CN109762059B - Novel glucagon analogue and application thereof - Google Patents

Abstract

The invention relates to a novel glucagon analogue and application thereof. Compared with wild human GLP1, the novel glucagon analogue has longer half-life and better insulin secretion promoting activity, and has strong long-acting hypoglycemic effect.

Description

Novel glucagon analogue and application thereof

Technical Field

The invention belongs to the technical field of polypeptide medicines. In particular to application of a novel GLP-1 receptor agonist medicament in promoting insulin secretion and reducing blood sugar and application in treating type II diabetes and obesity.

Background

Type II diabetes is a long-term metabolic disorder characterized by hyperglycemia, insulin resistance and a relative lack of insulin. The long-term complications caused by hyperglycemia of patients comprise heart disease, stroke, diabetic retinopathy, blindness, renal failure, limb blood flow disorder, amputation and the like. Type II diabetes accounts for approximately 90% of cases of diabetes. Since 1960, the incidence of type II diabetes has increased significantly. By 2017, approximately 4.5 million people were diagnosed with type II diabetes, in contrast to approximately 3000 million people in 1985. The world health organization predicts that more than 6 million people will suffer from type II diabetes worldwide by 2035.

Preproglucagon consists of 158 amino acids. Is cleaved at a different site to form glucagon-like peptide-1 (GLP-1). Glucagon-like peptide-1 (GLP-1) is a predominantly monopeptide compound that stimulates secretion of epidermal cells of the small intestine by food. GLP-1 can promote insulin secretion, protect islet beta cells, inhibit glucagon secretion, inhibit gastric emptying, and reduce appetite by stimulating its receptor. Therefore, it can be used for treating type II diabetes and obesity. GLP-1 with biological activity in human bodies is mainly GLP-1(7-36) amide and GLP-1(7-37), but is rapidly hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV) (half-life is less than 5min), and has no clinical use value. The prior GLP-1 receptor agonist drugs are obtained by structurally modifying GLP-1, eliminating or covering DPP-IV enzyme cutting sites and simultaneously retaining the pharmacological activity of the DPP-IV enzyme cutting sites, such as Liraglutide (Liraglutide) and Dulaglutide (Dulaglutide). In addition, native polypeptides that are structurally similar to GLP-1 may also have the same pharmacological activity. Exendin-4 is a glucagon analogue separated from lizard saliva, consists of 39 amino acids, has about 53 percent of homology with GLP-1, is not degraded by DPP-IV, and has longer half-life and stronger biological activity. Exenatide (Exenatide) synthesized based on the Exendin-4 sequence is marketed.

There is a need in the art for a novel glucagon analog having a longer half-life and better insulinotropic activity than wild-type human GLP 1.

Disclosure of Invention

After large-scale screening and verification, the inventor discovers a novel glucagon analogue.

Specifically, the present invention provides the following:

in one embodiment, the present invention provides a glucagon analog wherein the glucagon analog has a longer half-life and better insulinotropic activity compared to wild-type human GLP 1.

In a further embodiment, the present invention provides a glucagon analog, wherein the sequence of the glucagon analog is:

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

xaa17 is any one of Ser, His, Gln, Ala or Lys, Xaa20 is any one of Ser, His, Gln, Ala or Lys, and Xaa28 is any one of Ser, His, Asp, Ala or Lys.

In a further embodiment, the present invention provides a glucagon analog, wherein the sequence of the glucagon analog is:

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

xaa17 is any one of Ser, His, Gln, Ala or Lys, Xaa20 is any one of Ser, His, Gln, Ala or Lys, and Xaa28 is any one of Ser, His, Asp, Ala or Lys.

In a further embodiment, the present invention provides a glucagon analog wherein the sequence of the glucagon analog is selected from the group consisting of seq.id No. 1-16.

In a further embodiment, the present invention provides a pharmaceutically acceptable salt, solvate, prodrug, or any combination thereof of the glucagon analogs described above.

Experiments prove that the glucagon analogue is easy to synthesize, can effectively excite a GLP-1 receptor for a long time, promotes the secretion of insulin, enhances the sensitivity of the insulin, reduces the blood sugar and has better stability. Furthermore, the glucagon analogues have enhanced in vivo biological stability and prolonged half-life, resulting in more prolonged activity.

In another embodiment, the invention provides a dimer or multimer comprising two or more glucagon analogs of the invention.

In another embodiment, the invention provides a conjugate comprising a glucagon analog of the invention and a conjugate moiety.

In a further embodiment, the present invention provides a conjugate wherein the glucagon analog is fused to a heterologous peptide analog.

In a further embodiment, the present invention provides a pharmaceutical composition comprising a glucagon analog of the present invention, a dimer or multimer of the present invention, a conjugate of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the present invention provides a method of reducing weight gain or inducing weight loss in a subject in need thereof comprising administering to a patient in need thereof a pharmaceutical composition of the present invention in an amount effective to reduce weight gain or induce weight loss. In a further embodiment, the amount is effective to treat obesity in a subject in need thereof.

In another embodiment, the present invention provides a method of treating diabetes comprising administering to a patient in need thereof a pharmaceutical composition of the present invention.

In another embodiment, the invention provides a glucagon analogue of the invention, a dimer or multimer of the invention or a conjugate of the invention for use in the manufacture of a medicament for reducing weight gain or inducing weight loss in a subject in need thereof.

In another embodiment, the invention provides a use of a glucagon analogue of the invention, a dimer or multimer of the invention or a conjugate of the invention in the manufacture of a medicament for the treatment of diabetes.

Method for producing peptide

The glucagon analogs of the present invention can be obtained by methods known in the art. Suitable methods for resynthesizing peptides are described, for example, in 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; and U.S. patent No.5,449,752.

In some embodiments, the glucagon analogs or peptides described herein can be commercially synthesized by a company. In this regard, the peptide may be synthesized, recombinant, isolated and/or purified.

Furthermore, where the analogs of the invention do not contain any non-coding or unnatural amino acids, the glucagon analogs can be recombinantly produced using nucleic acids encoding the amino acid sequences of the analogs using standard recombinant methods. See, e.g., Sambrook et al, Molecular Cloning: a Laboratory Manual, 3 rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

In some embodiments, the glucagon analogs of the present invention are isolated. The term "isolated" as used herein means having been removed from its natural environment. In exemplary embodiments, the analogs are prepared via recombinant methods and the analogs are isolated from the host cell.

In some embodiments, the glucagon analogs of the present invention are purified. The term "purified" as used herein means that the molecule or compound is isolated in a form that is substantially free of contaminants (which in some aspects are typically associated with the molecule or compound in a natural or natural environment) and means that the purity has been increased as a result of separation from other components of the original composition. Purified peptides or compounds include, for example, peptides that are substantially free of nucleic acid molecules, lipids, and carbohydrates, or other starting materials or intermediates used or formed during chemical synthesis of the peptide. "purity" is recognized as a relative term and is not necessarily to be understood as absolute purity or absolute enrichment or absolute selection. In some aspects, the purity is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% (e.g., 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%).

Conjugates

The invention further provides conjugates comprising one or more of the glucagon analogs described herein conjugated to a heterologous moiety. The term "heterologous moiety" as used herein is synonymous with the term "conjugate moiety" and refers to any molecule (chemical or biochemical, naturally occurring or non-coding) that is different from the glucagon analogs described herein. Exemplary conjugate moieties that can be attached to any of the analogs described herein include, but are not limited to, heterologous peptides or polypeptides (including, e.g., plasma proteins), targeting agents, immunoglobulins or portions thereof (e.g., variable regions, CDRs, or Fc regions), diagnostic labels (such as radioisotopes, fluorophores, or enzyme labels), polymers (including water-soluble polymers), or other therapeutic or diagnostic agents. In some embodiments, conjugates are provided comprising an analog of the invention and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferrin, fibrinogen, and globulin. In some embodiments, the plasma protein portion of the conjugate is albumin or transferrin. In some embodiments, the conjugates comprise one or more glucagon analogs described herein and one or more of: peptides (which differ from the glucagon and/or GLP-1 receptor active glucagon analogs described herein), polypeptides, nucleic acid molecules, antibodies or fragments thereof, polymers, quantum dots, small molecules, toxins, diagnostic agents, carbohydrates, amino acids.

In some embodiments, the heterologous moiety is a peptide other than a glucagon analog described herein and the conjugate is a fusion peptide or a chimeric peptide. In some embodiments, the heterologous moiety is a peptide extension of 1-21 amino acids. In particular embodiments, the extension is linked to the C-terminus of the glucagon analog.

In some particular aspects, the extension is a single amino acid or a dipeptide. In particular embodiments, the extension comprises an amino acid selected from the group consisting of: charged amino acids (e.g., negatively charged amino acids (e.g., Glu), positively charged amino acids), amino acids comprising a hydrophilic moiety. In some aspects, the extension is Gly, Glu, Cys, Gly-Gly, Gly-Glu.

In some embodiments, the extension comprises the amino acid sequence of GPSSGAPPPS, GGPSSGAPPPS, KRNRNNIA or KRNR. In a particular aspect, the amino acid sequence is linked via the C-terminal amino acid of the glucagon analog. In some embodiments, the amino acid sequence of either GPSSGAPPPS, GGPSSGAPPPS, KRNRNNIA or KRNR is conjugated to the C-terminus of the glucagon analog via a peptide bond.

In some embodiments, the heterologous moiety is a polymer. In some embodiments, the polymer is selected from: a polyamide; a polycarbonate; polyalkylene (polyalkylene) and derivatives thereof including polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates; polymers of acrylates and methacrylates including poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate); polyvinyl polymers including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly (vinyl acetate), and polyvinyl pyrrolidone; polyglycolide; a polysiloxane; polyurethanes and copolymers thereof; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and sodium sulfate; polypropylene; polyethylene, including poly (ethylene glycol), poly (ethylene oxide), and poly (ethylene terephthalate); and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including synthetic biodegradable polymers (e.g., polymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) acid esters, polyurethanes, poly (butyric acid), poly (valeric acid), and poly (lactide-co-caprolactone)), and natural biodegradable polymers (e.g., alginates and other polysaccharides (including dextran and cellulose), collagen, chemical derivatives thereof (substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications commonly performed by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein (zein) and other prolamines (prolamines) and hydrophobic proteins)), and any copolymers or mixtures thereof. Typically, these materials degrade in vivo by enzymatic hydrolysis or exposure to water, by surface or bulk erosion (bulk oxidation).

In some aspects, the polymer is a bioadhesive polymer, such as a bioadhesive hydrogel (described by h.s.sawhney, c.p.pathak and j.a.hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein), polyhyaluronic acid, casein, gelatin, glutin (glutin), polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).

In some embodiments, the polymer is a water soluble polymer or a hydrophilic polymer. Hydrophilic polymers are further described herein under "hydrophilic moieties". Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl amylcellulose, methylcellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymer, polyhydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylic acid copolymer, polymethacrylic acid, polymethylmethacrylate, maleic anhydride/methylvinyl ether copolymer, polyvinyl alcohol, sodium and calcium polyacrylates, polyacrylic acid, and mixtures thereof, Acidic carboxyl polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymers, polymethylvinyl ether-co-maleic anhydride, carboxymethylamide, potassium methacrylate divinylbenzene copolymers, polyoxyethylene glycols, polyethylene oxide, and derivatives, salts and combinations thereof.

In particular embodiments, the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).

In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, guaiacum (callose), laminarin (laminarin), xylan, mannan, fucoidan (fucoidan), or galactomannan).

In some embodiments, the heterologous moiety is a lipid. In some embodiments, the lipid is a fatty acid, eicosanoid (eicosanoid), prostaglandin, leukotriene, thromboxane, N-acylethanolamine, glycerolipid (e.g., mono-, di-, tri-substituted glycerol), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid (prenol lipid), glycolipid, or polyketide, oil, wax, cholesterol, sterol, fat soluble vitamin, monoglyceride, diglyceride, triglyceride, phospholipid.

In some embodiments, the heterologous moiety is linked to the analog of the invention via non-covalent or covalent bonding. In exemplary aspects, the heterologous moiety is linked to the analog of the invention via a linker. Attachment may be achieved by covalent chemical bonds, physical forces such as electrostatic interactions, hydrogen bonding interactions, ionic interactions, van der waals interactions, or hydrophobic or hydrophilic interactions. A variety of non-covalent coupling systems may be used, including biotin-avidin, ligands/receptors, enzymes/substrates, nucleic acids/nucleic acid binding proteins, lipids/lipid binding proteins, cell adhesion chaperones; or any binding partners or fragments thereof that have affinity for each other.

In some embodiments, the glucagon analogs are attached to the conjugate moiety via a direct covalent bond by reacting the target amino acid residues of the analog with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of these target amino acids. Reactive groups on the analog or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimido, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride, or other reagents known in the art. Alternatively, the conjugate moiety may be indirectly linked to the analog via an intermediate carrier, such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids with other amino acids (e.g., serine) to impart desired solubility properties to the resulting loaded carrier.

Most commonly, the cysteinyl residue is reacted with an alpha-haloacetate (and corresponding amine), such as chloroacetic acid, chloroacetamide, to give a carboxymethyl or carboxamidomethyl derivative. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuriyl-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.

Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for histidyl side chains. Para-bromobenzoylmethyl bromide is also useful; the reaction is preferably carried out in 0.1M sodium cacodylate at pH 6.0.

Lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing the α -amino group-containing residue include imidoesters (such as methyl picolinoyl imidate), pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione, and transaminase-catalyzed reactions with glyoxylic esters.

Arginyl residues are modified by reaction with one or several conventional reagents, among which phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione and ninhydrin. Because the guanidine functional group has a high pKa, derivatization of arginine residues requires that the reaction be performed under basic conditions. In addition, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modifications of tyrosyl residues can be made, with particular attention being paid to the introduction of spectroscopic tags into tyrosyl residues by reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R-N ═ C ═ N-R '), where R and R' are different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-aza (azonia) -4, 4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Other modifications include: hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of a seryl or threonyl residue; methylation of the alpha-amino groups of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 (1983)); deamidation of asparagine or glutamine; acetylation of the N-terminal amine; and/or amidation or esterification of the C-terminal carboxylic acid group.

Another type of covalent modification involves chemical or enzymatic coupling of the glycoside to the analog. The sugar may be linked to: (a) arginine and histidine; (b) a free carboxyl group; (c) free sulfhydryl groups, such as cysteine; (d) free hydroxyl groups, such as serine, threonine, or hydroxyproline; (e) aromatic residues, such as tyrosine or tryptophan; or (f) the amide group of glutamine. These methods are described in WO87/05330, published on 9/11 1987; and Aplin and Wriston, CRC crit. rev. biochem., pp. 259-306 (1981).

In some embodiments, the glucagon analog is conjugated to the heterologous moiety via a covalent bond between an amino acid side chain of the glucagon analog and the heterologous moiety. In some embodiments, the glucagon analog is conjugated to the heterologous moiety via a side chain of an internal amino acid, a position within the C-terminal extension, or the C-terminal amino acid, or a combination of these positions.

In some embodiments, the conjugate comprises a linker joining the glucagon analog to the heterologous moiety. In some aspects, the linker comprises a chain of 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from C, O, N and S. The chain atoms and linker can be selected according to the desired solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that undergoes cleavage due to an enzyme or other catalyst or hydrolysis conditions present in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the potential for steric hindrance. If the linker is a covalent bond or a peptide bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such peptidyl linkers may be of any length. Exemplary linkers are about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length. Alternatively, such fusion proteins can be prepared by recombinant genetic engineering methods known to those of ordinary skill in the art.

Conjugate: fc fusions

As described above, in some embodiments, the analog is conjugated (e.g., fused) to an immunoglobulin or a portion thereof (e.g., a variable region, a CDR, or an Fc region). The known classes of immunoglobulins (Ig) include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain, which is responsible for binding to Fc receptors that perform activities such as recycling (which results in increased half-life), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

For example, according to some definitions, the human IgG heavy chain Fc region extends from Cys226 to the C-terminus of the heavy chain. The "hinge region" of human IgG1 typically extends from Glu216 to Pro230 (the hinge region of other IgG isotypes can be aligned with the IgG1 sequence by aligning the cysteines involved in cysteine bonding). The Fc region of IgG comprises two constant domains, CH2 and CH 3. The CH2 domain of the human IgG Fc region typically extends from amino acid 231 to amino acid 341. The CH3 domain of the human IgG Fc region typically extends from amino acid 342 to amino acid 447. Reference to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions is based on Kabat et al, Sequences of Proteins of Immunological Interest, u.s.department of Public Health, Bethesda, Md. In a related embodiment, the Fc region may comprise one or more native or modified constant regions (other than CH 1) from an immunoglobulin heavy chain, for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE.

Suitable conjugate moieties include those portions of an immunoglobulin sequence that comprise an FcRn binding site. FcRn (neonatal receptor) is responsible for recycling immunoglobulins and circulating them back into the blood. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al, 1994, Nature 372: 379). The primary interface of Fc to FcRn is near the junction of CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. The primary contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain structure.

Some conjugate moieties may or may not include an fcyr binding site. Fc γ R is responsible for ADCC and CDC. Examples of positions within the Fc region which are in direct contact with Fc γ R are the amino acids 234-. The lower hinge region of IgE has also been implicated in FcRI binding (Henry et al, Biochemistry 36, 15568-15578, 1997). Residues involved in IgA receptor binding are described in Lewis et al (J Immunol.175: 6694-701, 2005). Amino acid residues involved in IgE receptor binding are described in Sayers et al (J Biol chem.279 (34): 35320-5, 2004).

Amino acid modifications may be made to the Fc region of an immunoglobulin. Such variable Fc regions comprise at least one amino acid modification in the CH3 domain (residues 342-447) and/or at least one amino acid modification in the CH2 domain (residues 231-341) of the Fc region. Mutations believed to confer increased affinity to FcRn include T256A, T307A, E380A and N434A (Shields et al, 2001, j.biol.chem.276: 6591). Other mutations may reduce the binding of the Fc region to Fc γ RI, Fc γ RIIA, Fc γ RIIB, and/or Fc γ RIIIA without significantly reducing the affinity for FcRn. For example, substitution of Asn at position 297 of the Fc region with Ala or another amino acid removes a highly conserved N-glycosylation site and can lead to reduced immunogenicity with concomitant increase in half-life of the Fc region, and reduced binding to Fc γ R (Routledge et al, 1995, Transplantation 60: 847; Friend et al, 1999, Transplantation 68: 1632; Shields et al, 1995, J.biol.chem.276: 6591). Amino acid modifications at position 233-236 of IgG1 have been made which reduce binding to Fc γ R (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 U.S. patents 7,355,008 and 7,381,408, each of which is incorporated by reference herein in its entirety.

Conjugate: hydrophilic moieties

The glucagon analogs described herein can be further modified to improve their solubility and stability in aqueous solution at physiological pH while maintaining high biological activity relative to native glucagon. The hydrophilic moiety, such as a PEG group, can be attached to the analog under any suitable conditions for reacting the protein with the activated polymer molecule. Any means known in the art can be used, including via an acylation, reductive alkylation, Michael addition, mercaptoalkylation, or other chemoselective conjugation/attachment method to a reactive group (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimido, or hydrazino) on a target compound via a reactive group (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimido, or hydrazino) on a PEG moiety. Activating groups that can be used to attach the water-soluble polymer to one or more proteins include, but are not limited to, sulfones, maleimides, thiols (thiols), triflates, aziridines, oxiranes, 5-pyridyl, and α -haloacyl (e.g., α -iodoacetic acid, α -bromoacetic acid, α -chloroacetic acid). If attached to the analog by reductive alkylation, the polymer selected should have a single reactive aldehyde in order to control the degree of polymerization. See, e.g., Kinstler et al, adv.drug. Delivery rev.54: 477-; roberts et al, adv. drug Delivery rev.54: 459-476 (2002); and Zalipsky et al, adv. drug Delivery rev.16: 157-182(1995).

In particular aspects, the amino acid residue of the thiol-bearing analog is modified with a hydrophilic moiety such as PEG.

In some embodiments, the sulfhydryl group is modified with a haloacetyl activated PEG in a nucleophilic substitution reaction to result in the production of a pegylated analog comprising a thioether linkage.

Suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyalkylene oxides, polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, mono- (C1-C10) alkoxy-polyethylene glycol or mono- (C1-C10) aryloxy-polyethylene glycol, carboxymethylcellulose, polyacetal, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, poly (beta-amino acid) (homo-or random copolymers), poly (n-vinylpyrrolidone) polyethylene glycol, polyethylene glycol, Propylene glycol homopolymer (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, colonic acid (colonic acid) or other polysaccharide polymers, Ficoll or dextran and mixtures thereof. Dextran is a polysaccharide polymer of glucose subunits linked primarily by α 1-6 linkages. Dextrans are available in a variety of molecular weight ranges, for example, from about 1kD to about 100kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branched polymers are contemplated. The resulting conjugate formulation can be substantially monodisperse or polydisperse, and each analog can have about 0.5, 0.7, 1, 1.2, 1.5, or 2 polymer moieties.

In some or any embodiment, the glucagon analog is conjugated to the hydrophilic moiety via a covalent linkage between an amino acid side chain of the glucagon analog and the hydrophilic moiety. In some or any embodiment, the glucagon analog is conjugated to the hydrophilic moiety via an internal amino acid side chain, a position within the C-terminal extension, or the C-terminal amino acid, or a combination of these positions.

Conjugate: rPEG

In some or any embodiment, the conjugates of the invention comprise a glucagon analog having GIP receptor agonist activity fused to a helper analog capable of forming an extended configuration similar to chemical PEG (e.g., a recombinant PEG (rpeg) molecule), such as those described in international patent application publication No. WO2009/023270 and U.S. patent application publication No. US 20080286808. In some aspects, the rPEG molecule is a polypeptide comprising one or more of glycine, serine, glutamic acid, aspartic acid, alanine, or proline. In some aspects, the rPEG is a homopolymer, e.g., polyglycine, polyserine, polyglutamic acid, polyaspartic acid, polyalanine, or polyproline. In other embodiments, rPEG comprises two types of repeating amino acids, e.g., poly (Gly-Ser), poly (Gly-Glu), poly (Gly-Ala), poly (Gly-Asp), poly (Gly-Pro), poly (Ser-Glu), and the like. In some aspects, rPEG comprises three different types of amino acids, e.g., poly (Gly-Ser-Glu). In particular aspects, the rPEG increases the half-life of the glucagon and/or GLP-1 agonist analog. In some aspects, the rPEG comprises a net positive or negative charge. In some aspects, the rPEG lacks secondary structure. In some embodiments, the rPEG is greater than or equal to 10 amino acids in length and in some embodiments from about 40 to about 50 amino acids in length. In some aspects, the helper peptide is fused to the N-terminus or C-terminus of the analog of the invention via a peptide bond or protease cleavage site, or inserted into a loop of the analog of the invention. In some aspects, the rPEG comprises an affinity tag or is linked to a PEG of greater than 5 kDa. In some embodiments, the rPEG confers increased hydrodynamic radius, serum half-life, protease resistance or solubility to the analogs of the invention and in some aspects confers reduced immunogenicity to the analogs.

Conjugate: multimer

The invention further provides multimers or dimers of the analogs disclosed herein, including homo-or hetero-multimers or homo-or hetero-dimers. Two or more analogs can be linked together using standard linking agents and procedures known to those skilled in the art. For example, a dimer may be formed between two peptides through the use of a bifunctional thiol crosslinker and a bifunctional amine crosslinker, particularly for analogs that have been substituted with cysteine, lysine, ornithine, homocysteine, or acetylphenylalanine residues. The dimer may be a homodimer or alternatively may be a heterodimer. In exemplary embodiments, the linker connecting the two (or more) analogs is PEG, e.g., 5kDa PEG, 20kDa PEG. In some embodiments, the linker is a disulfide bond. For example, each monomer of the dimer may comprise a Cys residue (e.g., a Cys in a terminal or internal position) and the sulfur atom of each Cys residue is involved in the formation of a disulfide bond. In exemplary aspects, the monomers of the dimer are linked via a thioether linkage. In exemplary aspects, the epsilon amine of a Lys residue of one monomer is bonded to a Cys residue, which in turn is linked to the epsilon amine of a Lys residue of another monomer via a chemical moiety. Further described herein are methods of making such thioether-bonded dimers. In some aspects, the monomers are linked via a terminal amino acid (e.g., N-terminus or C-terminus), via an internal amino acid, or via a terminal amino acid of at least one monomer and an internal amino acid of at least another monomer. In particular aspects, the monomers are not linked via the N-terminal amino acid. In some aspects, monomers of a multimer are linked together in a "tail-to-tail" orientation in which the C-terminal amino acids of each monomer are linked together.

Prodrugs

The invention further provides prodrugs of the peptides and analogs described herein. The term "prodrug" as used herein is defined as any compound that undergoes chemical modification before exhibiting its full pharmacological effect.

In an exemplary embodiment, the prodrug is an amide-based peptide prodrug similar to those described in international patent application publication No. WO/2010/071807, published at 24/6/2010. Such prodrugs are intended to delay the onset of action and prolong the half-life of the drug. Delaying the onset of action is advantageous because it allows for systemic distribution of the prodrug prior to its activation. Thus, administration of the prodrug can eliminate complications arising from peak activity after administration and increase the therapeutic index of the parent drug.

In exemplary aspects, the prodrug comprises the structure: A-B-Q; wherein Q is a peptide or analog described herein; a is amino acid or hydroxy acid; b is an N-alkylated amino acid linked to Q via an amide bond between A-B and the amine of Q; wherein A, B or the amino acid of Q linked to A-B is a non-coding amino acid, and further wherein the half-life (t1/2) of chemical cleavage of A-B with Q in PBS under physiological conditions is at least about 1 hour to about 1 week. The term "hydroxy acid" as used herein refers to an amino acid that has been modified so as to replace the alpha carbon amino group with a hydroxyl group.

Pharmaceutical composition, use and kit

Salt (salt)

In some embodiments, the glucagon analog is in the form of a salt, e.g., a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein refers to salts of a compound that retain the biological activity of the parent compound and are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the analog, or separately by reacting the free base functionality with a suitable acid. Many of the compounds disclosed herein are capable of forming acidic and/or basic salts due to the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Salts derived from inorganic acids include salts of hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Salts derived from organic acids include salts of acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like. Examples of acids which can be used to form pharmaceutically acceptable acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid.

Base addition salts can also be prepared in situ during the final isolation and purification of the salicylic acid source, or by reacting the carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like; and nontoxic quaternary amines and amine cations including, inter alia, ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium. Other representative organic amines useful for forming base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines.

In addition, basic nitrogen-containing groups can be quaternized with the analogs of the invention, such as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides such as benzyl and phenethyl bromides and the like. Thus obtaining a water-or oil-soluble or dispersible product.

Preparation

According to some embodiments, there is provided a pharmaceutical composition, wherein the composition comprises a glucagon analog of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (such as oil/water or water/oil emulsions), and various types of wetting agents. The term also encompasses any agent approved by the U.S. Federal regulatory agency of the US Federal diet golf or used in the U.S. pharmacopoeia (US pharmacopoeia) in animals, including humans.

The pharmaceutical composition may comprise any pharmaceutically acceptable ingredient including, for example, acidulants, additives, adsorbents, aerosol propellants, air displacing agents (air displacement agents), alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, bactericides, binders, buffering agents, chelating agents, coating agents (coating agents), colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, odor enhancers, flavorants, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrating agents, Solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicity agents, viscosity enhancers, water absorbents, water-miscible co-solvents, water softeners, or humectants.

In some embodiments, the pharmaceutical composition comprises any one or combination of the following components: acacia, potassium acetamino sulfonate, tributyl acetylcitrate, triethyl acetylcitrate, agar, albumin, ethanol, anhydrous ethanol, denatured alcohol, diluted alcohol, elaeostearic acid, alginic acid, aliphatic polyester, alumina, aluminum hydroxide, aluminum stearate, amylopectin, alpha-amylose, ascorbic acid, ascorbyl palmitate, acanthalSpan, bacteriostatic water for injection, bentonite paste, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol, butyl hydroxyanisole, butylated hydroxytoluene, butyl paraben, sodium butylparaben, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, anhydrous calcium hydrogen phosphate, dehydrated calcium hydrogen phosphate, tricalcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate hemihydrate, canola oil (canola oil), carbomer (carbomer), carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, beta-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, sodium silicate, sodium alginate, sodium, Sodium carboxymethylcellulose, cetostearyl alcohol mixture (cetostearyl alcohol), cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons (CFC), chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, colorants, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrin, dextrates, dextrin, dextrose, anhydrous dextrose, diazoalkylimidazolium urea (diazolidinyl urea), dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl-beta-cyclodextrin, sodium lauryl sulfate, diethyl phthalate, diethyl sulfate, ethyl difluoride (HFC), dimethyl-beta-cyclodextrin, sodium lauryl, Cyclodextrin type compounds such as

Dimethyl ether, dimethyl phthalate, dipotassium edetate, disodium hydrogen phosphate, docusate calcium, potassium docusate, sodium docusate, dodecyl gallate, dodecyl trimethyl ammonium bromide, disodium edetate, ethylene diamine tetraacetic acid, meglumine, ethanol, ethyl cellulose, ethyl gallate, ethyl laurate, ethyl maltolEthyl oleate, ethyl p-hydroxymethylbenzoate, potassium ethyl p-hydroxymethylbenzoate, sodium ethyl p-hydroxymethylbenzoate, ethyl vanillin, fructose liquids, milled fructose, pyrogen-free fructose, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, glycerol ester mixtures of saturated vegetable fatty acids, glycerol behenate, glycerol monooleate, glycerol monostearate, self-emulsifying glycerol monostearate, glycerol palmitostearate, glycine, ethylene glycol, glycofurol (glycofurol), guar gum, Heptafluoropropane (HFC), cetyltrimethylammonium bromide, high fructose syrups, human serum albumin, Hydrocarbons (HC), dilute hydrochloric acid, hydrogenated vegetable oils of type II, hydroxyethyl cellulose, 2-hydroxyethyl-beta-cyclodextrin, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-beta-cyclodextrin, hydroxypropyl methylcellulose phthalate, imidurea, indigo, ion exchangers, iron oxide, isopropanol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin alcohols, anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesium carbonate, normal magnesium carbonate, anhydrous magnesium carbonate, basic magnesium carbonate, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, malt, maltitol solutions, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methyl methacrylate, methyl oleate, methyl p-methylolbenzoate, methyl p-methylol benzoate, methyl alcohol, sodium lauryl sulfate, magnesium hydroxide, magnesium silicate, magnesium stearate, potassium methyl p-hydroxymethylbenzoate, sodium methyl p-hydroxymethylbenzoate, microcrystalline cellulose and sodium carboxymethylcellulose, mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum and lanolin alcohols, pharmaceutical glazes, phenol, liquefied phenol, phenoxyethanol, phenoxypropanol, phenylethanol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin (polacrilin), potassium polacrilin, poloxamer (poloxamer), polydextrose, polyethylene glycol, polyepoxyEthane, polyacrylate, polyethylene-polyoxypropylene-block polymer, polymethacrylate, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene stearate, polyvinyl alcohol, polyvinylpyrrolidone, potassium alginate, potassium benzoate, potassium hydrogen carbonate, potassium hydrogen sulfate, potassium chloride, potassium citrate, anhydrous potassium citrate, potassium hydrogen phosphate, potassium metabisulfite, potassium dihydrogen phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol alginate, propyl gallate, propyl p-hydroxymethylbenzoate, potassium propylp-hydroxymethylbenzoate, sodium propylp-hydroxymethylbenzoate, protamine sulfate, rapeseed oil, Ringer's solution, saccharin ammonium saccharin, calcium saccharin, sodium saccharin, safflower oil, saponite, serum protein, sodium saccharin, sodium cyclamate, sodium stearate, sesame oil, silica gel, colloidal silicon dioxide, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfate, sodium chloride, sodium citrate anhydrous, sodium citrate dehydrate, sodium chloride, sodium cyclamate, sodium edetate, sodium lauryl sulfate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium propionate anhydrous, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty esters), sorbitol solution 70%, soybean oil, spermaceti, starch, corn starch, potato starch, pregelatinized starch, sterilized corn starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugar, compressible sugar, sugar for confectionery, sugar spheres, invert sugar, sucrose-invert sugar polymer (Sugartab), Sunset yellow FCF, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), cocoa butter (theobroma oil), thimerosal, titanium dioxide, alpha tocopherol, tocopherol acetate, alpha tocopherol succinate, beta tocopherol, delta tocopherol, gamma tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl-beta-cyclodextrin, trimethyltetradecylammonium bromide, tris buffer, trisodium edetate, vanillin, hydrogenated vegetable oil type I, water, soft water, hard water, carbonless water, pyrogen-free water, and mixtures thereofRaw water, water for injection, sterile water for inhalation, sterile water for injection, sterile water for rinsing, wax, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, suppository wax, white wax, yellow wax, white petrolatum, lanolin, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate, or Handbook of Pharmaceutical Excipients, 3 rd edition, a.h. kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980), which is incorporated by reference in its entirety, discloses various components used in formulating pharmaceutically acceptable compositions and known techniques for their preparation. Except insofar as any conventional agent is incompatible with the pharmaceutical composition, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

In some embodiments, one or more of the foregoing components may be present in the pharmaceutical composition at any concentration, such as, for example, at least a, wherein a is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, one or more of the foregoing components may be present in the pharmaceutical composition at any concentration, such as, for example, up to B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In other embodiments, one or more of the foregoing components may be present in the pharmaceutical composition in any concentration range, such as, for example, from about a to about B. In some embodiments, a is 0.0001% and B is 90%.

In some embodiments, the pharmaceutically acceptable ingredient is selected from: sugars (e.g., glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, melibiose (mellitiose), melezitose (melezitose), raffinose, mannotriose, stachyose (stachyyose), maltose, lactulose (1actulose), maltulose (maltulose) or isomaltulose, or a combination of these sugars), sugar alcohols (e.g., ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, dulcitol, iditol, isomaltose, maltitol, lactitol or glucitol, or a combination of these sugar alcohols), salts (e.g., sodium chloride), emulsifiers or surfactants (e.g., polysorbates, such as polyoxyethylene 20 sorbitan monooleate, or other block copolymers of ethylene oxide and propylene oxide), lyoprotectants, and mixtures thereof. For example, an excipient such as a sugar or sugar alcohol is present, for example, at a concentration of about 20mg/mL to about 40mg/mL, or 25mg/mL to 45mg/mL, such as 35 mg/mL.

The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 11, e.g., between 4 and 7, or between 4.5 and 5.5, depending on the formulation and route of administration. In exemplary embodiments, the pharmaceutical composition may comprise a buffer to achieve a physiologically compatible pH. The buffer may include any compound capable of buffering at a desired pH, such as phosphate buffer (e.g., PBS), triethanolamine, Tris, dihydroxyethylglycine (bicine), TAPS, trihydroxyethylglycine (tricine), HEPES, TES, MOPS, PIPES, dimethylarsinate (cacodylate), MES, acetate, citrate, succinate, histidine, or other pharmaceutically acceptable buffers. In exemplary embodiments, the buffer has an intensity of at least 0.5mM, at least 1mM, at least 5mM, at least 10mM, at least 20mM, at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 120mM, at least 150mM, or at least 200 mM. In some embodiments, the buffer has an intensity of no greater than 300mM (e.g., no greater than 200mM, no greater than 100mM, no greater than 90mM, no greater than 80mM, no greater than 70mM, no greater than 60mM, no greater than 50mM, no greater than 40mM, no greater than 30mM, no greater than 20mM, no greater than 10mM, no greater than 5mM, no greater than 1 mM). For example, the buffer concentration may be about 2mM to about 100mM, or about 10mM to about 50 mM.

Route of administration

The following discussion of routes of administration is provided merely to illustrate exemplary embodiments and should not be construed as limiting the scope in any way.

Formulations suitable for oral administration may consist of: (a) a liquid solution, such as an effective amount of an analog of the present invention dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges and troches, each containing a predetermined amount of the active ingredient, as a solid or as granules; (c) powder; (d) a suspension in a suitable liquid; and (e) a suitable emulsion. Liquid formulations may include diluents such as water and alcohols (e.g., ethanol, benzyl alcohol, and polyethylene glycol), with or without the addition of pharmaceutically acceptable surfactants. Capsule forms can be of the common hard or soft shell gelatin type containing, for example, surfactants, lubricants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch. The tablet form may include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents and other pharmacologically compatible excipients. Lozenge forms may comprise an analog of the invention in a flavoring agent, typically sucrose and acacia or tragacanth, as well as pastilles comprising an analog of the invention in an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing such excipients as are known in the art in addition to an analog of the invention.

The analogs of the invention, alone or in combination with other suitable components, can be delivered via pulmonary administration and can be formulated as aerosol formulations for administration via inhalation. These aerosol formulations can be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as medicaments for non-pressurized formulations, for example in a nebulizer or atomizer. Such spray formulations may also be used to spray mucous membranes. In some embodiments, the analogs are formulated as a powder blend or as microparticles or nanoparticles. Suitable pulmonary formulations are known in the art. See, e.g., Qian et al, Int J Pharm 366: 218-220 (2009); adjei and Garren, Pharmaceutical Research, 7 (6): 565 — 569 (1990); kawashima et al, J Controlled Release 62 (1-2): 279-287 (1999); liu et al, Pharm Res 10 (2): 228-; international patent application publication nos. WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means not via the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal or intravenous. The analogs of the present invention may be administered in a pharmaceutical carrier with a physiologically acceptable diluent, such as sterile liquids or liquid mixtures, including water, physiological saline, aqueous dextrose and related sugar solutions, alcohols (such as ethanol or cetyl alcohol), glycols (such as propylene glycol or polyethylene glycol), dimethyl sulfoxide, glycerol, ketals (such as 2, 2-dimethyl-1, 3-dioxolane-4-methanol), ethers, poly (ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants (such as soaps or detergents), suspending agents (such as pectins), carbomers, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils that may be used in the parenteral formulation include petroleum, animal, vegetable or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, paraffin oil, and mineral oil. Fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Soaps suitable for use in parenteral formulations include fatty alkali metal, ammonium and tetraalkanolamine salts, and suitable detergents include: (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- β -aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

Parenteral formulations will generally contain from about 0.5% to about 25% by weight of the analog of the invention in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters (such as sorbitan monooleate) and the high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. Parenteral formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient for injection, such as water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Injectable formulations are in accordance with the present invention. The requirement for an effective drug carrier for Injectable compositions is well known to those of ordinary skill in the art (see, e.g., pharmaceuticals and pharmaceutical Practice, J.B. Lippincott Company, Philadelphia, PA, Bank and Chalmers, eds. 238 and 250 (1982); and ASHP Handbook on Injectable Drugs, Toissel, 4 th edition, 622 and 630 (1986)).

In addition, the analogs of the present invention can be formulated as suppositories for rectal administration by mixing with various bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

It will be appreciated by those skilled in the art that in addition to the pharmaceutical compositions described above, the analogs of the invention may also be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

Dosage form

It is believed that the analogs of the invention are useful in methods of treating diseases or medical conditions in which GIP receptor agonism, GIP/GLP-1 receptor co-agonism, GIP/glucagon receptor co-agonism, or GIP/GLP-1/glucagon receptor triple agonism plays a role. For the purposes of the present invention, the amount or dose of the analog of the present invention administered should be sufficient to achieve, for example, a therapeutic or prophylactic response in a subject or animal within a reasonable time frame. For example, the analogs of the invention are dosed at a dose from when administered that is sufficient to stimulate secretion of cAMP from a cell as described herein or to reduce blood glucose levels, fat levels, food intake levels, or body weight in a mammal over a period of about 1 minute to 4 minutes, 1 hour to 4 hours, or 1 week to 4 weeks or more (e.g., 5 weeks to 20 weeks or more). In exemplary embodiments, the time period may be even longer. The dosage should be determined by the efficacy of the particular analog of the invention and the condition of the animal (e.g., human), as well as the weight of the animal (e.g., human) to be treated.

Many assays are known in the art for determining the administered dose. For purposes herein, an initial dose to be administered to a mammal can be determined using an assay that includes comparing the degree of reduction in blood glucose levels after a given dose of an analog of the invention is administered to a mammal (a group of mammals to which the mammal belongs are each given a different dose of the analog). The extent to which blood glucose levels are reduced after administration of a particular dose can be determined by methods known in the art.

The dosage of the analogs of the invention will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular analog of the invention. In general, the attending physician will determine the dosage of the analogs of the invention for use in treating each individual patient, taking into account a number of factors such as age, weight, general health, diet, sex, the analog of the invention to be administered, the route of administration, and the severity of the condition to be treated. By way of example and not intended to limit the invention, the dosage of the analogs of the invention may be from about 0.0001 to about 1g/kg body weight/day, from about 0.0001 to about 0.001g/kg body weight/day, or from about 0.01mg to about 1g/kg body weight/day of the subject being treated. In exemplary embodiments, the dose may be a total weekly dose of about 1mg to about 40mg, or about 4mg to about 30mg, or about 4 to about 20mg, or about 10 to about 20mg, or about 12mg to about 30 mg.

In some embodiments, the pharmaceutical composition comprises any of the analogs disclosed herein at a purity level suitable for administration to a patient. In some embodiments, the analog has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some aspects, the pharmaceutical composition comprises an analog of the invention at a concentration of at least A, 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/m1, 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 more than 25 mg/ml. In some embodiments, the pharmaceutical composition comprises the analog at a concentration of up to B, wherein B is 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 0.1 mg/ml. In some embodiments, the composition can contain the analog at a concentration ranging from A to Bmg/ml (e.g., from about 0.001mg/ml to about 30.0 mg/ml).

Targeting forms

One of ordinary skill in the art will readily appreciate that the analogs of the invention can be modified in any of a variety of ways such that the therapeutic or prophylactic efficacy of the analogs of the invention is increased via the modification. For example, an analog of the invention can be conjugated to a targeting moiety, either directly or indirectly via a linker. The manipulation of conjugating compounds, such as the glucagon analogs described herein, to targeting moieties is known in the art. See, for example, Wadhwa et al, J Drug Targeting, 3, 111-. The term "targeting moiety" as used herein refers to any molecule or agent that specifically recognizes and binds to a cell surface receptor such that the targeting moiety directs the delivery of the analogs of the invention to a population of cells that express the receptor (glucagon receptor, GLP-1 receptor) on the surface. Targeting moieties include, but are not limited to, antibodies or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligand that bind to cell surface receptors (e.g., Epithelial Growth Factor Receptor (EGFR), T Cell Receptor (TCR), B Cell Receptor (BCR), CD28, platelet derived growth factor receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.). A "linker" as used herein is a bond, molecule or group of molecules that binds two separate entities to each other. The linker may provide optimal spacing of the two entities or may further provide an unstable connection that allows the two entities to be separated from each other. Labile linkages include a photocleavable group, an acid labile moiety, a base labile moiety, and an enzyme cleavable group. In some embodiments, the term "linker" refers to any agent or molecule that bridges the analogs of the invention to the targeting moiety. One of ordinary skill in the art recognizes that the site on the analogs of the invention (which is not necessary for the function of the analogs of the invention) is an ideal site for attachment of a linker and/or targeting moiety, provided that the linker and/or targeting moiety, once attached to the analogs of the invention, does not interfere with the function of the analogs of the invention, i.e., the ability to stimulate cAMP secretion from cells to treat diabetes or obesity.

Controlled release formulations

Alternatively, the glucagon analogs described herein can be modified into depot forms such that the manner in which the analog of the present invention is released into the body to which it is administered is controlled in time and location within the body (see, e.g., U.S. Pat. No.4,450,150). Depot forms of the analogs of the invention can be, for example, implantable compositions comprising an analog of the invention and a porous or non-porous material, such as a polymer, by encapsulating or diffusing the analog of the invention into the material and/or degrading the non-porous material. The depot formulation is then implanted at a desired location in the body and the analog of the invention is released from the implant at a predetermined rate.

In exemplary aspects, the pharmaceutical compositions are modified to have any type of in vivo release profile. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or biphasic release formulation. Methods of formulating peptides for controlled release are known in the art. See, e.g., Qian et al, J Pharm 374: 46-52 (2009) and international patent application publication No. WO 2008/130158; WO 2004/033036; WO 2000/032218; and WO 1999/040942.

The present compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide an extended storage and/or delivery effect. The disclosed pharmaceutical formulations can be administered according to any regimen, including, for example, daily (1 time daily, 2 times daily, 3 times daily, 4 times daily, 5 times daily, 6 times daily), three times weekly, twice weekly, two days, three days, four days, five days, six days, weekly, two weeks, three weeks, monthly, or two months.

Combination of

The glucagon analogs described herein can be administered alone or in combination with other therapeutic agents that are intended to treat or prevent any of the diseases or medical conditions described herein. For example, a glucagon analog described herein may be co-administered (simultaneously or sequentially) with an anti-diabetic or anti-obesity agent. Antidiabetic agents known in the art or being studied include insulin, leptin, peptide yy (pyy), Pancreatic Peptide (PP), fibroblast growth factor 21(FGF21), Y2Y4 receptor agonists, sulfonylureas such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, micron, Glynase), glimepiride (amyl), or gliclazide (Diamicron); meglitinides such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos) or troglitazone (Rezulin), or other PPAR γ inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide; dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin (vildagliptin) or sitagliptin (sitagliptin); SGLT (sodium dependent glucose transporter 1) inhibitors; glucokinase activator (GKA); glucagon Receptor Antagonists (GRA); or an FBPase (fructose 1, 6-bisphosphatase) inhibitor.

Anti-obesity agents known or being investigated in the art include appetite suppressants including phenylethylamine type stimulants, phentermine (optionally with fenfluramine or dexfenfluramine), diethylamidopropyl

Phenyldimethylmorpholine

) Benzphetamine (benzphetamine)

Sibutramine (sibutramine)

Rimonabant (rimonabant)

Other cannabinoid receptor antagonists; oxyntomodulin; fluoxetine hydrochloride (profac); qnexa (topiramate and phentermine), Excalia (amfepramone and zonisamide) or Contrave (amfepramone and naltrexone)); or lipase inhibitors, like cenicrobial (XENICAL) (Orlistat) or Cetilistat (Cetilistat) (also known as ATL-962) or GT 389-255.

In some embodiments, the peptides described herein are co-administered with an agent for treating non-alcoholic fatty liver disease or NASH. Agents used to treat non-alcoholic fatty liver disease include ursodeoxycholic acid (also known as Actigall, URSO, and Ursodiol), metformin (Glucophage), rosiglitazone (Avandia), clofibrate, gemfibrozil, polymyxin B, and betaine.

In some embodiments, the peptides described herein are co-administered with an agent for treating a neurodegenerative Disease, such as Parkinson's Disease. Furthermore, anti-parkinson's disease agents are known in the art and include, but are not limited to, levodopa (levodopa), carbidopa (carbidopa), anticholinergic agents (anticholinergicergics), bromocriptine (bromocriptine), pramipexole (pramipexole) and ropinirole (ropinairole), amantadine (amantadine), and rasagiline (rasagiline).

In view of the foregoing, the present invention further provides pharmaceutical compositions and kits additionally comprising one of these other therapeutic agents. The additional therapeutic agent may be administered simultaneously or sequentially with an analog of the invention. In some aspects, the analog is administered before the additional therapeutic agent, while in other aspects, the analog is administered after the additional therapeutic agent.

Use of

Based on the information provided herein for the first time, it is contemplated that the compositions of the invention (e.g., related pharmaceutical compositions) can be used to treat a disease or medical condition in which, for example, lack of activity against the GIP receptor, the GLP-1 receptor, or against both receptors is a factor in the onset and/or progression of the disease or medical condition. Accordingly, the present disclosure provides methods of treating or preventing a disease or medical condition in a patient, wherein the disease or medical condition is one in which lack of GIP receptor activation and/or GLP-1 receptor activation is associated with the onset and/or progression of the disease or medical condition. The method comprises providing to the patient a composition or conjugate according to any of those described herein in an amount effective to treat or prevent the disease or medical condition.

In some embodiments, the disease or medical condition is metabolic syndrome. Metabolic syndrome (also known as metabolic syndrome X, insulin resistance syndrome or revenn's syndrome) is a condition that affects more than 5 million americans. Metabolic syndrome is generally characterized by the clustered occurrence of at least three or more of the following risk factors: (1) abdominal obesity (excess adipose tissue in and around the abdomen); (2) atherogenic dyslipidemia (dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol, which enhances the accumulation of plaque in the arterial wall); (3) hypertension; (4) insulin resistance or glucose intolerance; (5) a pre-embolic state (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood); and (6) proinflammatory state (e.g., elevated C-reactive protein in blood). Other risk factors may include aging, hormonal imbalances, and genetic predisposition.

Metabolic syndrome is associated with an increased risk of coronary heart disease and other conditions associated with vascular plaque accumulation, such as stroke and peripheral vascular disease (known as atherosclerotic cardiovascular disease (ASCVD)). Patients with metabolic syndrome may progress from an insulin resistant state at an early stage to fully developed type II diabetes, with a further increased risk of ASCVD. Without wishing to be bound by any particular theory, the relationship between insulin resistance, metabolic syndrome and vascular disease may involve one or more concurrent pathogenic mechanisms, including impaired insulin-stimulated vasodilation, reduced availability of NO associated with insulin resistance due to increased oxidative stress, and abnormalities in adipocyte-derived hormones, such as adiponectin (Lteif and Mather, can.j. carol.20 (supplement B): 66B-76B (2004)).

According to the american National Cholesterol Education Program Adult Treatment Panel (ATP III) 2001, any three of the following characteristics in the same individual meet the criteria for metabolic syndrome: (a) abdominal obesity (waist circumference of greater than 102em for men and greater than 88cm for women); (b) serum triglycerides (150mg/dl or above); (c) HDL cholesterol (40 mg/dl or less for men and 50mg/dl or less for women); (d) blood pressure (130/85 or above); and (e) fasting plasma glucose (110 mg/dl or above). According to the World Health Organization (WHO), individuals with high insulin levels (either high fasting blood glucose alone or high postprandial glucose) with at least two of the following criteria meet the criteria for metabolic syndrome: (a) abdominal obesity (waist to hip ratio greater than 0.9, body mass index of at least 30kg/m2, or waist measurement greater than 37 inches); (b) a group of cholesterol subjects exhibiting triglyceride levels of at least 150mg/dl or HDL cholesterol below 35 mg/dl; (c) blood pressure is 140/90 or above, or is treating hypertension. (Mathur, Ruchi, "Metabolic Syndrome,", Shiel, Jr., William C., ed. Medicine et. com., 2009, 11 d).

For purposes herein, an individual is considered to have metabolic syndrome if the individual meets either or both of the criteria set forth by the U.S. national cholesterol education program adult treatment panel or WHO in 2001.

Without being bound by any particular theory, the compositions and conjugates described herein may be used to treat metabolic syndrome. Accordingly, the present invention provides a method of preventing or treating metabolic syndrome or reducing one, two, three or more risk factors for metabolic syndrome in a subject, comprising providing to the subject an amount of a composition described herein effective to prevent or treat metabolic syndrome or a risk factor thereof.

In some embodiments, the method treats a hyperglycemic medical condition. In exemplary aspects, the hyperglycemic medical condition is diabetes, type I diabetes, type II diabetes, or gestational diabetes (insulin-dependent or non-insulin-dependent). In some aspects, the method treats a hyperglycemic medical condition by reducing one or more complications of diabetes, including nephropathy, retinopathy and vascular disease.

In some aspects, the disease or medical condition is obesity. In some aspects, the obesity is drug-induced obesity. In some aspects, the method treats obesity by preventing or reducing weight gain or increasing weight loss in the patient. In some aspects, the method treats obesity by reducing appetite, reducing food intake, reducing the fat level of the patient, or reducing the rate of food movement through the gastrointestinal system.

Because obesity is associated with the onset or progression of other diseases, methods of treating obesity are further useful in methods of reducing complications associated with obesity, including vascular disease (coronary artery disease, stroke, peripheral vascular disease, ischemia-reperfusion, etc.), hypertension, type II diabetes onset, hyperlipidemia, and musculoskeletal disease. The present disclosure thus provides methods of treating or preventing complications associated with these obesity.

In some embodiments, the disease or medical condition is nonalcoholic fatty liver disease (NAFLD). NAFLD refers to a variety of liver diseases ranging from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) to cirrhosis (irreversible late hepatic scarring). NAFLD at all stages has in common the accumulation of fat (fatty infiltration) in liver cells (hepatocytes). Simple fatty liver is an abnormal accumulation of a specific type of fat (triglycerides) in liver cells without inflammation or scarring. In NASH, fat accumulation is associated with different levels of inflammation (hepatitis) and scarring (fibrosis) of the liver. The inflammatory cells can destroy liver cells (hepatocyte necrosis). In the terms "steatohepatitis" and "liponecrosis", fat (steato) refers to fatty infiltration, hepatitis refers to liver inflammation, and necrosis refers to destruction of liver cells. NASH can eventually lead to scarring of the liver (fibrosis) and then irreversible late scarring (cirrhosis). Cirrhosis caused by NASH is the final and most severe stage in the range of NAFLD. (Mendler, Michel, "Fatty Liver: Nonalcholic fat Liver Disease (NAFLD) and Nonalcholic Steatohepatics (NASH)," Schoenfield, Leslie J. eds., MedicinneNet. com., 8.29.2005).

Alcoholic or alcohol-induced liver disease encompasses three pathologically distinct liver diseases associated with or caused by excessive consumption of alcohol: fatty liver (steatosis), chronic or acute hepatitis, and cirrhosis of the liver. Alcoholic hepatitis can range from mild hepatitis (abnormal laboratory tests being the only indicator of the disease) to severe liver dysfunction with complications such as jaundice (yellow skin due to bilirubin retention), hepatic encephalopathy (neurological dysfunction due to liver failure), ascites (fluid accumulation in the abdomen), bleeding esophageal varices (varices in the esophagus), abnormal blood clotting and coma. Alcoholic hepatitis has histologically a characteristic appearance of hepatocellular ballooning, neutrophilic inflammation and sometimes malorosomes (abnormal aggregation of intracellular filamin). Cirrhosis is characterized anatomically by extensive nodules and fibrosis in the liver. (Worman, Howard J., "Alcoholic Liver Disease", Columbia University Medical Center wet).

Without being bound by any particular theory, the compositions and conjugates described herein may be used to treat alcoholic liver disease, NAFLD, or any stage thereof, including, for example, steatosis, steatohepatitis, hepatitis, liver inflammation, NASH, cirrhosis, or complications thereof. Accordingly, the present disclosure provides a method of preventing or treating alcoholic liver disease, NAFLD, or any stage thereof in a subject comprising providing to the subject an amount of a composition described herein effective to prevent or treat alcoholic liver disease, NAFLD, or any stage thereof. Such treatment methods include reducing one, two, three or more of: liver fat content, the occurrence or progression of cirrhosis, the incidence of hepatocellular carcinoma, inflammatory conditions such as abnormal liver enzyme levels (e.g. aspartate aminotransferase AST and/or alanine aminotransferase ALT or LDH), elevated serum ferritin, elevated serum bilirubin and/or fibrotic conditions such as elevated TGF- β levels. In an exemplary embodiment, the composition is used to treat a patient who has progressed beyond simple fatty liver (steatosis) and exhibits signs of inflammation or hepatitis. Such methods may result in, for example, reduced AST and/or ALT levels.

GLP-1 and Exendin-4 have been shown to have some neuroprotective effects. The present disclosure also provides for the use of the compositions described herein in the treatment of neurodegenerative diseases, including but not limited to: alzheimer's disease (Alzheimer's disease), Parkinson's disease (Parkinson's disease), multiple Sclerosis, amyotrophic Lateral Sclerosis (Amylotrophic Lateral Sclerosis), other demyelination-related disorders, senile dementia, subcortical dementia, arteriosclerotic dementia, AIDS-related dementia or other dementias, central nervous system cancer, traumatic brain injury, spinal cord injury, stroke or cerebral ischemia, cerebrovascular disease, epilepsy, Huntington's disease, Tourette's syndrome, Guillain's syndrome, William's disease, Pick's disease, neuroinflammatory disorders, encephalitis, encephalomyelitis or meningitis of viral, fungal or bacterial origin, or other central nervous system infections, prion disorders, cerebellar ataxia, spinocerebellar syndrome, spinocerebellar degenerative syndrome, senile dementia, subcortical dementia, Alzheimer's disease, Alzheimer, Friedreich's ataxia (friedreich ataxia), ataxia telangiectasia, spinal muscular dystrophy, progressive supranuclear palsy, dystonia, muscle spasm, tremor, retinitis pigmentosa, striatal nigral degeneration, mitochondrial encephalomyopathy, neuronal waxy lipofuscinosis, hepatic encephalopathy, renal encephalopathy, metabolic encephalopathy, toxin-induced encephalopathy, and radiation-induced brain injury.

In some embodiments, the composition is used in conjunction with parenteral administration of nutrients for non-diabetic patients in a hospital setting, e.g., for patients receiving parenteral nutrition or total parenteral nutrition. Non-limiting examples include surgical patients; patients in coma; patients with the following diseases: digestive tract diseases, or non-functional gastrointestinal tract (e.g., due to surgical resection, occlusion, or impaired absorptive capacity), Crohn's disease, ulcerative colitis, gastrointestinal obstruction, gastrointestinal fistula, acute pancreatitis, intestinal ischemia, major gastrointestinal surgery, certain congenital gastrointestinal abnormalities, prolonged diarrhea, or short bowel syndrome resulting from surgery; patients in shock, and those undergoing a healing process, often receive parenteral administration of carbohydrates, as well as various combinations of lipids, electrolytes, minerals, vitamins and amino acids. The composition comprising a GIP agonist peptide and a glucagon antagonist peptide as described herein and the parenteral nutritional composition may be administered simultaneously, at different times, one after the other, provided that the composition exerts the desired biological effect upon digestion of the parenteral nutritional composition. For example, parenteral nutrition may be administered 1, 2 or 3 times per day, while the composition is administered once every other day, three times per week, twice per week, once per 2 weeks, once per 3 weeks or once per month.

The term "treating" as used herein includes preventing a particular disorder or condition, or alleviating a symptom associated with a particular disorder or condition, and/or preventing or eliminating the symptom. For example, the term "treating diabetes" as used herein will generally refer to changing blood glucose levels in the direction of normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.

As used herein, an "effective" amount or "therapeutically effective amount" of a glucagon peptide refers to a nontoxic but sufficient amount of the peptide to provide the desired effect. For example, one desired effect would be to prevent or treat hypoglycemia, as measured, for example, by an increase in blood glucose levels. The desired effect of the replacement of glucagon peptides of the present disclosure would include treating hyperglycemia, for example, as measured by changes in blood glucose levels closer to normal; or inducing weight loss/preventing weight gain, e.g., as measured by weight loss; or preventing or reducing weight gain; or normalize body fat distribution. The "effective" amount will vary from subject to subject depending on the age and general condition of the individual, the mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount". However, an appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

Test subject

With respect to the above treatment methods, the patient is any host. In some embodiments, the host is a mammal. The term "mammal" as used herein refers to any vertebrate of the mammalian species, including but not limited to any of the monoforamen, marsupials and placental species. In some embodiments, the mammal is one of Rodentia (Rodentia) mammals, such as mice and hamsters, and lagomorpha (Logomorpha) mammals, such as rabbits. In exemplary embodiments, the mammal is from the order carnivora (Carn ivora), including felines (cats) and canines (dogs). In exemplary embodiments, the mammal is from the order Artiodactyla, including bovines (cows) and porcines (pigs), or from the order Perssodactyla, including equines (horses). In certain instances, the mammal is of the order Primates (Primates), Ceboids or Simoids (monkeys) or of the order Anthropoids (humans and apes). In a particular embodiment, the mammal is a human.

Reagent kit

The glucagon analogs of the present invention may be provided as part of a kit according to one embodiment. Thus, in some embodiments, a kit for administering a glucagon analog to a patient in need thereof is provided, wherein the kit comprises a glucagon analog as described herein.

In one embodiment, a kit is provided having a device for administering a glucagon analog to a subject. The device is in some aspects a syringe needle, pen device, jet injector, or other needle-less injector. The kit may alternatively or additionally comprise one or more containers, e.g., vials, tubes, bottles, single or multi-chamber prefilled syringes, cartridges, infusion pumps (external or implantable), jet injectors, prefilled pen devices, and the like, optionally containing the glucagon analog in lyophilized form or in aqueous solution. In some embodiments, the kit comprises instructions for use. According to one embodiment, the device of the kit is an aerosol dispensing device, wherein the composition is pre-packaged in the aerosol device. In another embodiment, the kit comprises a syringe and a needle, and in one embodiment the sterile glucagon composition is prepackaged in the syringe.

In some embodiments, the kit comprises instructions for use. In some aspects, the instructions include instructions for use according to any of the methods described herein. The instructions may additionally include instructions for maintaining a healthy diet and/or a physical exercise program. The instructions may be in the form of a paper booklet or in electronic form, such as a computer readable storage device containing the instructions.

Drawings

FIG. 1 shows that SEQ ID NO.1 and SEQ ID NO.2 can effectively agonize GLP-1 receptor and resist hydrolysis by DPP4 in vivo.

FIG. 2 shows the effect of SEQ ID NO.1 and SEQ ID NO.2 on insulin secretion from pancreatic BETA cells. The SEQ ID NO.1 and the SEQ ID NO.2 do not promote insulin secretion when the sugar content is low, so that hypoglycemia is avoided, and the safety is good; when the glucose is increased, the BETA cell can be promoted to secrete insulin, and when the glucose concentration is higher, the BETA cell can play a role (11uM glucose), and compared with hGLP1, the BETA cell is more sensitive to blood sugar change and has a better blood sugar reducing effect.

FIG. 3 shows that SEQ ID NO.1 and SEQ ID NO.2 modulate blood glucose levels and lower blood glucose in diabetic obese mice (BKS db) (0-60 min monitoring).

FIG. 4 shows that SEQ.ID NO.1 and SEQ.ID NO.2 modulate insulin secretion (monitored for 0-60 minutes) in diabetic obese mice (BKS db).

FIG. 5 shows that SEQ ID NO.1 and SEQ ID NO.2 can modulate blood glucose levels and lower blood glucose levels in diabetic obese mice (BKS db) for long periods of time (0-240 min monitoring).

FIG. 6 shows that SEQ. ID No.1 and SEQ. ID No.2 can modulate insulin secretion in diabetic obese mice (BKS db) for long periods of time (0-240 min monitoring).

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1, the newly discovered 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-Gly-Leu-Glu) and SEQ.ID NO.2 (His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Lys-Ser-Leu-Glu) are effective at agonizing the GLP-1 receptor and may be resistant to the in the human body Hydrolysis of DPP4

To verify whether the sequences seq.d No.1 and seq.d No.2 can act as GLP-1 receptor agonists, we chemically synthesized polypeptides with a purity of greater than 95%. The polypeptide was diluted to a concentration of 100nM in Hank's balanced salt solution (HBSS, pH 7.4) containing 20mM HEPES and 2.5mM oxybenzenesulfenpropylamine. To test whether the synthesized GLP-1 polypeptide could activate the GLP-1 receptor, ready-to-use GLP-1 detection cells (cat. HTS163RTA) and corresponding complete medium were purchased from Millipore. The cell is derived from the Chem-9 cell line and further expresses the GLP-1 receptor and the G-a protein. The purchased cells were stored in liquid nitrogen. When in use, the mixture is melted in a water bath at 37 ℃. The cells were transferred to a 15ml centrifuge tube containing 6ml of complete medium, centrifuged at 1000rpm for 5 minutes, and the supernatant was removed. Cells were resuspended in 10ml complete medium and seeded into 96-well plates at 200 ul/well. The cells were incubated in a 5% CO2 incubator at 37 ℃ for 24 hours. The 96-well plate was removed from the incubator prior to analysis. Cells from each well were washed with Hank's Balanced salt solution (HBSS, pH 7.4) containing 20mM HEPES and 2.5mM oxybenzenesulfenpropylamine. Then, 100ul Hank's balanced salt solution containing 5mM Fluo-8NW (ABD Bioquest 21080) calcium indicator, 20mM HEPES and 2.5mM sulfolan-oxybenzene was added per well. Incubate for 20 min in a 5% CO2, 37 ℃ incubator. Cells from each well were washed with 100ul Hank's Balanced salt solution (HBSS, pH 7.4) containing 20mM HEPES and 2.5mM sulfopropyl oxybenzene. Before observation under a fluorescence microscope, Hank's balanced salt solution in the corresponding hole is removed, and 50ul of GLP-1 polypeptide to be detected is added. The dynamic change in cell fluorescence was recorded with a camera using a GFP filter. Since this cell line overexpresses the GLP-1 receptor and the G-alpha protein, calcium influx can be initiated after the GLP-1 receptor is activated. Whether the GLP-1 receptor is activated or not can be reflected by the change in fluorescence. As shown in table one and fig. 1: the peptide segments SEQ.ID NO.1 and SEQ.ID NO.2 can rapidly activate a GLP-1 receptor, and the addition of DPP4 does not affect the activity of the peptide segments, which shows that the peptide segments SEQ.ID NO.1 and SEQ.ID NO.2 can resist the hydrolysis of DPP4 in human body. Meanwhile, the results show that the activities of the peptide segments of SEQ.ID NO.1 and SEQ.ID NO.2 are more durable compared with the activity of human GLP 1.

TABLE 1 changes in cellular Ca signals caused by SEQ ID NO.1 and SEQ ID NO.2

Example 2, SEQ.ID NO.1 and SEQ.ID NO.2 can modulate insulin secretion

The INS-1832/13 cell line was derived from rat beta cells transformed with the human proinsulin gene. Expresses the GLP1 receptor, and has increased insulin secretion after being stimulated by active GLP1 or an analogue thereof and under the condition of high concentration of glucose. We have demonstrated through calcium influx experiments that GLP1 analogs to be tested can activate GLP-1R. The INS-1832/13 cell line was used to test whether the GLP1 analogs to be tested could promote insulin release by activating GLP-1R.

The complete medium of the INS-1832/13 cell line was RPMI 1640 and supplemented with 10% fetal bovine serum, 50IU/ml penicillin, 50mg/L streptomycin, 10mM HEPES, 2mM L-glutamine, 1mM sodium pyruvate, and 50uM β -mercaptoethanol. INS-1832/13 cells were passaged twice weekly and cultured in a 5% CO2 incubator at 37 ℃.

Each well of a 24-well plate was seeded with 1X105 INS-1832/13 cells. Culturing in 5% CO2 incubator at 37 deg.C, changing culture medium every other day, and culturing for 6 days. The medium was removed and the cells washed twice with 400ul of sugarless KRB solution (116mM NaCl, 1.8mM CaCl2, 0.8mM MgSO4, 5.4 mM KCl, 1mM NaH2PO4, 26mM NaHCO3, 0.5% BSA, pH 7.4). 400ul of sugarless KRB solution was added to each well and placed in a 5% CO2 incubator at 37 ℃ for 1 hour. KRB was removed and cells were washed twice with 400ul of sugarless KRB solution. 400ul of KRB solution containing 2.2mM or 16.8mM glucose or 100nM GLP1 polypeptide was added. The mixture was placed in an incubator at 37 ℃ for 2 hours under 5% CO 2. Cell culture supernatant was taken and analyzed for insulin content using ELISA kit.

Rat insulin ELISA kits were purchased from Thermo Fisher. For each well of INS-1832/13 cells, 100ul of cell culture supernatant was added to the corresponding well of the ELISA plate. A blank control and varying concentrations of rat insulin were added as references. The 96-well plate was sealed and incubated at room temperature for 2.5 hours. The plate was washed 4 times. Then 100ul of biotin-labeled anti-rat insulin antibody was added to each well and the 96-well plate was sealed. Incubate at room temperature for 1 hour. The plate was washed 4 times. 100ul of streptavidin-labeled horseradish peroxidase was added to each well. Incubate for 45 minutes at room temperature. The plate was washed 4 times. 50ul of TMB substrate was added to each well. Incubate in the dark at room temperature for 30 minutes. 50ul of reaction stop solution was added to each well. The absorbance at 450nm and 550nm was read with a plate reader, and the read values were corrected with a blank control well. The reading at 550nm is subtracted from the reading at 450 nm. Standard curves were prepared according to rat reference insulin wells (700uIU/ml, 300uIU/ml, 150uIU/ml, 75uIU/ml, 37.5uIU/ml, 18.75uIU/ml, 9.38uIU/ml, and 0 uIU/ml). The insulin concentration of the sample corresponding to each well was calculated. The results show that under low sugar conditions (2.8uM glucose), none of hGLP1, SEQ. D No.1, SEQ. D No.2 promoted insulin secretion, that under high sugar conditions (16.7uM glucose), all of hGLP1, SEQ. ID No.1, SEQ. ID No.2 promoted insulin secretion (P < 0.01), and that under higher sugar conditions (11uM glucose), both of SEQ. ID No.1, SEQ. ID No.2 promoted insulin secretion (P < 0.01). This shows that SEQ ID No.1 and SEQ ID No.2 do not work at low sugar, thereby avoiding causing hypoglycemia and having good safety. Meanwhile, the BETA cells can be promoted to secrete insulin by the SEQ ID NO.1 and the SEQ ID NO.2 when the glucose is increased, and the BETA cells can play a role when the glucose concentration is higher (11uM glucose), are more sensitive to blood sugar change compared with hGLP1 (table 2 and figure 2), and have better blood sugar reducing effect.

Table 2 influence of seq.id No.1 and seq.id No.2 on insulin secretion.

Example 3, SEQ.ID NO.1 and SEQ.ID NO.2 modulate insulin levels and lower blood glucose in obese mice (BKS db).

To verify whether The polypeptides SEQ ID No.1 and SEQ ID No.2 are able to promote insulin secretion and lower blood glucose concentration in type 2 diabetic model animals, we chose BKS. Cg-Dock7< m > +/+ Lepr < db >/J (db/db) male obese mice as subjects (The Jackson Laboratory). The experimental animals were first starved for 18 hours and then injected intraperitoneally at a polypeptide concentration of 25nmol/kg (SEQ. ID No.1, SEQ. ID No.2, Exendin-4) and a glucose concentration of 18 mmol/kg. The control group was injected with physiological saline. Blood glucose concentrations were measured prior to injection and then every 15 minutes (0 min, 15 min, 30 min, 45 min, 60 min). Collecting blood from rat tail, analyzing and measuring insulin concentration by enzyme-linked immunosorbent assay (ELISA), and measuring glucose concentration by blood glucose tester. It can be found that the polypeptides SEQ.ID No.1 and SEQ.ID No.2 are effective in lowering blood glucose levels (Table 3, FIG. 3) and increasing blood insulin levels (Table 4, FIG. 4).

To further verify whether seq.id No.1 and seq.id No.2 could increase insulin sensitivity, regulate insulin secretion, lower blood glucose concentration for a long period of time in type 2 diabetic model animals, we continued to select bks.cg-Dock7< m > +/+ Lepr < db >/J (db/db) male obese mice as subjects (The Jackson Laboratory) with 25nmol/kg polypeptide concentration (seq.id No.1, seq.id No.2, Exendin-4) and 18mmol/kg glucose concentration and performed intraperitoneal injections, and The control group was injected with physiological saline. The normally bred experimental animals were subjected to intraperitoneal injection, and blood glucose concentration was measured before injection, and then blood from the tail was collected at intervals of 60 minutes (0 minute, 60 minutes, 120 minutes, 180 minutes, 240 minutes) to measure insulin concentration by enzyme-linked immunosorbent assay (ELISA), and glucose concentration was measured by a blood glucose meter. It can be found that the polypeptides SEQ.ID No.1 and SEQ.ID No.2 are effective in lowering blood glucose levels (Table 5, FIG. 5) and increasing insulin sensitivity (Table 6, FIG. 6).

Example 4, SEQ ID NO.1 and SEQ ID NO.2 modified sequences SEQ ID NO.3 and SEQ ID NO.4 can modulate insulin levels and lower blood glucose in obese mice (BKS db) for a long time.

The C-terminal amino acid of the polypeptide is further modified to increase the in vivo biological stability and prolong the half-life of the polypeptide, and a novel long-acting GLP-1 derivative is prepared, and the sequence is as follows:

SEQ.ID NO.3：

His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gl y-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-Xa a17-Ala-Ala-Xaa20-Glu-Phe-Val-Ala-Trp-Leu-Val-Xaa28-Ser-Leu-Glu。

wherein Xaa17 is any one of Ser, His, Gln, Ala or Lys, Xaa20 is any one of Ser, His, Gln, Ala or Lys, and Xaa28 is any one of Ser, His, Asp, Ala or Lys.

To verify whether The polypeptides SEQ ID NO.3 and SEQ ID NO.4 are able to promote insulin secretion and lower blood glucose levels for a long time in type 2 diabetic model animals, we continued to select BKS.Cg-Dock7< m > +/+ Lepr < db >/J (db/db) male obese mice as subjects (The Jackson Laboratory). Physiological saline of a negative control group, Liraglutide (Liraglutide) and Exenatide (Exenatide) of a positive control group (25nmol/kg), a compound group (25 nmol/kg):

SEQ.ID NO.5， His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Ly s-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-Ly s-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-Al a-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-Gl y-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-Ly s-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-Gl y-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-Gl y-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-Gl y-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-Gl y-Asp-Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Ala-Ser-Leu-Glu

SEQ.ID NO.14，

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

when mice were normally drunk and fed, the compound was administered at 0h, and blood glucose was measured by a glucometer at 0, 3, 6, 12, 24, and 48h, it was found that the polypeptides 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, and SEQ ID NO.16 could lower blood glucose levels for a long period of time (Table 7).

In conclusion, the novel GLP-1 seq.id No.1 and seq.id No.2 and their modified sequences seq.id No.3 and seq.id No.4 can regulate insulin levels in obese mice (BKS db), lower blood glucose, and have a long-acting mechanism compared to Liraglutide and Exenatide. Therefore, the compound can be used as a hypoglycemic drug for the type II diabetes.

Sequence listing

<110> pure Zi Biotechnology (Shenzhen) Limited

Zhang Xiang Min;

the horse danjun;

<120> novel glucagon analogue and application thereof

<130> QZSW201901

<160> 16

<170> PatentIn version 3.5 edition

<210> 1

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<223> glucagon analogues

<400> 1

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

1 5 10 15

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

20 25 30

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

1 5 10 15

Lys Ala Ala Lys Glu Phe Val Ala Trp Leu Val Lys Ser Leu Glu

20 25 30

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<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> Xaa17 is any one amino acid of Ser, His, Gln, Ala or Lys

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> Xaa20 is any one amino acid of Ser, His, Gln, Ala or Lys

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> Xaa28 is any one amino acid Ser, His, Asp, Ala or Lys

<400> 3

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

1 5 10 15

Xaa17 Ala Thr Xaa20 Phe Ile Ala Trp Leu Val Xaa28 Gly Leu Glu

20 25 30

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<222> (17)..(17)

<223> Xaa17 is any one amino acid of Ser, His, Gln, Ala or Lys

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> Xaa20 is any one amino acid of Ser, His, Gln, Ala or Lys

<220>

<221> MISC_FEATURE

<222> (28)..(28)

<223> Xaa28 is any one amino acid Ser, His, Asp, Ala or Lys

<400> 4

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

1 5 10 15

Xaa17 Ala Ala Xaa20 Glu Phe Val Ala Trp Leu Val Xaa28 Ser Leu Glu

20 25 30

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<400> 5

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

1 5 10 15

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

20 25 30

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<400> 6

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

1 5 10 15

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

20 25 30

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<400> 7

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

1 5 10 15

Ala Ala Thr Ala Glu Phe Ile Ala Trp Leu Val Ala Gly Leu Glu

20 25 30

<210> 8

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<400> 8

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

1 5 10 15

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

20 25 30

<210> 9

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<400> 9

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

1 5 10 15

Lys Ala Ala Lys Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

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<400> 10

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

1 5 10 15

Ala Ala Ala Lys Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

<210> 11

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<400> 11

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

1 5 10 15

Ala Ala Ala Ala Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

<210> 12

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<400> 12

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

1 5 10 15

Lys Ala Ala Asp Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

<210> 13

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<400> 13

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

1 5 10 15

Asp Ala Ala Lys Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

<210> 14

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<400> 14

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

1 5 10 15

Asp Ala Ala His Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

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

1 5 10 15

His Ala Ala Asp Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

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

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Ser Ala Ala Asp Glu Phe Val Ala Trp Leu Val Ala Ser Leu Glu

20 25 30

Claims (8)

1. A glucagon analog, wherein the sequence of said glucagon analog is selected from the group consisting of SEQ ID No.1-2, 5-16, and pharmaceutically acceptable salts, solvates, prodrugs, or any combination thereof.

2. The glucagon analog of claim 1, wherein the glucagon analog has a longer half-life and better insulinotropic activity than wild-type human GLP1, and pharmaceutically acceptable salts, solvates, prodrugs, or any combination thereof.

3. A dimer or multimer comprising two or more glucagon analogs of claim 1 or 2.

4. A conjugate comprising the glucagon analog of claim 1 or 2, and pharmaceutically acceptable salts, solvates, or prodrugs thereof, and a conjugate moiety.

5. The conjugate of claim 4, wherein the glucagon analog and pharmaceutically acceptable salts, solvates, or prodrugs thereof are fused to a heterologous peptide analog.

6. A pharmaceutical composition comprising the glucagon analog of claim 1 or 2, and pharmaceutically acceptable salts, solvates, prodrugs, or any combination thereof, the dimer or multimer of claim 3, the conjugate of claim 4 or 5, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

7. Use of the glucagon analog of claim 1 or 2, and pharmaceutically acceptable salts, solvates, prodrugs, or any combination thereof, the dimer or multimer of claim 3, or the conjugate of claim 4 or 5, in the preparation of a medicament for reducing blood glucose in a subject in need thereof.

8. Use of a glucagon analogue of claim 1 or 2, and pharmaceutically acceptable salts, solvates, prodrugs or any combination thereof, a dimer or multimer of claim 3, or a conjugate of claim 4 or 5, for the preparation of a medicament for the treatment of type II diabetes.

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