Glucagon analogues and their medical uses

Abstract

The present invention relates to a glucagon analog and its medical use. Specifically, the glucagon analog has significantly improved in vitro activity, excellent physical / chemical stability and good solubility, and is applicable to the treatment of metabolic diseases such as hypoglycemia, obesity, and diabetes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a Chinese patent application filed on June 18, 2021, bearing application number 202110676681.3.

[0002] The present disclosure relates to glucagon analogs and their pharmaceutical uses. The glucagon analogs of the present disclosure are applicable to the treatment or alleviation of symptoms of metabolic disorders such as hypoglycemia, obesity, and diabetes. [Background technology]

[0003] Controlling blood glucose balance in the body is very important for the body's metabolism. Normally, human blood glucose levels are maintained in dynamic balance through the regulation of various polypeptide hormones (mainly insulin and glucagon). Blood glucose imbalance may cause complications. When blood glucose concentration falls below normal levels, hypoglycemia occurs. The pancreas in the islets produces glucagon to raise blood glucose levels to the normal range. Non-severe hypoglycemia generally causes spontaneous or neurological hypoglycemic symptoms, and patients can recognize the onset of hypoglycemia, so no auxiliary treatment by others is required. On the other hand, when blood glucose levels are reduced by other drugs, or by disease, or by hormonal or enzymatic deficiencies (e.g., a range of clinically severe hypoglycemic conditions resulting from the treatment of diabetes, renal failure, certain tumors, liver disease, hypothyroidism, inborn errors of metabolism, sepsis, and reactive hypoglycemia), the body's glucagon is insufficient to restore blood glucose, which can even be life-threatening, and is usually manifested as cognitive or physical dysfunction, limited ability to self-treat, and necessitating assisted therapy by another person to restore normal blood glucose.

[0004] Currently, in the prevention and treatment of hypoglycemia, fully conscious hypoglycemic patients are most preferably treated with oral administration of carbohydrate foods, while those with impaired consciousness need to be treated with intravenous injection of 50% glucose solution or intramuscular injection of 0.5-2 mg of glucagon with the help of non-medical personnel or medical personnel. Non-medical personnel have difficulty administering intravenous injection of 50% glucose solution. If the injection dose is too high, it will result in rebound hyperglycemia, and if the glucose solution seeps out of the blood vessels, it will cause local tissue damage. Therefore, in non-hospital settings, the optimal treatment strategy for severely hypoglycemic patients with impaired consciousness is subcutaneous or intramuscular injection of glucagon. Glucagon is also used in clinical settings as an emergency drug for severe hypoglycemia, and based on the lipid-lowering and energy consumption-promoting effects of glucagon, dual receptor agonists with incretin also provide additional benefits to obese diabetic patients.

[0005] Glucagon is a linear polypeptide consisting of 29 amino acids secreted by pancreatic α-cells and controls the production of glucose and ketone bodies by the liver. Glucagon is secreted at night and between meals and increases glucose production from amino acid precursors (gluconeogenesis) and promotes the breakdown of glycogen into glucose (glycolysis), thereby increasing hepatic glucose output and improving blood glucose concentration. Glucagon, together with insulin secreted by pancreatic β-cells, precisely controls blood glucose balance in the body. In addition to its therapeutic effect against hypoglycemia, glucagon has been shown to have an anti-obesity effect by suppressing appetite and activating hormone-sensitive lipase in adipocytes to promote lipolysis.

[0006] Glucagon is an important component of the hypoglycemic defense mechanism, and low doses of glucagon can prevent insulin-induced hypoglycemia and improve the ability to recover from hypoglycemia. Low doses of glucagon can also suppress appetite by inducing satiety, and can activate hormone-sensitive lipase in adipocytes to promote lipolysis, so that it has potential anti-obesity effects and can be applied to the treatment of overweight or obesity in many patients with type 2 diabetes.

[0007] However, natural glucagon has very low solubility in aqueous solutions at neutral pH. In addition, glucagon contains amino acids or amino acid sequences that cause side chain deamidation, oxidation, and formation of cyclic imide intermediates of amino acid residues, and also contains amino acids or amino acid sequences that cause isomerization, peptide chain cleavage, etc., and cannot maintain stability for a long period of time, forming gels or fibrils within a few hours to a few days, and has very poor chemical and physical stability. Therefore, in order to meet the demands of the development of injectable formulations, it is necessary to develop new glucagon analogs with excellent solubility and good physical / chemical stability.

[0008] First generation glucagon injection preparations, such as Glucagon Rescue Reagent Kit (Glucagon (登録商標) , Eli Lilly), Glucagon Reagent Kit (Glucagon (登録商標) glucagon is limited by its undesirable druggability, which means that it must be stored in the form of a freeze-dried powder and then reconstituted with a solvent before being injected subcutaneously or intramuscularly, making it difficult to use for patients and non-medical personnel.

[0009] Second generation glucagon injection preparations, such as human glucagon nasal powder (BAQSIMI (登録商標) , Eli Lilly), prefilled human glucagon injection (GVOKE HYPOPEN (登録商標) BAQSIMI (XERIS) has made targeted improvements to the formulation to ensure therapeutic efficacy while significantly improving convenience for patients and non-medical personnel. (登録商標) GVOKE HYPOPEN is administered as an intranasal spray, which slows the onset of action, requires three times the dose compared to intramuscular injection, and has adverse reactions. (登録商標)is formulated in DMSO and can be directly injected subcutaneously, but discomfort at the injection site and other side effects are evident. In addition, Dasiglucagon (developed by Zealand Pharma) is based on natural human glucagon and has seven amino acid modifications that improve solubility and physical / chemical stability without affecting activity, allowing it to be directly injected subcutaneously in an injectable liquid form. Dasiglucagon was recently approved for marketing by the U.S. Food and Drug Administration.

[0010] There is still a need in the field for glucagon analogs with high activity, good solubility, and good physical / chemical stability. The present disclosure provides glucagon analogs with high activity (enhanced activity compared to native glucagon), good solubility in aqueous solutions (especially at physiological pH), and improved stability (including physical and chemical stability). Compared with glucagon analogs in the field, the glucagon analogs of the present disclosure have stronger glucagon receptor agonist activity in vitro, which greatly helps to reduce the volume and frequency of subcutaneous administration, improve the ease of use for hypoglycemic patients, and reduce the economic burden on patients. The glucagon analogs of the present disclosure can be used alone or in combination with other therapeutic agents in methods for treating metabolic diseases or conditions such as hypoglycemia, obesity, and diabetes. Summary of the Invention

[0011] The present disclosure provides glucagon analogs or pharma- ceutically acceptable salts and / or solvates thereof, pharmaceutical compositions comprising same, polynucleotides encoding said glucagon analogs, vectors comprising said polynucleotides, host cells comprising said polynucleotides or vectors, as well as methods and related pharmaceutical uses of said glucagon analogs or pharma- ceutically acceptable salts and / or solvates thereof or pharmaceutical compositions comprising same in the treatment of disease, alleviation of medical conditions (e.g., metabolic diseases or conditions such as hypoglycemia, obesity, diabetes, etc.).

[0012] Glucagon analogs or pharma- ceutically acceptable salts and / or solvates thereof The present disclosure provides glucagon analogs having the structure represented by formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof: TIFF2024525144000001.tif27156

[0013] The present disclosure further provides a glucagon analog comprising the structure of formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof, wherein R1 and / or R2 are absent: Among them, R1 is hydrogen, C 1-4 an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group, or pGlu; R2 is -OH or -NH2; X3, X 15 , X 16 , X 20 , X 21 , X 24 , X 27 and X 28 are each independently selected from any natural or non-natural amino acid residue, 17 is Aib.

[0014] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0015] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0016] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0017] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0018] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28 is Ser.

[0019] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0020] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28is selected from Asn, Glu, or Ser.

[0021] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28 is Ser.

[0022] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0023] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28is Ser.

[0024] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0025] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0026] In some embodiments, X3 is selected from His or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0027] In some embodiments, R1 is hydrogen and R2 is -OH or -NH2. In some embodiments, R1 is hydrogen and R2 is -OH.

[0028] The pharma- ceutically acceptable salts and / or solvates of the present disclosure are selected from inorganic salts or organic salts.

[0029] The glucagon analogs of the present disclosure can be reacted with acidic or basic compounds to produce the corresponding salts.

[0030] "Reacting with an acidic compound to produce the corresponding salt" refers to a salt formed with an inorganic or organic acid that is capable of retaining the biological effectiveness of the free base without other adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc., and organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0031] "Reacting with a basic compound to produce the corresponding salt" refers to a salt formed with an inorganic or organic base that is capable of retaining the biological effectiveness of the free acid without other adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Salts derived from organic bases include, but are not limited to, primary amines, secondary amines, tertiary amines, substituted amines, cyclic amines, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, ethylenediamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, etc. Suitable organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0032] The term "solvate" refers to a complex formed of a glucagon analog of the present disclosure, or a pharma- ceutically acceptable salt thereof, and a suitable solvent. Non-limiting examples of solvents include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate. In a specific embodiment, the solvate is a hydrate.

[0033] In some embodiments, the present disclosure provides a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, wherein the glucagon analog is a compound represented by any one of SEQ ID NOs: 1 to 49.

[0034] In some specific embodiments, the disclosure provides a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, the glucagon analog comprising: H-HSQGTFTSDYSKYLDTAibRAQEFVQWLEST-OH (SEQ ID NO: 39), H-HSQGTFTSDYSKYLDLAibRAQEFVQWLEST-OH (SEQ ID NO: 40), H-HSQGTFTSDYSKYLDVAibRAQEFVQWLEST-OH (SEQ ID NO: 41), H-HSQGTFTSDYSKYLDIAibRAQEFVQWLEST-OH (SEQ ID NO: 42), H-HSHGTFTSDYSKYLDLAibRAQEFVQWLEST-OH (SEQ ID NO: 43), H-HSHGTFTSDYSKYLDLAibRAQEFVEWLEST-OH (SEQ ID NO: 47), It is any one compound selected from the following:

[0035] In the sequence of each glucagon analog of the present disclosure, the C-terminal "-OH" moiety can be replaced with a C-terminal "-NH2."

[0036] In some specific embodiments, the present disclosure provides a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, the glucagon analog comprising any one of the structures selected from SEQ ID NOs: 39, 40, 41, 42, 43, and 47, wherein the N-terminal H and / or the C-terminal OH of SEQ ID NOs: 39, 40, 41, 42, 43, and 47 is absent.

[0037] The present disclosure provides glucagon analogs having the structure represented by formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof: TIFF2024525144000002.tif27156

[0038] The present disclosure further provides a glucagon analog comprising the structure of formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof, wherein R1 and / or R2 are absent: Among them, R1 is hydrogen, C 1-4 an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group, or pGlu; R2 is -OH or -NH2; X3, X 17 , X 20 , X 21 , X 24 , X 27 and X 28 are each independently selected from any natural or non-natural amino acid residue, 15 is Asp, and X 16 is selected from Ser, Thr, Leu, Val, Ile or α-methyl-Ser, for example selected from Thr, Leu, Val or Ile.

[0039] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is selected from Aib or Ala, and X 20 is not Glu, but X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0040] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is selected from Aib or Ala, and X 20 is not Glu, but X21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0041] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0042] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0043] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17is Aib, and X 20 is not Glu, but X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0044] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is not Glu, but X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28 is Ser.

[0045] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is not Glu, but X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0046] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is selected from Ala, Gln, Glu, Ser, Thr or Lys, 21 is selected from Asp or Glu, and X 24is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0047] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0048] In some embodiments, X3 is selected from His or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0049] In some embodiments, R1 is hydrogen and R2 is -OH or -NH2. In some embodiments, R1 is hydrogen and R2 is -OH.

[0050] The present disclosure provides a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, the glucagon analog comprising the structure shown in formula (I): TIFF2024525144000003.tif27156

[0051] The present disclosure further provides a glucagon analog comprising the structure of formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof, wherein R1 and / or R2 are absent: Among them, R1 is hydrogen, C 1-4 an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group, or pGlu; R2 is -OH or -NH2; X3, X 15 , X 16 , X 17 , X 21 , X 24 , X 27 and X 28 are each independently selected from any natural or non-natural amino acid residue, 20 is Gln.

[0052] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0053] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0054] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0055] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28 is Ser.

[0056] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0057] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, 27 is Glu, and X 28 is Ser.

[0058] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0059] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is selected from Aib or Ala, and X 20 is Gln, and X 21 is selected from Asp or Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0060] In some embodiments, X3 is selected from His or Gln; 15 is Asp, and X16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0061] In some embodiments, R1 is hydrogen and R2 is -OH or -NH2. In some embodiments, R1 is hydrogen and R2 is -OH.

[0062] The present disclosure provides glucagon analogs having the structure represented by formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof: TIFF2024525144000004.tif27156

[0063] The present disclosure further provides a glucagon analog comprising the structure of formula (I) or a pharma- ceutically acceptable salt and / or solvate thereof, wherein R1 and / or R2 are absent: Among them, R1 is hydrogen, C 1-4 an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group, or a pGlu; R2 is -OH or -NH2; X3, X 15 , X 16 , X 17 , X 21 , X 27 and X 28 are each independently selected from any natural or non-natural amino acid residue, 20 is Glu, and X 24 is Arg.

[0064] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is Glu, and X 21 is selected from Asp or Glu, and X 24 is Arg and X 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0065] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Glu, and X 21 is selected from Asp or Glu, and X 24 is Arg and X 27 is selected from Met, Glu, Nle, Leu or Ser, and X 28 is selected from Asn, Glu, or Ser.

[0066] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is selected from Asp or Glu, and X 16 is selected from Ser, Thr, Leu, Val, Ile, or α-methyl-Ser; 17 is Aib, and X 20 is Glu, and X 21 is selected from Asp or Glu, and X 24 is Arg and X 27 is Glu, and X 28 is Ser.

[0067] In some embodiments, X3 is selected from His, Dap(Ac) or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Glu, and X 21is selected from Asp or Glu, and X 24 is Arg and X 27 is Glu, and X 28 is Ser.

[0068] In some embodiments, X3 is selected from His or Gln; 15 is Asp, and X 16 is selected from Thr, Leu, Val or Ile, and X 17 is Aib, and X 20 is Gln, and X 21 is Glu, and X 24 is selected from Gln or Glu, 27 is Glu, and X 28 is Ser.

[0069] In some embodiments, R1 is hydrogen and R2 is -OH or -NH2. In some embodiments, R1 is hydrogen and R2 is -OH.

[0070] The glucagon analogs of the present disclosure, or their pharma- ceutically acceptable salts and / or solvates, have glucagon receptor (GCGR) agonist activity, and their binding to GCGR can be used as an indication of agonist activity. In some optional embodiments, intracellular signaling caused by the binding of the compound to the receptor can also be measured. For example, activation of GCGR by glucagon receptor agonists stimulates the formation of intracellular cyclic adenosine monophosphate (cAMP). Thus, the activity of the receptor can be monitored by the production of cAMP in suitable cells expressing the receptor. For example, it can be measured under the above conditions using the materials shown in Example 2 (e.g., CHO-K1 / GCGR / CRE-Luc stable transformed cell line and Cisbio-cAMP-Gs Dynamic kit). EC 50 The EC value can be used as a numerical metric of agonist efficacy against GCGR. 50 The value is the concentration of the compound required to achieve half of the compound's maximal activity for the receptor under consideration in a particular measurement.

[0071] In some embodiments, the glucagon receptor is a mammalian glucagon receptor, for example a human glucagon receptor.

[0072] In some embodiments, the agonist activity of the glucagon analogs of the present disclosure, or pharma- ceutically acceptable salts and / or solvates thereof, on human GCGR is at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0-fold, or at least 3.1-fold that of native glucagon.

[0073] In some embodiments, the glucagon analogs or pharma- ceutically acceptable salts and / or solvates thereof of the present disclosure have no or negligible agonist activity at the human GLP-1R and / or GIPR.

[0074] In some embodiments, the glucagon analogs or pharma- ceutically acceptable salts and / or solvates thereof of the present disclosure have improved solubility and / or stability compared to native glucagon.

[0075] In some embodiments, improved solubility may include or refer to having improved solubility compared to native glucagon at pH 4 (e.g., in acetate buffer at pH 4), pH 5 (e.g., in acetate buffer at pH 5), pH 6 (e.g., in phosphate buffer at pH 6), pH 7 (e.g., in phosphate buffer at pH 7), and / or pH 7.5 (e.g., in phosphate buffer at pH 7.5). As an example, it can be measured under the conditions shown in Example 5. A solubility of ≧1 mg / mL may be desired.

[0076] In some embodiments, the solubility of a glucagon analog of the present disclosure in phosphate buffer at pH 7.4, e.g., as calculated by Beer's law measuring absorbance at 280 nm, is ≧1.00 mg / mL, ≧1.10 mg / mL, ≧1.20 mg / mL, ≧1.30 mg / mL, ≧1.40 mg / mL, ≧1.50 mg / mL, ≧1.60 mg / mL, ≧1.70 mg / mL, ≧1.80 mg / mL, ≧1.90 mg / mL, ≧2.00 mg / mL, ≧2.10 mg / mL, ≧2.20 mg / mL, ≧2.30 mg / mL, ≧2.40 mg / mL, ≧2.50 mg / mL, ≧2.60 mg / mL, ≧2.70 mg / mL, ≧2.80 mg / mL, ≧2.90 mg / mL, ≧3.00 mg / mL, ≧3.10 mg / mL, ≧3.20 mg / mL, ≧3.30 mg / mL, ≧3.40 mg / mL, ≧3.50 mg / mL, ≧3.60 mg / mL, ≧3.70 mg / mL, ≧3.80 mg / mL, ≧3.9 ... mg / mL, 3.20 mg / mL, 3.30 mg / mL, 3.40 mg / mL, 3.50 mg / mL, 3.60 mg / mL, 3.70 mg / mL, 3.80 mg / mL, 3.90 mg / mL, 4.00 mg / mL, 4.10 mg / mL, 4.20 mg / mL, 4.30 mg / mL, 4.40 mg / mL, 4.50 mg / mL, 4.60 mg / mL, 4.70 mg / mL, 4.80 mg / mL, 4.90 mg / mL, or 5.00 mg / mL.

[0077] In some embodiments, improved stability may include or refer to improved physical stability and / or improved chemical stability compared to native human glucagon.

[0078] In some embodiments, improved physical stability may include or refer to a decreased tendency to aggregate (e.g., form soluble or insoluble aggregates (e.g., fibrils)). The glucagon analog may be dissolved in 0.1 N HCl and aggregation (e.g., form fibrils) may be measured at a concentration of 1 mg / mL at 37° C. Any suitable period may be used, for example, 24 hours, 48 ​​hours, 96 hours, or 120 hours. Aggregation may be measured by a ThT fluorescence assay. Aggregation may be measured under the conditions set forth in Example 4.

[0079] In some embodiments, improved chemical stability may include or refer to a reduced tendency of the peptide to be cleaved or degraded in aqueous buffer (generally in the absence of contaminating protease or peptidase activity). Stability may be measured, for example, by dissolving the glucagon analog in phosphate buffer at pH 7.4 at a concentration of 1 mg / mL at 4°C, 25°C or 40°C. Evaluation may include measuring stability after a suitable period of incubation, for example, 1 day, 7 days, 14 days, 21 days or 28 days. This may include measuring the purity of the compound, i.e., the percentage area of ​​the main peak relative to the total area of ​​all integrated peaks in each chromatogram, as limited in Example 4. Stability may be measured under the conditions set forth in Example 4.

[0080] Polynucleotides The present disclosure provides polynucleotides encoding precursor peptides of the glucagon analogs of the present disclosure, which may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the polynucleotides of the present disclosure are essentially isolated polynucleotides.

[0081] The precursor peptide is a peptide that, when modified, can provide a glucagon analog of the present disclosure. For example, the X 17 is Arg or other natural amino acid, and when modified, X 17 is obtained a glucagon analog of the present disclosure wherein is Aib.

[0082] The polynucleotides of the present disclosure may also be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e., a vector that allows for the in vitro and / or in vivo expression of the glucagon analogue (i.e., in a suitable host cell, host organism, and / or expression system). Such an expression vector usually comprises at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is within the skill of the art to select the above elements and their sequences for expression in a particular host.

[0083] In some embodiments, the vector comprises the following elements operably linked in a 5' to 3' direction: a promoter to drive expression of the nucleic acid fragment, an optional polynucleotide encoding a leader peptide to enable secretion (into the extracellular phase or into the periplasm, as appropriate), a polynucleotide encoding a precursor peptide, and an optional polynucleotide encoding a terminator.

[0084] The polynucleotides of the disclosure can be prepared or obtained by known methods (eg, automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of the precursor peptide of the glucagon analog of the disclosure.

[0085] host cell The present disclosure provides recombinant host cells that express a precursor peptide of a glucagon analog of the present disclosure or that contain a polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0086] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0087] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0088] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0089] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0090] The cells of the present disclosure are incapable of developing into complete plants or animal organisms.

[0091] Method of production or preparation The present disclosure provides a method for preparing the glucagon analogs of the present disclosure, which comprises, for example, (a) synthesizing a glucagon analogue by a method of chemical synthesis (e.g., solid-phase or liquid-phase synthesis) and recovering the synthesized glucagon analogue thus obtained; or (b) a method in which a precursor peptide can be expressed by a polynucleotide or vector encoding the precursor peptide, the expression product can be recovered, and the precursor peptide can be modified, for example by introducing one or more unnatural amino acids (e.g., Aib), such that a glucagon analog of the disclosure is produced; Includes.

[0092] In one embodiment, - culturing a host cell of the present disclosure under conditions allowing expression of the precursor peptide; - recovering the precursor peptide from the culture; - modifying said precursor peptide to obtain a glucagon analog of the present disclosure; Optionally, purifying the precursor peptide.

[0093] Methods and reagents for recombinantly producing polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, techniques for isolating and purifying polypeptides, which are applied in methods for producing precursor peptides, are well known to those skilled in the art. Methods for solid-phase or liquid-phase synthesis are known in the art, and reference may be made, for example, to WO98 / 11125, in particular Fields, GB et al., 2002, "Principles and practice of solid-phase peptide synthesis". In: Synthetic Peptides (2nd ed.), and the Examples in the present disclosure.

[0094] In some embodiments, there is further provided a method of forming pharma- ceutically acceptable salts and / or solvates of glucagon analogs, for example, the glucagon analogs of the present disclosure can be reacted with pharma- ceutically acceptable acidic or basic compounds to form salts.

[0095] In some embodiments, the glucagon analogs of the present disclosure may be prepared by a method of recombinant expression in a microorganism or by a method of Fmoc solid phase synthesis (e.g., an SPPS method that includes a method for deprotecting the amino acids, a method for cleaving the peptide from the resin, and a method for purifying it).

[0096] In some embodiments, when using the method of Fmoc solid phase synthesis, the synthesis vector is Fmoc-L-Thr(tBu)-Wang resin (German Chemical, loading 0.533 mmol / g). In some embodiments, the amino acid derivatives used in the synthesis process are Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile ...Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc- c-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- Including L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH, Fmoc-Aib-OH, Fmoc-L-Nle-OH, etc. In some embodiments, the synthesis process includes the following: after washing the solid-phase synthesis vector, first remove the Fmoc protecting group in the α-amino of the solid-phase synthesis vector with a solution of N,N-dimethylformamide (DMF) containing 20% ​​4-methylpiperidine; then, if in excess, activate the solid-phase synthesis vector and the next amino acid derivative in the sequence with HCTU / 4-methylmorpholine, and then condense to form an amide bond, thereby extending the peptide chain; repeat the operations of condensation → washing → deprotection → washing → condensation of the next amino acid to achieve the polypeptide chain length to be synthesized; finally, react the mixed solution of trifluoroacetic acid: water: triisopropylsilane (90:5:5, v:v:v) with the resin to cut the polypeptide from the solid-phase vector; further precipitate with frozen methyl tert-butyl ether to obtain a solid crude product of the polypeptide derivative; dissolve the solid crude product of the polypeptide in a mixed solution of acetonitrile / water containing 0.1% trifluoroacetic acid, and then purify and isolate by a C-18 reverse phase preparative chromatography column, and obtain the pure product of the polypeptide and its derivative.

[0097] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising a prophylactically, therapeutically or palliatively effective amount of a glucagon analogue of the present disclosure or a pharma- ceutically acceptable salt and / or solvate thereof, and one or more pharma- ceutically acceptable vectors, diluents, buffers or excipients.

[0098] In some embodiments, a unit dose of the pharmaceutical composition may contain 0.01-99% by weight of the glucagon analog or pharma- ceutical acceptable salt and / or solvate of the present disclosure. In some further embodiments, the content of the glucagon analog or pharma- ceutical acceptable salt and / or solvate of the present disclosure in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0099] In some embodiments, the pharmaceutical composition further comprises at least one compound or substance having therapeutic activity for a metabolic disorder.

[0100] In some embodiments, the compound or substance having metabolic disease therapeutic activity is one or more selected from glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) receptor agonist, insulin, enteroglucagon, neuropeptide Y5 receptor antagonist, acetyl-coenzyme A carboxylase inhibitor, leptin receptor agonist, glinide, α-glucosidase inhibitor (AGi), thiazolidinedione (TZD), dipeptide peptidase-4 inhibitors (DPP-IV), sodium glucose cotransporter 2 inhibitor (SGLT-2), farnesol X receptor (FXR) agonist, and obestatin.

[0101] The pharmaceutical compositions of the present disclosure can be administered parenterally, as by subcutaneous, intramuscular, intraperitoneal or intravenous injection with a syringe (optionally a pen-type syringe). Alternatively, parenteral administration can be by infusion pump. Alternatively, the composition can be a solution or suspension administering the glucagon peptide in the form of a nasal or pulmonary spray, or it can be administered transdermally, such as by needle-free injection or by a patch, optionally an iontophoretic patch, or it can be administered transmucosally (e.g., buccal).

[0102] Reagent kit (or kit) The present disclosure further provides a kit comprising a glucagon analog of the present disclosure, or a pharma- ceutically acceptable salt and / or solvate thereof, or a pharmaceutical composition comprising same, and an administration device.

[0103] In some embodiments, the device includes a syringe and a needle, hi some specific embodiments, the device is a pre-filled syringe.

[0104] In some embodiments, the glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof of the present disclosure, or a pharmaceutical composition comprising same, is contained in a pre-filled syringe.

[0105] Methods for preventing and treating diseases or conditions and pharmaceutical uses The disclosure further provides the use of the glucagon analogue or a pharma- ceutically acceptable salt and / or solvate thereof, and / or the pharmaceutical composition, in the preparation of a medicament for preventing or treating a disease or condition.

[0106] The present disclosure further provides the glucagon analogue or a pharma- ceutically acceptable salt and / or solvate thereof, and / or the pharmaceutical composition as described above, for use in the prophylaxis or treatment of a disease or condition.

[0107] The present disclosure further provides a method for preventing or treating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a glucagon analog of the present disclosure, or a pharma- ceutically acceptable salt and / or solvate thereof, and / or a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0108] In some embodiments, the disease or condition is selected from hypoglycemia, hyperglycemia, type 2 diabetes, type 1 diabetes, coronary heart disease, atherosclerosis, beta-blocker toxicity, insulinoma and von Gierke's disease, and the disease or condition is selected from glucose intolerance, dyslipidemia, hypertension, overweight, hyperphagia, hepatic steatosis and obesity.

[0109] In some embodiments, the hypoglycemia is selected from pathological hypoglycemia or non-pathological hypoglycemia.

[0110] In some embodiments, the hypoglycemia is selected from diabetic hypoglycemia, non-diabetic hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, acute insulin-induced hypoglycemia, gastric bypass-induced hypoglycemia, alcohol-induced hypoglycemia, reactive hypoglycemia, and pregnancy-induced hypoglycemia.

[0111] In some embodiments, a method is provided for rapidly increasing glucose levels to normalize blood glucose levels, stabilize blood glucose levels, or prevent or treat hypoglycemia, comprising administering to a subject in need thereof an effective amount of a glucagon analog of the present disclosure, or a pharma- ceutical composition comprising the same, or a pharma- ceutical salt and / or solvate thereof.

[0112] In some embodiments, a method for reducing weight gain or causing weight loss is provided, which comprises administering to a subject in need thereof an effective amount of a glucagon analog of the present disclosure or a pharma- ceutically acceptable salt and / or solvate thereof, or a pharmaceutical composition comprising the same. The method for reducing weight gain or causing weight loss is expected to treat obesity due to various causes, including drug-induced obesity, and reduce obesity-related complications, including vascular disease (e.g., coronary artery disease, stroke, peripheral vascular disease, ischemia-reperfusion, etc.), hypertension, type 2 diabetes, hyperlipidemia, and musculoskeletal disease. The present disclosure provides pharmaceutical uses of the corresponding glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, or a pharmaceutical composition comprising the same, for use in the preparation of a medicament for preventing, treating, or alleviating obesity.

[0113] In some embodiments, a method for treating hyperglycemia or diabetes is provided, which comprises co-administering insulin with a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof of the present disclosure, or a pharmaceutical composition comprising the same. Co-administration of insulin (e.g., basal, rapid-acting, or long-acting insulin) with a glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof of the present disclosure, or a pharmaceutical composition comprising the same, can reduce nocturnal hypoglycemia and / or alleviate the excessive blood glucose lowering effect of insulin. The present disclosure provides a pharmaceutical use of the corresponding glucagon analog or a pharma- ceutically acceptable salt and / or solvate thereof, or a pharmaceutical composition comprising the same, for use in the preparation of a medicament for preventing, treating, or alleviating hyperglycemia or diabetes.

[0114] In some embodiments, the method is for use in a human subject and the pharmaceutical use is for the preparation of a medicament for a human subject. [Brief description of the drawings]

[0115] [Figure 1] FIG. 1 shows the physical stability of glucagon analogues assessed by the ThT fibrillation assay. [Diagram 2]FIG. 2 shows the efficacy results of glucagon analog 40 and dasiglucagon in a normoglycemic rat model. [Diagram 3] FIG. 3 shows the efficacy results of glucagon analog 40 and dasiglucagon in a hypoglycemic rat model. [Figure 4] FIG. 4A shows the hemolytic activity of Dasiglucagon, and FIG. 4B shows the hemolytic activity of Glucagon analog 40. [Diagram 5] 5A and 5B show the stability results of glucagon analog 40 and Dasiglucagon in human liver microsomes and human kidney microsomes, respectively. [Figure 6] FIG. 6 shows the blood concentration-time curves of glucagon analogue 40 and dasiglucagon after subcutaneous or intravenous administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] definition In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0117] The term "glucagon analog" refers to a compound that has at least some, all, or more of the agonist activity of native glucagon at the glucagon receptor. For example, glucagon analogs include, but are not limited to, polypeptides that contain any amino acid substitutions, additions, or deletions based on the amino acid sequence set forth in SEQ ID NO:51, or that are post-translationally or otherwise chemically modified, provided that the analog has activity in stimulating the glucagon receptor, as measured, for example, by the production of cAMP using the assay described in Example 2.

[0118] In the present disclosure, glucagon analogs also include peptides with modified amino and / or carboxy termini. For example, they include peptides in which the terminal carboxylic acid is replaced with an amide group. For example, glucagon analogs of the present disclosure may introduce an "H-" moiety at the amino end (N-terminus) of the sequence and an "-OH" or "-NH2" moiety at the carboxy end (C-terminus) of the sequence. In this case, unless otherwise specified, the "H-" moiety at the N-terminus of the sequence under consideration represents a hydrogen atom [i.e., in formula (I), R1 = hydrogen = H, corresponding to a free primary or secondary amino at the N-terminus], while the "-OH" or "-NH2" moiety at the C-terminus of the sequence represents a hydroxy group [e.g., in formula (I), R2 = OH, corresponding to a carboxy group (COOH) at the C-terminus] or an amino [e.g., in formula (I), R2 = NH2, corresponding to an amide group (CONH2) at the C-terminus]. In each sequence of the present disclosure, the C-terminal "-OH" moiety may be replaced with a C-terminal "-NH2", and vice versa.

[0119] "Native glucagon" refers to native human glucagon having the following sequence: H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH (SEQ ID NO: 51).

[0120] "Dasiglucagon" refers specifically to a glucagon analog developed by Zealand Pharma and whose amino acid sequence is: H-HSQGTFTSDYSKYLDAibARAEEFVKWLEST-OH (SEQ ID NO:52).

[0121] In this disclosure, unless further interpreted, the term "GLP-1" refers to human GLP-1(7-36 or 7-37) and the term "GIP" refers to human GIP(1-42).

[0122] The three-letter and one-letter codes of amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968). Unless otherwise specified, the configuration of all amino acid residues in this disclosure is L-type. In addition, Aib is 2-aminoisobutyric acid (also called α-aminoisobutyric acid), α-methyl-Ser (α-methylserine) is serine with methyl substituted for H at the α position, and pGlu is pyroglutamic acid.

[0123] "Natural amino acids" are any of the 20 common amino acids (i.e., alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

[0124] "Unnatural amino acids" are amino acids that are not naturally encoded or expressed in the genetic code of any organism. They may be, for example, purely chemically synthesized compounds. Examples of unnatural amino acids are hydroxyproline, γ-carboxyglutamic acid, O-phosphate serine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, N- Examples of "unnatural amino acids" include, but are not limited to, ethylasparagine, pipecolic acid, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine (Nle), ornithine (Orn), D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline. "Unnatural amino acids" also include derivatives obtained by chemically modifying the C-terminal carboxyl group, the N-terminal amino group, and / or the side chain functional groups of natural or unnatural amino acids.

[0125] The term "agonist" is defined as a substance or ligand that activates the receptor type under consideration. For example, the term GLP-1 or GIP or glucagon (receptor) agonist as used in the context of the present disclosure refers to a substance or ligand capable of activating the GLP-1 receptor or the GIP receptor or the glucagon receptor.

[0126] The term "pharmaceutically acceptable salt" refers to a salt that is harmless to the patient or subject being treated. Such salts are generally acid addition salts or base salts.

[0127] The term "solvate" refers to a complex formed by a stoichiometrically defined amount of solute (in this case a glucagon analog of the present disclosure or a pharma- ceutically acceptable salt thereof) and solvent.

[0128] The term "pharmaceutical composition" refers to a mixture of one or more glucagon analogs of the present disclosure, or physiologically / pharmaceutical acceptable salts or prodrugs thereof, with other chemical components, and other components such as physiologically / pharmaceutical acceptable vectors and excipients.

[0129] The term "pharmaceutically acceptable vector" includes any standard pharmaceutically acceptable vector or diluent, such as those used in compositions or formulations suitable for oral, pulmonary, rectal, nasal, topical, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, transdermal or vaginal administration.

[0130] The term "treatment" refers to a method for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of a disease condition, reduction in the extent of a disease, stabilization of a disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of a disease state, and remission (partial or total), whether detectable or not. "Treatment" may also refer to an increase in survival compared to expected survival in the absence of treatment. "Treatment" is an intervention undertaken to prevent the onset of a disease or to alter the pathological state of a disease. Thus, "treatment" refers to both therapeutic treatments and prophylactic or preventative measures. When used in the context of prophylactic or preventative measures, a compound may not completely prevent the onset of a disease or condition. Subjects in need of treatment include those suffering from a disease or those in need of preventing the onset of a disease. "Treatment" also refers to inhibiting or reducing the increase in a pathological condition or symptom (e.g., weight gain or hypoglycemia) compared to the absence of treatment, and does not imply a complete termination of the associated condition.

[0131] The term "effective amount" or "therapeutically effective amount" refers to an amount or dosage sufficient to cure, reduce or partially prevent or otherwise promote a given condition (disorder, disease) or injury, and preferably cure or reverse complications thereof. The amount or dosage effective for a particular purpose will depend on the severity of the disease or condition, and the weight and general condition of the subject or patient to be treated. Determination of the appropriate amount or dosage is within the capabilities of a trained, generally skilled physician (or veterinarian).

[0132] The term "subject" may be used interchangeably with "patient," "individual," "subject," etc., and refers to a human or non-human animal, including, but not limited to, mammals, such as humans, primates, farm animals (e.g., cows, pigs), pets (e.g., dogs, cats), and rodents (e.g., mice and rats). EXAMPLES

[0133] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the standard polypeptide Fmoc solid phase synthesis manual, Cold Spring Harbor antibody technology experimental manual, molecular cloning manual, etc., or follow conditions recommended by raw material or product manufacturers. Reagents for which a specific source is not specified are standard commercially available reagents.

[0134] Main experimental reagents (sources) in this disclosure: Fmoc-Thr(tBu)-Wang resin (TCI Chemicals), HCTU (Haofan Biosciences), Fmoc-Aib-OH (TCI Chemicals), N,N-dimethylformamide (TCI Pharmaceuticals), dichloromethane (TCI Pharmaceuticals), trifluoroacetic acid (TCI Chemicals), triisopropylsilane (TCI Chemicals), acetonitrile (Merck-Millipore), diisopropylethylamine (Sigma), 4-methylpiperidine (TCI Chemicals), methyl tert-butyl ether (TCI Chemicals), 4-methylmorpholine (TCI Chemicals), Fmoc-Nle-OH (TCI Chemicals). DMEM / F12 (Gibco 11330032), Casein, 3-isobutyl-1-methylxanthine (Sigma I7018-250MG), cAMP-Gs Dynamic kit (Cisbio 62AM4PEC, 20000 tests), 384-well plate (Sigma CLS4514-50EA), 96V base plate (PS) (Axygen WIPP02280), 96-well plate (Cisbio 66PL96100), Countess® Cell Counting Chamber Slides (Invitrogen C10228), Cisbio-cAMP-Gs Dynamic Reagent Kit (Cisbio 62AM4PEC), 0.25% Trypsin-EDTA and Phenol Red (ThermoFisher 25200-114), Fetal Bovine Serum (Gibco TM, ThermoFisher 10099-141), and human glucagon (Abmole M9312).

[0135] Main experimental equipment (sources) of this disclosure: H-CLASS analytical ultra-high performance liquid chromatography (WATERS), Agilent 1290-6530 ultra-high performance liquid chromatography-mass spectrometer (Agilent Technologies), Prep-150 preparative high performance liquid chromatography (WATERS), Prelude-X multichannel polypeptide solid phase synthesis instrument (Protein Technologies), microplate reader (BioTek H1MFD, Tecan-Infinite F Plex).

[0136] Example 1. Synthesis of glucagon analogues In this example, the compounds in Table 1 were designed and synthesized by the following method.

[0137] [Table 1-1] [Table 1-2] [Table 1-3]

[0138] 1. Chemical synthesis of compound 1 1.1 Synthesis of the Polypeptide Backbone 0.1 mmol of Fmoc-Thr(tBu)-Wang resin, a solid-phase synthesis vector, was weighed and placed in a disposable polypropylene polypeptide synthesis solid-phase reaction tube, DMF (10 mL) was added to swell the resin for 10 min, DMF was removed by suction under vacuum, and DMF (10 mL) was added to wash the resin, and washing was repeated twice. The synthesis of the polypeptide backbone was achieved by a Prelude-X fully automated polypeptide synthesizer, where the amide bond condensation was performed by using a 10-fold excess of the corresponding Fmoc-protected amino acid, activating it with HCTU / 4-methylmorpholine, and then reacting it at room temperature for 30 min to condense the amino acid. The deprotection of the Fmoc group was performed by using an N,N-dimethylformamide solution containing 20% ​​4-methylpiperidine, and reacting it twice at room temperature for 10 min each time. In addition, the condensation of the N-terminal amino acid next to Aib requires two or three condensations at room temperature, with the condensation time being 30 to 60 min each time, which is very important for improving the purity of the crude peptide.

[0139] 1.2 Cleavage of the resin peptide The resin peptide obtained in the above step was washed three times with DMF and DCM, respectively, and then dried under vacuum. Then, 10 mL of freshly prepared cleavage solution (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was added and reacted at room temperature for 3 hours with shaking. After the reaction was completed, the resin was filtered and washed twice with trifluoroacetic acid. The filtrate was combined and a large amount of frozen methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the supernatant was removed to obtain the crude polypeptide product, which is compound 1.

[0140] 1.3 Purification of crude peptides by reversed-phase liquid chromatography The crude peptide was dissolved in a mixed solvent containing 0.1% (v / v) trifluoroacetic acid and 20% (v / v) acetonitrile / water, filtered through a 0.22 μm membrane, and then separated by a WATERS Prep150 LC reversed-phase high-performance liquid chromatography system, with mobile phases A (0.1% trifluoroacetic acid, 10% acetonitrile, aqueous solution, v / v) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution, v / v). Here, the chromatography column was an X-SELECT OBD C-18 (WATERS) reversed-phase chromatography column, and during purification, the detection wavelength of the chromatograph was set at 220 nm and the flow rate was 20 mL / min. The product-related distillate was collected and lyophilized to obtain a pure polypeptide, compound number 1, with a yield of 20%. The purity and compound identity of the pure polypeptide were confirmed by a combination of analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, where the purity was 96.09% and the molecular weight was correct.

[0141] 2. Chemical synthesis of compounds 2–49 Polypeptide compounds, which are compounds 2 to 49 of the present disclosure, were synthesized according to the experimental design of compound 1, and the purity and molecular weight of the compounds were confirmed by a combination of analytical ultra-high performance liquid chromatography and liquid chromatography / mass spectrometry, where the compounds have the correct molecular weight and the purity as shown in Table 2.

[0142] [Table 2-1] [Table 2-2]

[0143] In the following examples, the glucagon analogues synthesized in Example 1 were subjected to biological tests and evaluations.

[0144] Example 2. Evaluation of the agonist activity of glucagon analogues on the glucagon receptor (GCGR), glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) 1. Purpose of the experiment The agonist activity of the glucagon analogs of the present disclosure against the human glucagon receptor (GCGR), glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) was measured. Activation of such compounds to GCGR stimulates the formation of intracellular cyclic adenosine monophosphate (cAMP). Thus, receptor activity can be monitored by the production of cAMP in suitable cells expressing the receptor.

[0145] 2. Experimental Method Measurement of GCGR agonist activity: The frozen CHO-K1 / GCGR / CRE-Luc stable transfected cell line was taken out from the liquid nitrogen container, quickly thawed in a water bath pot at 37°C, resuspended in DMEM / F12 medium (Sigma Cat#D8437), centrifuged, and then seeded into a T75 culture flask. The cells were passaged at least once before being used for the next test. Before the test, cells were digested with pancreatin, centrifuged, washed once, resuspended in serum-free DMEM / F12 to adjust the cell density, and seeded in a 96-well plate at a density of 2,500 cells / 5 μL / well. 1× buffer in the Cisbio-cAMP-Gs Dynamic Reagent Kit was prepared, IBMX was added to a final concentration of 0.5 mM, and 5 μL of the polypeptide gradient diluted with the buffer was added to each well. The mixture was gently shaken to mix uniformly, and incubated at 37°C for 30 minutes. cAMP-d2 and Anti-cAMP-Eu were added. 3+ -Cryptate was diluted 20-fold with cAMP Lysis & Detection buffer and mixed uniformly. 5 μL of diluted cAMP-d2 solution was added to each well. 3+5 μL of -Cryptate solution was added, gently shaken to mix uniformly, and incubated at room temperature in the dark for 1 hour.

[0146] Detection of GLP-1R agonist activity: This method differs from the above-mentioned detection method of GCGR agonist activity in that a CHO-GLP-1R stably transformed cell line was used.

[0147] Detection of GIPR agonist activity: The method differs from the above detection method of GCGR agonist activity in that a CHO-K1 / GIPR stably transformed cell line was used and the cell density was 3,000 cells / 5 μL / well.

[0148] 3. Experimental Results The HTRF signal was read using a Tecan-Infinite F Plex microplate reader with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm / 620 nm × 10,000) was calculated and the EC was calculated by nonlinear fitting of the signal ratio and the sample concentration with a four-variable equation. 50 The values ​​obtained are shown in Tables 3 and 4.

[0149] [Table 3]

[0150] [Table 4]

[0151] According to the results in Table 3, the glucagon analogs of the present disclosure have significantly better agonist activity against human GCGR than Dasiglucagon. According to the results in Table 4, compared with native glucagon, the glucagon analogs of the present disclosure have very low and negligible agonist activity against GLP-1R and GIPR, i.e., the glucagon analogs of the present disclosure have good specificity for GCGR.

[0152] Example 3. Evaluation of the solubility of glucagon analogues in physiological buffers 1. Purpose of the experiment Native glucagon has a solubility much lower than 1 mg / mL at neutral pH 7.4, limiting its direct use as a therapeutic agent. The purpose of this example is to evaluate the solubility of the glucagon analogs of the present disclosure in buffers at neutral pH, and hopefully have a higher solubility than native glucagon.

[0153] 2. Experimental Method Saturated solutions of some of the disclosed glucagon analogs were prepared in phosphate buffer at pH 7.4, sonicated for 5 min at room temperature, then centrifuged (10000 g, 5 min) and the supernatant was aspirated to ensure no precipitation was observed. The absorbance of the solutions at 280 nm (Abs λ=280 nm ) was measured and the molar extinction coefficient at 280 nm (ε = 8250 M) of the polypeptide was determined based on the tryptophan and tyrosine content in the glucagon analog sequence. -1 cm -1 ) was calculated and the maximum solubility in neutral pH buffer for each test glucagon analogue was calculated according to Beer's law.

[0154] 3. Experimental Results The various glucagon analogs of the present disclosure have significantly improved solubility compared to native glucagon, as shown in Table 5. Comparing compounds 2 to 5, it was found that compound 3, which contains Aib at position 17, has improved solubility compared to compound 4, which contains Aib at position 18, and compound 3, which contains Aib at position 17, has improved solubility compared to compounds 2 and 4, which contain α-methyl-Ser at positions 16 and 19. It can be seen that the inclusion of Aib at position 17 in glucagon analogs plays an important role in improving the solubility of the polypeptide.

[0155] [Table 5]

[0156] Example 4. Assessment of the physical and chemical stability of glucagon analogues Native glucagon has very low physical and chemical stability in solution. Aqueous solutions of native glucagon form gels or fibrous precipitates within hours to days, and become even more susceptible to gelation when high polypeptide concentrations, disturbances, and salts are present. The chemical instability of native glucagon is mainly due to isomerization (aspartic acid residues), deamidation (asparagine and glutamine residues), and oxidation (methionine residues) of its sequence.

[0157] 1. Purpose of the experiment The purpose of this example is to evaluate the physical and chemical stability in solution of the glucagon analogs of the present disclosure.

[0158] 2. Physical stability evaluation method In this experiment, the formation of fibrils was monitored by Thioflavin-T (ThT) fluorescence assay. The lyophilized glucagon analog was dissolved in 0.1 N HCl aqueous solution at a concentration of 1 mg / mL. The polypeptide solution was incubated in a thermostatic shaker for 120 h (37°C, 300 rpm), and the fibril content in the solution was detected at 24 h, 48 h, and 120 h, respectively. For the ThT fluorescence assay, 8 mg of ThT powder was accurately weighed and dissolved in 50 mL of PBS buffer (1×), filtered through a 0.22 μm filter membrane, and then stored at 4°C in the dark for use. When used, the working solution was prepared by diluting with PBS buffer (1x) at a ratio of 1:500. 100 μL of the working solution and 5 μL of the polypeptide solution to be measured were added to each well of a 96-well plate, mixed uniformly, and incubated at room temperature for 20-30 min. The ThT fluorescence signal was read using a Tecan-Infinite F Plex microplate reader, with an excitation wavelength of 450 nm and an emission wavelength of 482 nm. Each polypeptide test included four biological replicates.

[0159] 3. Chemical stability evaluation method Lyophilized powders of glucagon analogs were dissolved in phosphate buffer (10 mM sodium dihydrogen phosphate, 15 mg / mL propylene glycol, 0.01% Tween-80, pH 7.4) to a final concentration of 1 mg / mL (determined by measuring the absorbance of the solution at 280 nm), and the samples were incubated in glass bottles at 4°C and 25°C for 30 days. After sampling on days 0, 7, 14, 21, and 28, the purity and degradation products of the samples were detected by ultra-performance liquid chromatography, which utilized a C-18 reversed-phase chromatography column with UV=214 nm. The purity of the compound was defined as the area percentage of the main peak relative to the total area of ​​all integrated peaks in each chromatogram, which was normalized for the purity of a given polypeptide at different times based on the purity of the sample on day 0.

[0160] 4. Experimental Results Through the above experimental methods, some of the glucagon analogs of the present disclosure have physical stability in aqueous solution as shown in FIG. 1, and chemical stability in phosphate buffer as shown in Table 6.

[0161] [Table 6]

[0162] Studies have demonstrated that the glucagon analogs of the present disclosure exhibit superior physical and chemical stability compared to native glucagon, and among them, compound 40 has superior chemical stability in phosphate buffer to Dasiglucagon.

[0163] Example 5. Evaluation of the efficacy of glucagon analogues in a euglycemic rat model 1. Purpose of the experiment This example is to comparatively evaluate the in vivo hyperglycemic efficacy of glucagon analog 40, native glucagon, and dasiglucagon in a normal rat model according to the present disclosure.

[0164] 2. Experimental Method Male Sprague-Dawley rats aged 7-8 weeks were adapted for one week. They were divided into groups according to body weight, and the mean body weight and standard deviation of the rats in each group were ensured to be similar after grouping. They were fasted for 4 hours before and during the experiment, and water was administered as usual. After fasting for 4 hours, the rats in the experimental group were subcutaneously administered a single dose of 0.5 nmol / kg, 2 nmol / kg, or 6 nmol / kg glucagon analog 40, or 2 nmol / kg native glucagon, or 2 nmol / kg Dasiglucagon, respectively, and the rats in the control group were subcutaneously administered a phosphate buffer solution prepared in the same manner as the glucagon analog. Blood was collected from the tail before administration to measure blood glucose at T0, and then blood glucose was measured at 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min, respectively.

[0165] 3. Experimental Results The increase in blood glucose level in rats by glucagon analogue 40 was dose-dependent. Although both natural glucagon and dasiglucagon have the characteristic of producing their effects in a short period of time, glucagon analogue 40 at the same dose increased blood glucose level more significantly. The specific results are shown in Figure 2 and Table 7.

[0166] [Table 7]

[0167] 4. Conclusion of the experiment The results show that after subcutaneous injection, glucagon analogue 40, natural glucagon and dasiglucagon can all rapidly increase the blood glucose level in rats. The effect of glucagon analogue 40 at a dose of 0.5 nmol / kg and dasiglucagon at a dose of 2 nmol / kg on increasing blood glucose is similar, and at the same dose (2 nmol / kg), glucagon analogue 40 has a significantly better efficacy than dasiglucagon.

[0168] In a normoglycemic rat model, glucagon analog 40 of the present disclosure elevates blood glucose levels to a higher level and has better efficacy than Dasiglucagon under the same dosage conditions.

[0169] Example 6. Evaluation of the efficacy of glucagon analogues in a hypoglycemic rat model Rats were induced to suffer from hypoglycemia by subcutaneous injection of insulin to simulate the condition that occurs in humans, and were then treated with a subcutaneous glucagon analogue to rapidly restore blood glucose to normal levels.

[0170] 1. Purpose of the experiment The efficacy of glucagon analog 40 of the present disclosure was evaluated in vivo in a hypoglycemic rat model in comparison to Dasiglucagon.

[0171] 2. Experimental Method Male SD rats (Sprague-Dawley rats) aged 7 to 8 weeks were adapted and reared for one week. They were divided into groups according to blood glucose levels, and after grouping, the mean blood glucose and standard deviation of the rats in each group were ensured to be similar. They were fasted for 4 hours before and during the experiment, and water was administered as usual. After fasting for 4 hours, blood was collected from the tail to measure blood glucose at T0 point, and the rats in the model construction group were immediately subcutaneously injected with 0.65 IU / kg insulin, and the rats in the non-model construction group were subcutaneously injected with the same volume of physiological saline. Blood glucose was then measured at 15 min, 30 min, and 45 min. At 45 min, the rats in the model construction group were subcutaneously administered a single dose of 6 nmol / kg, 12 nmol / kg, or 20 nmol / kg of glucagon analog 40 or 12 nmol / kg of dasiglucagon, respectively, while the rats in the control group and the rats in the non-model construction group were subcutaneously administered a phosphate buffer solution prepared in the same manner as glucagon analog 40. Then, the blood glucose of the rats was measured at 60 min, 75 min, 90 min, 105 min, 120 min, 150 min, and 180 min.

[0172] 3. Experimental Results After insulin treatment, the rats were treated with glucagon analog 40 of the present disclosure at the blood glucose nadir, which can rapidly restore the blood glucose of the rats and shows dose-dependency. Both glucagon analog 40 of the present disclosure and dasiglucagon have the characteristic of producing effects in a short time, but at the same dose, glucagon analog 40 of the present disclosure can increase blood glucose levels to a higher extent. The specific results are shown in Figure 3 and Table 8.

[0173] [Table 8]

[0174] 4. Conclusion of the experiment The results show that in insulin-induced hypoglycemic rat model, after subcutaneous administration, glucagon analogue 40, native glucagon and dasiglucagon can all rapidly restore and increase the blood glucose level of rats within 15 minutes. At the same dose (12 nmol / kg), glucagon analogue 40 has a significantly better and longer-lasting blood glucose increasing effect than dasiglucagon.

[0175] In an insulin-induced hypoglycemic rat model, glucagon analog 40 was more effective than Dasiglucagon under the same dose conditions.

[0176] Example 7. Hemolytic Experimental Evaluation of Glucagon Analogues Hemolysis means that the red blood cell membrane is destroyed, the transparency is increased, and the color is deep red. Some drug ingredients and additives contain hemolytic ingredients, which can cause hemolysis in the human body and cause adverse reactions such as local swelling and blood circulation disorders. Based on the principle that hemoglobin released by the destruction of red blood cells has absorption in the visible light wavelength band, the test compound solution was added to the rat red blood cell suspension, incubated, and the absorbance value was measured with a microplate reader to evaluate the degree of hemolysis.

[0177] 1. Purpose of the experiment It was observed whether glucagon analog 40 induces hemolysis.

[0178] 2. Hemolysis test method Preparation of red blood cell suspension: 100 μL of fresh rat whole blood was collected, then 900 μL of 1× PBS solution was added, placed on a plate shaker, shaken at 30 rpm for 5 min, then centrifuged at 1000 g for 5 min, the supernatant was discarded, and the above washing steps were repeated until the supernatant no longer showed red color, and the blood was prepared for testing.

[0179] Preparation of test sample solutions: Add an appropriate amount of 1x PBS solution to the solid content of the test sample, dissolve it to obtain a standard stock solution, and then dilute it with 1x PBS to obtain test sample solutions with concentrations of 1 μg / mL, 3 μg / mL, 10 μg / mL, 30 μg / mL, 100 μg / mL and 300 μg / mL. At the same time, blank 1x PBS was used as a negative control, and 1x PBS solution containing 0.1% Triton X-100 (1 μg / mL) was used as a positive control.

[0180] Incubation process: 500 μL of the test solution was added to the red blood cell suspension, and the suspension was shaken at 30 rpm for 5 min on a plate shaker to ensure thorough and uniform mixing. The suspension was then incubated at 37°C for 1 h, and then centrifuged at 1000 g for 5 min. 100 μL of the supernatant was taken and placed into one well of a microplate to measure the absorbance at 540 nm.

[0181] 3. Experimental Results The formula for calculating the hemolysis rate (%) is (test sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) x 100%. If the result was less than 5%, it was determined that there was no hemolysis, and if the result was more than 5%, the sample waiting to be measured was determined to have hemolysis.

[0182] The results of the hemolytic assay of the glucagon analogs of the present disclosure are shown in Figures 4A, 4B and Table 9. The results show that the glucagon analog 40 and Dasiglucagon of the present disclosure both meet the prescribed requirements in the hemolytic assay at a concentration range of 1-300 g / mL, and no significant risk of hemolytic side effects is observed.

[0183] [Table 9-1] [Table 9-2]

[0184] Example 8. Evaluation of human liver and kidney microsomal stability of glucagon analogues The liver and kidney are the most important metabolic organs in the human body, rich in phase 1 and phase 2 metabolic enzymes, which affect the metabolic rate of drugs, play an important role in the degradation and clearance of drugs, and affect the blood concentration and half-life. Commonly used models for hepatic and renal metabolism include hepatic and renal microsomes, S9 components, hepatocytes, tissue homogenates, etc. In vitro hepatic and renal metabolic stability is one of the main indicators for predicting in vivo pharmacokinetic and pharmacodynamic properties. As a hepatic and renal microsome model, microsomes extracted from the liver or kidney are adopted, and a reduced coenzyme (NADPH) regeneration system is added to simulate metabolic reactions in a physiological environment, and the in vitro metabolic stability of test compounds is evaluated, which can be used to predict the in vivo degradation and clearance rate of drugs. Under ideal conditions, glucagon analogs require rapid absorption and clearance, have a relatively fast degradation rate in the body, and hypoglycemia is restored while hyperglycemia toxicity and side effects are avoided.

[0185] 1. Purpose of the experiment The metabolic stability of glucagon analogues was comparatively evaluated in human liver and kidney microsomal in vitro metabolic systems.

[0186] 2. Experimental methods for detecting human liver microsomes and kidney microsomes Preparation of solutions: Pure compounds were weighed in appropriate amounts, and dissolved in appropriate amounts of DMSO to obtain 1 mM test solutions of Dasiglucagon and 40, respectively. Preparation of MgCl2-PBS buffer solution: 200 μL of 200 mM PBS buffer was taken, 106 μL of H2O and 40 μL of MgCl2 solution (50 mM) were added in order, and mixed uniformly by vortexing to obtain MgCl2-PBS buffer solution.

[0187] Stability Experimental Procedure: Samples are treated as follows: (1) Preparation of incubation system a) Add NADPH: 10 μL of 20 mg / mL liver microsomes and 40 μL of 10 mM NADPH solution were taken and added to the incubation system, so that the final concentrations of liver microsomes and NADPH were 0.5 mg / mL and 1 mM, respectively. b) No NADPH added: 10 μL of 20 mg / mL liver microsomes and 40 μL of H2O were added to the incubation system.

[0188] (2) 4 μL of 100 μM test compound solution and positive control compound (Verapamil was used for human liver microsomes, and benzydamine hydrochloride was used for human kidney microsomes) were added to the incubation reaction system, and the final concentration of the incubation system was 1 μM and the incubation temperature was 37°C, where Verapamil and benzydamine hydrochloride are known substrates of hepatic and renal metabolic enzymes, respectively (see Pauli-Magnus C et al., J Pharmacol Exp Ther. 2000 May;293(2):376-82.), in order to confirm the enzyme reaction activity of the microsome incubation system.

[0189] (3) 50 μL samples were taken at 0, 15, 30, 60, and 120 min, respectively, and the reaction was stopped by adding 4 volumes of acetonitrile solution containing an internal standard and 3% formic acid. The samples were further centrifuged at 3220 g for 40 min, and 100 μL of the supernatant was taken and mixed with an equal volume of H2O for HPLC-MS / MS analysis.

[0190] 3. Test Results: The stability results of the glucagon analogs of the present disclosure in human liver and kidney microsomes are shown in Figures 5A and 5B and Tables 10 and 11.

[0191] [Table 10]

[0192] [Table 11]

[0193] 4. Conclusion of the experiment First, Dasiglucagon and glucagon analog 40 are metabolized slower in liver microsomes than in kidney microsomes, and the metabolism is independent of NADPH. Second, glucagon analog 40 is degraded slightly faster than Dasiglucagon (in vitro T1 / 2: 68.76 min vs. 161.74 min for Dasiglucagon).

[0194] Regarding renal metabolism, first, Dasiglucagon and glucagon analog 40 are metabolized in renal microsomes, but the metabolism is independent of NADPH, and second, glucagon analog 40 is degraded more quickly than Dasiglucagon (in vitro T1 / 2: 35.73 min vs. 51.27 min for Dasiglucagon).

[0195] In any case, during the liver-kidney microsome incubation process, the glucagon analog 40 in the present disclosure has a faster metabolic rate than Dasiglucagon, and is therefore more easily cleared in the body, with a lower risk of hyperglycemic side effects, which is more beneficial for glycemic control in diabetic patients.

[0196] Example 9. Pharmacokinetic evaluation of glucagon analogues in rats The general method of animal pharmacokinetic research is to administer a fixed dose of a test compound to an animal via a clinically proposed administration route, and then use analytical instruments such as HPLC, GC, and HPLC-MS to measure the change in drug concentration over time in biological matrices such as blood and urine, thereby understanding the dynamic change law of the compound in the body, determining the blood concentration-time curve, and obtaining the pharmacokinetic parameters of the drug to elucidate the processes and characteristics of the absorption, distribution, metabolism, and excretion of the compound.

[0197] 1. Purpose of the experiment Male SD rats were used as test animals, and Dasiglucagon and Glucagon Analogue 40 were administered by a single subcutaneous injection to study the pharmacokinetic characteristics of Dasiglucagon and Glucagon Analogue 40 in SD rats (plasma).

[0198] 2. Experimental Method (1) The animals were divided into groups as shown in Table 12, and the rats were allowed to freely access water and food before administration.

[0199] [Table 12]

[0200] (2) Blood collection Sampling times were 0 min (predose), 2 min, 4 min, 7 min, 10 min, 20 min, 40 min, 1 h, and 2 h after administration. Blood was collected by inserting a cannula into the jugular vein of the rats.

[0201] (3) Collection of samples 1) At each time point, 0.2 mL of whole blood was drawn and transferred to a centrifuge tube containing EDTA-K2 anticoagulant. 2) The blood samples were centrifuged at 4000 g for 5 min at 4° C. to obtain plasma. 3) Plasma samples were frozen and stored at -80°C.

[0202] (4) Biological sample analysis Sample pretreatment method: 100 μL of plasma sample was taken, 300 μL of methanol solution containing 1 ng / mL of internal standard (verapamil) was added, vortexed for 5 minutes, centrifuged at low temperature for 10 minutes, 100 μL of the supernatant was taken, 100 μL of 0.1% formic acid solution was added, mixed uniformly, and analyzed by the instrument. Liquid chromatography and mass spectrometry: LC-30A liquid chromatography system (Shimadzu, Japan) and API 5500 type liquid chromatography-triple quadrupole tandem mass spectrometer (Analyst Software data processing system, AB, USA) were used. The chromatography column was a nanocro Unisil C18aq (4.6 mm × 150 mm, 5 μm) chromatography column, the column temperature was 40 ° C, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution, the flow rate was 0.5 mL / min, the injection volume was 20 μL, and the gradient conditions are as shown in Table 13 below. Mass spectrometry employed an electrospray ion source (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning mode.

[0203] [Table 13]

[0204] 3. Experimental Results The blood concentration of each target substance in plasma was measured by the above HPLC-MS / MS method, the blood concentration-time curve was drawn, and the pharmacokinetic parameters were calculated by PKSolver software.By the above experimental method, the pharmacokinetic (PK) parameters of the glucagon analogues of the present disclosure are as shown in Table 14 and Figure 6.

[0205] [Table 14]

[0206] 4. Conclusion of the experiment The results showed that after subcutaneous administration, glucagon analog 40 had a significantly shorter T1 / 2 than dasiglucagon (T 1 / 2 is 0.41 h, and T of Dasiglucagon 1 / 2 The T was 0.51 h, indicating that glucagon analog 40 had a faster clearance rate. maxis also shorter than Dasiglucagon (40 T max is 0.117 h, and T of Dasiglucagon max The absorption rate of glucagon analogue 40 was significantly faster than that of dasiglucagon (0.167 h).

[0207] Regardless, compared to Dasiglucagon, Glucagon analog 40 has superior pharmacokinetic properties, being able to be absorbed and produce effects more quickly and being cleared more rapidly.

Claims

1. A glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof, represented by the structure of formula (I), wherein among them, R １ is hydrogen, C １－４ is an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group or pGlu, R ２ is -OH or -NH ２ and X ３ , X １５ , X １６ , X ２０ , X ２１ , X ２４ , X ２７ and X ２８ are each independently selected from any natural amino acid residue or unnatural amino acid residue, and X １７ is Aib, A glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof, represented by the structure of formula (I).

2. X ３ is selected from His, Dap(Ac) or Gln, X １５ is selected from Asp or Glu, X １６ is selected from Ser, Thr, Leu, Val, Ile or α-methyl-Ser, X ２０ is selected from Ala, Gln, Glu, Ser, Thr or Lys, X ２１ is selected from Asp or Glu, X ２４ is selected from Ala, Gln, Ser, Glu, α-methyl-Ser or Arg, X ２７ is selected from Met, Glu, Nle, Leu or Ser, X ２８ is selected from Asn, Glu or Ser, The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to Claim 1.

3. X １５ is Asp, X １６ is selected from Thr, Leu, Val or Ile, The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to Claim 1.

4. X ２０ The glucagon analogue or a pharmaceutically acceptable salt and / or solvate thereof according to claim 1, wherein X is Gln.

5. X ２４ The glucagon analog according to claim 1, or a pharmaceutically acceptable salt and / or solvate thereof, wherein X is selected from Gln or Glu.

6. X ２７ is Glu, and X ２８ is Ser, the glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to claim 1.

7. X ３ is selected from His or Gln, X １５ is Asp, X １６ is selected from Thr, Leu, Val or Ile, X １７ is Aib, X ２０ is Gln, X ２１ is Glu, X ２４ is selected from Gln or Glu, X ２７ is Glu, X ２８ is Ser, The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to Claim 1.

8. R １ is hydrogen, and R ２ is -OH or -NH ２ and -OH is preferred. The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to Claim 7.

9. The glucagon analog is H-HSQGTFTSDYSKYLDLAibRAQEFFVQWLEST-OH (SEQ ID NO: 40), H-HSQGTFTSDYSKYLDT AibRAQEFFVQWLEST-OH (SEQ ID NO: 39), H-HSQGTFTSDYSKYLDLVAibRAQEFFVQWLEST-OH (SEQ ID NO: 41), H-HSQGTFTSDYSKYLDI AibRAQEFFVQWLEST-OH (SEQ ID NO: 42), H-HSHGTFTSDYSKYLDL AibRAQEFFVQWLEST-OH (SEQ ID NO: 43), H-HSHGTFTSDYSKYLDL AibRAQEFFEWLEST-OH (SEQ ID NO: 47), The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to Claim 1, which is any one compound selected from the above.

10. The glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to any one of Claims 1 to 9, and One or more pharmaceutically acceptable vectors, diluents, buffers or excipients, and A pharmaceutical composition comprising the same.

11. The pharmaceutical composition according to Claim 10, further comprising at least one compound having metabolic disease treatment activity.

12. The compound having the metabolic disease treatment activity is one or more selected from glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) receptor agonist, insulin, entero-glucagon, neuropeptide Y5 receptor antagonist, acetyl coenzyme A carboxylase inhibitor, Leptin receptor agonist, glinide, α-glucosidase inhibitor (AGi), thiazolidinedione (TZD), dipeptidyl peptidase-4 inhibitors (DPP-IV), sodium glucose co-transporter 2 inhibitor (SGLT-2), farnesol X receptor (FXR) agonist and obestatin, the pharmaceutical composition according to claim 11.

13. A pharmaceutical composition for treating a disease or medical condition, comprising a glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to any one of claims 1 to 9, Preferably, the disease or medical condition is selected from hypoglycemia, hyperglycemia, type 2 diabetes, type 1 diabetes, coronary heart disease, atherosclerosis, β-blocker poisoning, insulinoma, von Gierke's disease, impaired glucose tolerance, dyslipidemia, hypertension, overweight, bulimia and hepatic steatosis. Pharmaceutical composition.

14. The pharmaceutical composition according to claim 13, wherein the hypoglycemia is pathological hypoglycemia or non-pathological hypoglycemia.

15. The pharmaceutical composition according to claim 13, wherein the hypoglycemia is selected from diabetic hypoglycemia, non-diabetic hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, acute insulin-induced hypoglycemia, gastric bypass-induced hypoglycemia, alcohol-induced hypoglycemia, reactive hypoglycemia and pregnancy hypoglycemia.

16. A pharmaceutical composition according to claim 11 for treating a disease or medical condition, Preferably, the disease or medical condition is selected from hypoglycemia, hyperglycemia, type 2 diabetes, type 1 diabetes, coronary heart disease, atherosclerosis, β-blocker poisoning, insulinoma, von Gierke's disease, impaired glucose tolerance, dyslipidemia, hypertension, overweight, bulimia and hepatic steatosis. Pharmaceutical composition.

17. The pharmaceutical composition according to claim 16, wherein the hypoglycemia is pathological hypoglycemia or non-pathological hypoglycemia.

18. The hypoglycemia is selected from diabetic hypoglycemia, non-diabetic hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, acute insulin-induced hypoglycemia, gastric bypass-induced hypoglycemia, alcohol-induced hypoglycemia, reactive hypoglycemia, and pregnancy hypoglycemia. The pharmaceutical composition according to Claim 16.

19. The pharmaceutical composition according to Claim 12 for treating a disease or medical condition, preferably, the disease or medical condition is selected from hypoglycemia, hyperglycemia, type 2 diabetes, type 1 diabetes, coronary heart disease, atherosclerosis, beta-blocker poisoning, insulinoma, von Gierke's disease, impaired glucose tolerance, dyslipidemia, hypertension, overweight, bulimia, and hepatic steatosis. Pharmaceutical composition.

20. The hypoglycemia is pathological hypoglycemia or non-pathological hypoglycemia. The pharmaceutical composition according to Claim 19.

21. The hypoglycemia is selected from diabetic hypoglycemia, non-diabetic hypoglycemia, fasting hypoglycemia, drug-induced hypoglycemia, acute insulin-induced hypoglycemia, gastric bypass-induced hypoglycemia, alcohol-induced hypoglycemia, reactive hypoglycemia, and pregnancy hypoglycemia. The pharmaceutical composition according to Claim 19.

22. A method for preparing a glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof according to any one of Claims 1 to 9, the method comprising preparing the glucagon analog or a pharmaceutically acceptable salt and / or solvate thereof by a method of solid-phase synthesis, liquid-phase synthesis, or cell recombinant expression.

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