feeding, 0h give compound, measure blood glucose with blood glucose meter 0,3,6,12,24,48h, can be with It was found that polypeptide 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 can reduce blood glucose level (table 7) for a long time.

In conclusion novel glp-1-1SEQ.ID NO.1 and SEQ.ID NO.2 and its modification sequence SEQ.ID NO.3 and Obesity mice (BKS db) insulin level is adjusted in SEQ.ID NO.4, reduces blood glucose, and and Liraglutide (Liraglutide) compare with that peptide (Exenatide) of Ethiopia with permanent mechanism.Therefore it can be used as the drop of type-II diabetes Sugared drug.

Sequence table

SEQ.ID NO.1 glucagon analogue

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

SEQ.ID NO.2 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Lys-Ser-Leu-Glu

SEQ.ID NO.3 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly- Xaa17-Ala-Thr-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gl y-Leu-Glu,

Wherein Xaa17 is Ser, His, Gln, any one of Ala or Lys amino acid, and Xaa20 is Ser, His, Gln, Any one of Ala or Lys amino acid, Xaa28 are Ser, His, Asp, any one of Ala or Lys amino acid.

SEQ.ID NO.4 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly- Xaa17-Ala-Ala-Xaa20-Glu-Phe-Val-Ala-Trp-Leu-Val-Xaa28-Se r-Leu-Glu,

Wherein Xaa17 is Ser, His, Gln, any one of Ala or Lys amino acid, and Xaa20 is Ser, His, Gln, Any one of Ala or Lys amino acid, Xaa28 are Ser, His, Asp, any one of Ala or Lys amino acid.

SEQ.ID NO.5 glucagon analogue

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

SEQ.ID NO.6 glucagon analogue

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

SEQ.ID NO.7 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Ala- Ala-Thr-Ala-Glu-Phe-Ile-Ala-Trp-Leu-Val-Ala-Gly-Leu-Glu

SEQ.ID NO.8 glucagon analogue

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

SEQ.ID NO.9 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.10 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Ala- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.11 glucagon analogue

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

SEQ.ID NO.12 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys- Ala-Ala-Asp-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.13 glucagon analogue

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Asp- Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.14 glucagon analogue

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

SEQ.ID NO.15 glucagon analogue

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

SEQ.ID NO.16 glucagon analogue

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

Claims (10)

1. a kind of glucagon analogue and its pharmaceutically acceptable salt, solvate, prodrug or their any group It closes, wherein the glucagon analogue has longer half-life period compared with Wild type human GLP1 and preferably promotees pancreas islet Plain secretion activity.

2. the glucagon analogue of claim 1 and its pharmaceutically acceptable salt, solvate, prodrug or they Any combination, wherein the sequence of the glucagon analogue are as follows:

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Xaa17- Ala-Thr-Xaa20-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa28-Gly-Leu- Glu (SEQ.ID NO.3),

Xaa17 is Ser, His, Gln, and any one of Ala or Lys amino acid, Xaa20 is Ser, His, Gln, Ala or Lys Any one of amino acid, Xaa28 be Ser, His, Asp, any one of Ala or Lys amino acid.

3. the glucagon analogue of claim 1 and its pharmaceutically acceptable salt, solvate, prodrug or they Any combination, wherein the sequence of the glucagon analogue are as follows:

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Xaa17- Ala-Ala-Xaa20-Glu-Phe-Val-Ala-Trp-Leu-Val-Xaa28-Ser-Leu- Glu (SEQ.ID NO.4),

Xaa17 is Ser, His, Gln, and any one of Ala or Lys amino acid, Xaa20 is Ser, His, Gln, Ala or Lys Any one of amino acid, Xaa28 be Ser, His, Asp, any one of Ala or Lys amino acid.

4. the glucagon analogue of any one of preceding claims and its pharmaceutically acceptable salt, solvate, Prodrug or their any combination, wherein the sequence of the glucagon analogue is selected from SEQ.ID NO.1-16.

5. it includes the glucagon analogues of any one of two or more preceding claims.

6. conjugate, it includes the glucagon analogue of any one of preceding claims and conjugate fractions.

7. the conjugate of claim 6, wherein the glucagon analogue is merged with heterologous peptide analogue.

8. pharmaceutical composition, it includes the glucagon analogues of any one of preceding claims, the dimerization of claim 5 Body or polymer, claim 6 or 7 conjugate, or combinations thereof and pharmaceutically acceptable carrier, diluent or excipient.

9. the dimer or polymer or right of the glucagon analogue of any one of claim 1-4, claim 5 are wanted 6 or 7 conjugate is asked to prepare for being lost weight in increase or the drug for inducing weight loss in subject in need Purposes.

10. the dimer or polymer or right of the glucagon analogue of any one of claim 1-4, claim 5 It is required that 6 or 7 conjugate is preparing the purposes in the drug for treating diabetes.