WO2022262837A1 - 胰高血糖素类似物及其医药用途 - Google Patents

胰高血糖素类似物及其医药用途 Download PDF

Info

Publication number
WO2022262837A1
WO2022262837A1 PCT/CN2022/099357 CN2022099357W WO2022262837A1 WO 2022262837 A1 WO2022262837 A1 WO 2022262837A1 CN 2022099357 W CN2022099357 W CN 2022099357W WO 2022262837 A1 WO2022262837 A1 WO 2022262837A1
Authority
WO
WIPO (PCT)
Prior art keywords
glucagon
glu
ser
gln
pharmaceutically acceptable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/099357
Other languages
English (en)
French (fr)
Inventor
刘伟兵
黄旭超
张晓谦
吴方舟
王雷
曲亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Tuo Jie Biopharmaceutical Co Ltd
Original Assignee
Beijing Tuo Jie Biopharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Tuo Jie Biopharmaceutical Co Ltd filed Critical Beijing Tuo Jie Biopharmaceutical Co Ltd
Priority to KR1020247000856A priority Critical patent/KR20240022551A/ko
Priority to EP22824309.3A priority patent/EP4357358A4/en
Priority to CA3221829A priority patent/CA3221829A1/en
Priority to CN202280032659.8A priority patent/CN117242087A/zh
Priority to MX2023015068A priority patent/MX2023015068A/es
Priority to JP2023576365A priority patent/JP2024525144A/ja
Priority to US18/570,551 priority patent/US20240279298A1/en
Publication of WO2022262837A1 publication Critical patent/WO2022262837A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to glucagon analogs and their medical uses.
  • the glucagon analogues of the present disclosure can be used to treat metabolic diseases such as hypoglycemia, obesity, diabetes, or alleviate symptoms.
  • Controlling the balance of blood sugar in the body is extremely important for the body's metabolism. Under normal circumstances, the blood sugar level of the human body maintains a dynamic balance under the regulation of a variety of different polypeptide hormones (mainly insulin and glucagon). Blood sugar imbalances can lead to complications. Hypoglycemia occurs when blood sugar levels are lower than normal. The pancreas in the islets produces glucagon, which increases blood sugar levels to the normal range. Non-severe hypoglycemia usually produces autonomic or neurological symptoms of hypoglycemia, and the patient can recognize the onset of hypoglycemia, so there is no need for additional treatment.
  • polypeptide hormones mainly insulin and glucagon
  • the glucagon in the body is not enough to restore blood sugar, and even life-threatening, usually manifested as impaired cognitive or physical function, limited self-healing ability , so need the help of others to treat to restore normal blood sugar.
  • oral sugar food is the first choice for fully awake hypoglycemic patients; for those with impaired consciousness, they need to receive intravenous injection of 50% glucose solution or intramuscular injection of 0.5% glucose with the help of non-medical or medical personnel. to 2 mg glucagon therapy. It is difficult for non-medical personnel to carry out intravenous injection of 50% glucose solution. If the injection dose is too high, it will cause rebound hyperglycemia, and if the glucose solution leaks out of the blood vessel, it will cause local tissue damage. Therefore, in a nonhospital setting, the optimal management strategy for severely hypoglycemic patients with impaired consciousness is glucagon by subcutaneous or intramuscular injection. Glucagon has also been used clinically as an emergency drug for severe hypoglycemia; and based on the lipid-lowering and energy-promoting effects of glucagon, its dual receptor agonist with incretin also makes obese diabetic patients benefit further.
  • Glucagon is a linear polypeptide composed of 29 amino acids secreted by pancreatic ⁇ -cells, which controls the production of glucose and ketone bodies in the liver.
  • Glucagon secreted at night and between meals, increases glucose efflux from the liver by increasing glucose production from amino acid precursors (gluconeogenesis) and promoting the breakdown of glycogen to glucose (glycolysis), leading to increased hepatic glucose efflux, thereby enhancing blood sugar concentration.
  • Glucagon works with insulin secreted by the beta cells of the pancreas to precisely control the balance of blood sugar in the body. In addition to its therapeutic effect on hypoglycemia, glucagon also suppresses appetite and activates hormone-sensitive lipase in adipocytes to promote lipolysis, thereby exhibiting an anti-obesity effect.
  • Glucagon is an important part of the defense mechanism against hypoglycemia, and low doses of glucagon prevent insulin-induced hypoglycemia and improve the ability to recover from hypoglycemia. Low doses of glucagon can also cause satiety, suppress appetite, and at the same time activate hormone-sensitive lipase in adipocytes to promote lipolysis, thus having potential anti-obesity effects and can be used to treat many patients with type 2 diabetes overweight or obese.
  • native glucagon has extremely low solubility in aqueous solutions at neutral pH.
  • glucagon contains amino acids or amino acid sequences that can produce amino acid residue side chain deamidation, oxidation, and formation of cyclic imine intermediates, as well as amino acids or amino acid sequences that can undergo isomerization, peptide chain cleavage, etc.
  • the sequence cannot be kept stable for a long time, and gel or fibril will be formed within a few hours to a few days, and the chemical and physical stability is extremely poor. Therefore, it is necessary to develop new glucagon analogues with excellent solubility and good physical/chemical stability to meet the requirements of injection formulation development.
  • glucagon injectable preparations such as the Glucagon Emergency Kit ( Eli Lilly), Glucagon Kit ( Novo Nordisk A/S), due to the unsatisfactory pharmaceutical properties of glucagon, it is stored in the form of freeze-dried powder, and it needs to be re-dissolved with a solvent before subcutaneous/muscular injection, which is inconvenient for patients and non-medical care Caregivers use.
  • Second-generation glucagon injection preparations such as human glucagon nasal spray powder ( Eli Lilly), prefilled human glucagon injection (GVOKE XERIS) has made targeted improvements to the preparation, which not only ensures the therapeutic effect, but also significantly improves the convenience of operation for patients and non-medical staff. but, Administered by nasal spray, the onset of effect is slow, and three times the dose is required compared with intramuscular injection, and there are adverse reactions. GVOKE With DMSO as the preparation, although it can be directly injected subcutaneously, there are obvious discomfort at the injection site and other side effects.
  • Dasiglucagon developed by New Zealand Pharma
  • Dasiglucagon has modified 7 amino acid sites on the basis of natural human glucagon, improving solubility and physical/chemical stability without affecting activity. It can be injected directly under the skin in the form of an injection solution. Dasiglucagon has recently been approved by the US Food and Drug Administration.
  • Glucagon analogues with high activity, excellent solubility, and good physical/chemical stability are still urgently needed in the art.
  • the present disclosure provides a class of glucagon with high activity (increased activity compared to native glucagon), high solubility in aqueous solution (especially at physiological pH) and improved stability (including physical stability and chemical stability).
  • glucagon analogues Compared with glucagon analogues in the art, the glucagon analogues disclosed in the present disclosure have stronger in vitro glucagon receptor agonist activity, which can reduce the volume and frequency of subcutaneous administration and improve low blood pressure. The convenience of use for blood sugar patients and the reduction of financial burden on patients are of great help.
  • the glucagon analogues of the present disclosure can be used alone or in combination with other therapeutic drugs in the treatment of metabolic diseases or disorders such as hypoglycemia, obesity, and diabetes.
  • the present disclosure provides a glucagon analog or a pharmaceutically acceptable salt and/or solvate thereof, a pharmaceutical composition comprising the same, a polynucleotide encoding the glucagon analog, and a vector containing the polynucleotide .
  • the glucagon analogue or its pharmaceutically acceptable salt and/or solvate or the pharmaceutical composition comprising it is used for treating disease, alleviating disease (such as hypoglycemia, obesity, diabetes and other metabolic diseases or diseases) ) methods and related pharmaceutical uses.
  • the present disclosure provides a glucagon analogue or a pharmaceutically acceptable salt and/or solvate thereof as shown in formula (I):
  • the present disclosure also provides a glucagon analogue comprising a structure represented by formula (I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein R 1 and/or R 2 do not exist;
  • R is hydrogen, C 1-4 alkyl, acetyl, formyl , benzoyl, trifluoroacetyl or pGlu;
  • R 2 is -OH or -NH 2 ;
  • X 3 , X 15 , X 16 , X 20 , X 21 , X 24 , X 27 and X 28 are independently selected from any natural amino acid residue or unnatural amino acid residue;
  • X 17 is Aib.
  • 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 ⁇ -methyl-Ser;
  • X 17 is Aib;
  • 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, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle , Leu or Ser;
  • X is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is Aib ;
  • X 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, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X is selected from Asn , Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 is Aib;
  • X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser ;
  • X 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 is Aib;
  • 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 Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser; 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 is Aib;
  • 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, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • X 3 is selected from His, Dap(Ac) 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 selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is Aib ;
  • X is selected from Ala, Gln,
  • Glu X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is Aib ;
  • X 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, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 is Aib;
  • 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 Gln or Glu;
  • X 27 is Glu;
  • X 28 is Ser.
  • X 3 is selected from His, Dap(Ac) 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 selected from Asp or Glu; X 24 is selected from Gln or Glu; X 27 is Glu; X 28 is Ser.
  • 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.
  • R1 is hydrogen; R2 is -OH or -NH2 . In some embodiments, R1 is hydrogen ; R2 is -OH.
  • the pharmaceutically acceptable salts and/or solvates of the present disclosure are selected from: inorganic salts or organic salts.
  • glucagon analogs of the present disclosure can react with acidic or basic compounds to form corresponding salts.
  • reaction with an acidic compound to form a corresponding salt refers to a salt formed with an inorganic acid or an organic acid that can retain the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include but not limited to: hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include but not limited to: formate, acetate, 2,2-dichloroethane trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, Adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oil salt, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate,
  • “Reacting with a basic compound to form a corresponding salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Salts derived from organic bases include, but are not limited to: primary amines, secondary amines, tertiary amines, substituted amines, cyclic amines; for example ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, Ethylenediamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, Lucaine, choline, betaine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • solvate means a complex of a glucagon analog of the present disclosure, or a pharmaceutically acceptable salt thereof, with a suitable solvent.
  • suitable solvents include: water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.
  • the solvate is a hydrate.
  • the present disclosure provides a glucagon analog or a pharmaceutically acceptable salt and/or solvate thereof, wherein the glucagon analog is selected from any of SEQ ID NO: 1-49 compound of.
  • the present disclosure provides a glucagon analog or a pharmaceutically acceptable salt and/or solvate thereof, the glucagon analog being a compound selected from the group consisting of:
  • the present disclosure provides a glucagon analog or a pharmaceutically acceptable salt and/or solvate thereof, said glucagon analog comprising a compound selected from the group consisting of SEQ ID NO: 39, 40, 41, The structure of any one of 42, 43, 47, wherein said SEQ ID NO: 39, 40, 41, 42, 43, 47 N-terminal H and/or C-terminal OH does not exist.
  • the present disclosure provides a glucagon analogue or a pharmaceutically acceptable salt and/or solvate thereof as shown in formula (I):
  • the present disclosure also provides a glucagon analogue comprising a structure represented by formula (I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein R 1 and/or R 2 do not exist;
  • R is hydrogen, C 1-4 alkyl, acetyl, formyl , benzoyl, trifluoroacetyl or pGlu;
  • R 2 is -OH or -NH 2 ;
  • X 3 , X 17 , X 20 , X 21 , X 24 , X 27 and X 28 are independently selected from any natural amino acid residue or unnatural amino acid residue;
  • X 15 is Asp;
  • X 16 is selected from Ser, Thr, Leu, Val, He or ⁇ -methyl-Ser, for example selected from Thr, Leu, Val or He.
  • X 3 is selected from His, Dap(Ac) or Gln;
  • X 15 is Asp;
  • X 16 is selected from Ser, Thr, Leu, Val, Ile or ⁇ -methyl-Ser;
  • X 17 is selected from Aib or Ala;
  • X 20 is not Glu;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser ;
  • X 28 is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is selected from Aib or Ala;
  • 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is Aib ;
  • X 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, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X is selected from Asn , Glu or Ser.
  • X 3 is selected from His, Dap(Ac) 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 selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X 3 is selected from His, Dap(Ac) or Gln;
  • X 15 is Asp;
  • X 16 is selected from Thr, Leu, Val or Ile;
  • X 17 is Aib;
  • X 20 is not Glu;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X 3 is selected from His, Dap(Ac) or Gln;
  • X 15 is Asp;
  • X 16 is selected from Thr, Leu, Val or Ile;
  • X 17 is Aib;
  • X 20 is not Glu;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • X 3 is selected from His, Dap(Ac) or Gln; X 15 is Asp; X 16 is selected from Thr, Leu, Val or Ile; X 17 is Aib; X 20 is not Glu; X 21 is selected from Asp or Glu; X 24 is selected from Gln or Glu; X 27 is Glu; X 28 is Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is Aib ;
  • X is selected from Ala, Gln, Glu , Ser, Thr or Lys;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is Glu;
  • X 28 is Ser.
  • X 3 is selected from His, Dap(Ac) 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 selected from Asp or Glu; X 24 is selected from Gln or Glu; X 27 is Glu; X 28 is Ser.
  • 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.
  • R1 is hydrogen; R2 is -OH or -NH2 . In some embodiments, R1 is hydrogen ; R2 is -OH.
  • the present disclosure provides a glucagon analog or a pharmaceutically acceptable salt and/or solvate thereof, the glucagon analog comprising a structure as shown in formula (I):
  • the present disclosure also provides a glucagon analogue comprising a structure represented by formula (I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein R 1 and/or R 2 do not exist;
  • R is hydrogen, C 1-4 alkyl, acetyl, formyl , benzoyl, trifluoroacetyl or pGlu;
  • R 2 is -OH or -NH 2 ;
  • X 3 , X 15 , X 16 , X 17 , X 21 , X 24 , X 27 and X 28 are independently selected from any natural amino acid residue or unnatural amino acid residue;
  • X 20 is Gln.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser .
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is selected from Aib or Ala;
  • 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is selected from Aib or Ala;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is selected from Aib or Ala;
  • 21 is selected from Asp or Glu;
  • X 24 is selected from Ala, Gln, Ser, Glu, ⁇ -methyl-Ser or Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Gln;
  • X 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is Glu;
  • X 28 is Ser.
  • X is selected from His, Dap(Ac) or Gln ;
  • X is Asp;
  • X is selected from Thr , Leu, Val or Ile;
  • X is selected from Aib or Ala;
  • 21 is selected from Asp or Glu;
  • X 24 is selected from Gln or Glu;
  • X 27 is Glu;
  • X 28 is Ser.
  • 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.
  • R1 is hydrogen; R2 is -OH or -NH2 . In some embodiments, R1 is hydrogen ; R2 is -OH.
  • the present disclosure provides a glucagon analogue with a structure represented by formula (I) or a pharmaceutically acceptable salt and/or solvate thereof:
  • the present disclosure also provides a glucagon analogue comprising a structure represented by formula (I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein R 1 and/or R 2 do not exist;
  • R is hydrogen, C 1-4 alkyl, acetyl, formyl , benzoyl or trifluoroacetyl or pGlu;
  • R 2 is -OH or -NH 2 ;
  • X 3 , X 15 , X 16 , X 17 , X 21 , X 27 and X 28 are independently selected from any natural amino acid residue or unnatural amino acid residue;
  • X 20 is Glu, and
  • X 24 is Arg.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Glu;
  • X 21 is selected from Asp or Glu;
  • X 24 is Arg;
  • X 27 is selected from Met, Glu, Nle, Leu or Ser;
  • X 28 is selected from Asn, Glu or Ser.
  • X 3 is selected from His, Dap(Ac) or Gln; X 15 is Asp; X 16 is selected from Thr, Leu, Val or Ile; X 17 is Aib; X 20 is Glu; X 21 is selected from Asp or Glu; X 24 is Arg; X 27 is selected from Met, Glu, Nle, Leu or Ser; X 28 is selected from Asn, Glu or Ser.
  • 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 ⁇ -methyl-Ser;
  • X 17 X 20 is Glu;
  • X 21 is selected from Asp or Glu;
  • X 24 is Arg;
  • X 27 is Glu;
  • X 28 is Ser.
  • X 3 is selected from His, Dap(Ac) or Gln; X 15 is Asp; X 16 is selected from Thr, Leu, Val or Ile; X 17 is Aib; X 20 is Glu; X 21 is selected from Asp or Glu; X 24 is Arg; X 27 is Glu; X 28 is Ser.
  • 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.
  • R1 is hydrogen; R2 is -OH or -NH2 . In some embodiments, R1 is hydrogen ; R2 is -OH.
  • Glucagon analogs of the present disclosure have glucagon receptor (GCGR) agonist activity, and their binding to GCGR can be used as an indicator of agonist activity.
  • intracellular signal transduction resulting from the binding of a compound to a receptor may also be measured.
  • activation of GCGR by glucagon receptor agonists will stimulate the formation of cellular cyclic adenosine monophosphate (cAMP).
  • cAMP production in appropriate receptor-expressing cells can be used to monitor receptor activity.
  • Example 2 the materials given in Example 2 (such as CHO-K1/GCGR/CRE-Luc stable cell line and Cisbio-cAMP-Gs Dynamic kit) can be used and assayed under the conditions.
  • the EC50 value can be used as a numerical measure of agonist potency against GCGR.
  • the EC50 value is the concentration of the compound required to achieve half-maximal activity of the compound at the receptor in question in a particular assay.
  • the glucagon receptor is a mammalian glucagon receptor, such as a human glucagon receptor.
  • the glucagon analogs of the present disclosure or pharmaceutically acceptable salts and/or solvates thereof have agonist activity on human GCGR that is at least 1.5 times, at least 1.6 times, at least 1.7 times that of native glucagon. times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, At least 3.0 times, or at least 3.1 times.
  • a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof, has no or negligible agonist activity on human GLP-1R and/or GIPR.
  • a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof, has increased solubility and/or stability compared to native glucagon.
  • increased solubility can include or refer to: at pH 4 (e.g., in acetate buffer at pH 4), at pH 5 (e.g., in acetate buffer at pH 5), pH 6 (e.g., in a phosphate buffer of pH 6), pH 7 (e.g., in a phosphate buffer of pH 7), and/or pH 7.5 (e.g., in a phosphate buffer of pH 7.5), Has improved solubility compared to native glucagon.
  • the measurement can be performed under the conditions given in Example 5. Solubility > 1 mg/mL may be expected.
  • the solubility of the glucagon analogs of the present disclosure in phosphate buffered saline solution at pH 7.4 is ⁇ 1.00 mg/mL, ⁇ 1.10mg/mL, ⁇ 1.20mg/mL, ⁇ 1.30mg/mL, ⁇ 1.40mg/mL, ⁇ 1.50mg/mL, ⁇ 1.60mg/mL, ⁇ 1.70mg/mL, ⁇ 1.80mg/mL, ⁇ 1.90mg /mL, ⁇ 2.00mg/mL, ⁇ 2.10mg/mL, ⁇ 2.20mg/mL, ⁇ 2.30mg/mL, ⁇ 2.40mg/mL, ⁇ 2.50mg/mL, ⁇ 2.60mg/mL, ⁇ 2.70mg/mL , ⁇ 2.80mg/mL, ⁇
  • increased stability may include or refer to: increased physical stability and/or increased chemical stability compared to native human glucagon.
  • increased physical stability can include or refer to: a reduced tendency to aggregate (eg, form soluble or insoluble aggregates (eg, fibrils)).
  • Glucagon analogs can be dissolved in 0.1 N HCl and assayed for aggregation (eg, fibril formation) at a concentration of 1 mg/mL at 37°C. Any suitable period of time may be used, eg, 24 hours, 48 hours, 96 hours or 120 hours.
  • Aggregation can be determined using a ThT fluorescence assay. Aggregation can be determined under the conditions given in Example 4.
  • increased chemical stability may include or refer to a reduced tendency of the peptide to be cleaved or degraded in aqueous buffers (typically in the absence of contaminating protease or peptidase activity).
  • Stability can be measured at 4°C, 25°C or 40°C by dissolving the glucagon analog in, for example, pH 7.4 phosphate buffer at a concentration of 1 mg/mL.
  • Evaluation can include determining stability after incubation for a suitable period of time, eg, 1 day, 7 days, 14 days, 21 days, or 28 days. This may include determining compound purity as defined in Example 4, ie the area percentage of the main peak in each chromatogram relative to the total area of all integrated peaks. Stability can be determined under the conditions given in Example 4.
  • the present disclosure provides polynucleotides encoding glucagon analog precursor peptides 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 substantially isolated polynucleotides.
  • the precursor peptide is a peptide that has been modified to yield the glucagon analogs of the present disclosure.
  • X 17 of the precursor peptide is Arg or other natural amino acids, and after modification, X 17 is Aib to obtain the glucagon analog of the present disclosure.
  • a polynucleotide of the present disclosure may also be in the form of, may be present in, and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, ie a vector providing for expression of the glucagon analogue in vitro and/or in vivo, ie in a suitable host cell, host organism and/or expression system.
  • the expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). It is common knowledge of those skilled in the art to select said elements and their sequences for expression in a particular host.
  • the vector comprises the following elements in the 5' ⁇ 3' direction and operably linked: a promoter for driving the expression of the nucleic acid fragment, coding for secretion (secretion to the extracellular phase or appropriate secreted into the periplasm (periplasma), an optional polynucleotide encoding a leader peptide, a polynucleotide encoding a precursor peptide, and an optional polynucleotide encoding a terminator.
  • polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on the information on the amino acid sequence of the glucagon analog precursor peptide of the present disclosure.
  • the present disclosure provides recombinant host cells expressing a glucagon analog precursor peptide of the present disclosure or containing a polynucleotide or vector of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Fungal cells include, for example, cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen A cell of a species of Saccharomyces Hansenula
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure can also be used with amphibian cells, insect cells, plant cells, and any other cell used in the art to express heterologous proteins.
  • the cells of the present disclosure do not develop into finished plants or individual animals.
  • the present disclosure provides methods of preparing glucagon analogs of the present disclosure, including, for example, the following methods:
  • a step of purifying the precursor peptide is included.
  • polypeptides such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods of inducing protein expression, culture conditions, etc. are known in the art.
  • polypeptide isolation and purification techniques suitable for use in methods of making precursor peptides are well known to those skilled in the art.
  • Methods of solid-phase or liquid-phase synthesis are known in the art, for example, reference may be made to WO98/11125, especially Fields, GB et al., 2002, "Principles and practice of solid-phase peptide synthesis". In: Synthetic Peptides (Second Edition), and Examples in this Disclosure.
  • glucagon analogue to a pharmaceutically acceptable salt and/or solvate thereof are also provided.
  • a glucagon analog of the present disclosure can be reacted with a pharmaceutically acceptable acidic or basic compound to form a salt.
  • the glucagon analogs of the present disclosure can be prepared by recombinant expression of microorganisms, or can be prepared by Fmoc solid-phase synthesis (for example, SPPS method, including amino acid deprotection method, from resin A method for cutting peptides and a method for purifying them).
  • Fmoc solid-phase synthesis for example, SPPS method, including amino acid deprotection method, from resin A method for cutting peptides and a method for purifying them).
  • the synthetic carrier is Fmoc-L-Thr(tBu)-Wang resin (Gill Biochemical, loading capacity: 0.533mmol/g).
  • amino acid derivatives used during synthesis include: 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-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-L-
  • the synthesis process includes: after washing the solid-phase synthesis support, first remove the solid-phase synthesis support ⁇ - The Fmoc protecting group on the amino group, then the solid phase support and the next amino acid derivative in the sequence are condensed to form an amide bond after activation with HCTU/4-methylmorpholine in excess to extend the peptide chain; repeat condensation ⁇ washing ⁇ Deprotection ⁇ Washing ⁇ The next round of amino acid condensation operation to achieve the length of the polypeptide chain to be synthesized, and finally use a mixture of trifluoroacetic acid: water: triisopropylsilane (90:5:5, v:v:v) The solution is reacted with the resin to crack the polypeptide from the solid phase carrier, and then the solid crude product of the polypeptide derivative is obtained after precipitation by freezing methyl tert-butyl ether; the solid crude product of the polypeptide is prepared from a mixed solution of acetonitrile/water containing 0.1% trifluor
  • the present disclosure provides a pharmaceutical composition, which contains an effective amount of the glucagon analog or pharmaceutically acceptable salt and/or solvate of the present disclosure for prevention, treatment or alleviation, and one or more pharmaceutically acceptable carriers, Diluents, buffers or excipients.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the glucagon analog or pharmaceutically acceptable salt and/or solvate of the present disclosure. In some other embodiments, the amount of the glucagon analog or the pharmaceutically acceptable salt and/or solvate of the present disclosure in the unit dosage of the pharmaceutical composition is 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.
  • the pharmaceutical composition further includes at least one compound or substance having metabolic disease therapeutic activity.
  • the compound or substance having therapeutic activity for metabolic diseases is selected from one or more of the following: glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP- 1) Receptor agonists, insulin, glucagon, neuropeptide Y5 receptor antagonists, acetyl-CoA carboxylase inhibitors, Leptin receptor agonists, glinide, ⁇ -glucosidase inhibitors (AGi ), thiazolidinedione (TZD), dipeptidyl peptidase-4 inhibitors (DPP-IV), sodium glucose cotransporter 2 inhibitor (SGLT-2), farnesoid X receptor (FXR) agonist Drugs and Obestatin.
  • GIP glucose-dependent insulinotropic peptide
  • GLP-1 glucagon-like peptide-1
  • Receptor agonists Receptor agonists
  • insulin glucagon
  • neuropeptide Y5 receptor antagonists acetyl-CoA carboxylase inhibitors
  • compositions of the present disclosure can be administered parenterally, with a syringe (optionally a pen-type syringe), by subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • parenteral administration can be performed with an infusion pump.
  • the composition may be administered as a solution or suspension of the glucagon peptide in the form of a nasal or pulmonary spray; alternatively, it may be administered transdermally, such as by needle-free injection or via a patch, An iontophoretic patch is optional; alternatively, it may be transmucosal (eg, buccal) administration.
  • Kit (or kit)
  • the present disclosure also provides a kit comprising the glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof or a pharmaceutical composition comprising the same and an administration device.
  • the device comprises a syringe and a needle. In some embodiments, the device is a prefilled needle.
  • a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof, or a pharmaceutical composition comprising the same is in a prefilled syringe.
  • the present disclosure also provides the use of the above-mentioned glucagon analog or its pharmaceutically acceptable salt and/or solvate, and/or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing or treating a disease or a disease.
  • the present disclosure also provides the above-mentioned glucagon analogs or pharmaceutically acceptable salts and/or solvates thereof, and/or the above-mentioned pharmaceutical compositions, which are used for preventing and treating diseases or conditions.
  • the present disclosure also provides a method for preventing, treating a disease or a disorder, comprising administering a therapeutically effective amount of a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof to a subject in need thereof, and/or A therapeutically effective amount of a pharmaceutical composition of the present disclosure is administered.
  • the disease or condition is selected from the group consisting of hypoglycemia, hyperglycemia, type 2 diabetes, type 1 diabetes, coronary heart disease, atherosclerosis, beta-blocker poisoning, insulinoma, and von Gil disease; the disease or disease is selected from impaired glucose tolerance, dyslipidemia, hypertension, overweight, binge eating disorder, hepatic steatosis, obesity.
  • the hypoglycemia is selected from pathological hypoglycemia or non-pathological hypoglycemia.
  • 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 hypoglycemia, reactive hypoglycemia, and hypoglycemia during pregnancy.
  • a method of rapidly increasing glucose levels, normalizing blood glucose levels, stabilizing blood glucose levels, or preventing or treating hypoglycemia comprising administering to a subject in need thereof an effective amount of a hyperglycemic agent of the present disclosure.
  • a glucagon analogue or a pharmaceutically acceptable salt and/or solvate thereof or a pharmaceutical composition comprising the same.
  • a method of reducing weight gain or causing weight loss comprising administering to a subject in need thereof an effective amount of a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvent thereof compounds or pharmaceutical compositions containing them.
  • Methods of reducing weight gain or causing weight loss are expected to be useful in the treatment of obesity of various causes, including drug-induced obesity, and in reducing obesity-related complications, including vascular disease (e.g., coronary artery disease, stroke, peripheral vascular disease, blood reperfusion, etc.), hypertension, type II diabetes, hyperlipidemia and skeletal muscle disease.
  • the present disclosure provides the pharmaceutical use of the corresponding glucagon analogue or its pharmaceutically acceptable salt and/or solvate or a pharmaceutical composition containing it for preparing a medicine for preventing, treating and alleviating obesity.
  • a method of treating hyperglycemia or diabetes comprising administering insulin in combination with a glucagon analog of the present disclosure or a pharmaceutically acceptable salt and/or solvate thereof or a medicament comprising the same combination.
  • the present disclosure provides corresponding glucagon analogues or their pharmaceutically acceptable salts and/or solvates or pharmaceutical compositions containing them for the preparation of pharmaceuticals for preventing, treating, and alleviating hyperglycemia or diabetes.
  • the above-mentioned method is for a human subject
  • the above-mentioned pharmaceutical use is for the preparation of a medicament for a human subject.
  • Figure 1 shows the results of evaluating the physical stability of glucagon analogs by ThT fibril formation assay.
  • Fig. 2 is the pharmacodynamic results of glucagon analogue 40 and Dasiglucagon in a normoglycemic rat model.
  • Fig. 3 is the pharmacodynamic results of glucagon analogue 40 and Dasiglucagon in the hypoglycemia rat model.
  • Figure 4A is the hemolytic test results of Dasiglucagon
  • Figure 4B is the hemolytic test results of glucagon analog 40.
  • Figure 5A and Figure 5B are the stability results of glucagon analog 40 and Dasiglucagon in human liver microsomes and human kidney microsomes, respectively.
  • Figure 6 shows the results of the drug-time curves of glucagon analog 40 and Dasiglucagon after subcutaneous or intravenous injection.
  • glucagon analog refers to a compound having at least a portion, all, or greater agonist activity of native glucagon at the glucagon receptor.
  • glucagon analogs include, but are not limited to: polypeptides containing any amino acid substitutions, additions or deletions, or post-translational modifications or other chemical modifications based on the amino acid sequence shown in SEQ ID NO: 51, provided that Analogs have glucagon receptor stimulating activity, as determined, for example, by cAMP production using the assay described in Example 2.
  • glucagon analogs also include peptides with modified amino and/or carboxy termini.
  • peptides comprising a terminal carboxylic acid substituted with an amide group.
  • the glucagon analogs of the present disclosure may incorporate an "H-" moiety at the amino terminus (N-terminus) of the sequence and an "-OH” moiety or " -NH2 " at the carboxyl terminus (C-terminus) of the sequence. "part.
  • the C-terminal "-OH" moiety can be replaced with C-terminal "-NH 2 ", and vice versa.
  • Native glucagon refers to native human glucagon having the following sequence:
  • Depicglucagon specifically refers to a glucagon analog developed by Zealand Pharma, and its amino acid sequence is: H-HSQGTFTSDYSKYLDAibARAEEFVKWLEST-OH (SEQ ID NO: 52).
  • GLP-1 refers to human GLP-1 (7-36 or 7-37); the term “GIP” refers to human GIP (1-42).
  • Aib is 2-aminoisobutyric acid (also known as ⁇ -aminoisobutyric acid), ⁇ -methyl-Ser ( ⁇ -methylserine) is a serine whose ⁇ -position H is replaced by a methyl group, and pGlu is pyroglutamine acid.
  • Natural amino acids refer to the 20 conventional 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), Pro amino acid (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine ( Y).
  • Unnatural amino acid refers to an amino acid that is not naturally encoded or found in the genetic code of any organism. They can be, for example, purely chemically synthesized compounds. Examples of unnatural amino acids include, but are not limited to: hydroxyproline, ⁇ -carboxyglutamic acid, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta -Alanine, Alanine, 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
  • agonist is defined as a substance or ligand that activates the type of receptor in question.
  • GLP-1 or GIP or glucagon (receptor) agonist as used in the context of this disclosure refers to a substance or ligand that can activate the GLP-1 receptor or the GIP receptor or the glucagon receptor.
  • pharmaceutically acceptable salt refers to a salt that is not deleterious to the patient or subject to be treated. Such salts are generally acid addition or base salts.
  • solvate refers to a complex formed by a defined stoichiometry of a solute (in this case a glucagon analog of the present disclosure or a pharmaceutically acceptable salt thereof) and a solvent.
  • composition means a mixture containing one or more glucagon analogs of the present disclosure or physiologically/pharmaceutically acceptable salts or prodrugs thereof and other chemical components, as well as other components such as physiological / Pharmaceutically acceptable carrier and excipient.
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier or diluent, for example, for oral, pulmonary, rectal, nasal, topical, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, transdermal or Those used in compositions or formulations for intravaginal administration.
  • treatment refers to a method used to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, lessening of disease extent, stabilization (i.e., not worsening) of disease state, delay or slowing of disease progression, amelioration or palliation of disease state, and (partial or Totally) remission, whether detectable or not.
  • Treatment can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • Treatment is an intervention for the purpose of preventing the occurrence of a disease or changing the pathological condition of a disease.
  • treatment refers to both therapeutic treatment and prophylactic or preventive measures.
  • a compound does not necessarily completely prevent a disease or condition from occurring.
  • Those in need of treatment include those already with the disease or those in which the disease is to be prevented.
  • Treating also means inhibiting or reducing an increase in a pathological condition or symptom (eg, weight gain or hypoglycemia) as compared to the absence of treatment, and does not imply complete cessation of the associated condition.
  • an effective amount or “therapeutically effective amount” means sufficient to cure, alleviate, or partially arrest, or otherwise promote the healing or rehabilitation of a given condition (disorder, disease) or injury and preferably complications resulting therefrom amount or dosage. Amounts or dosages 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 an appropriate amount or dosage is within the skill of a trained physician (or veterinarian) of ordinary skill.
  • subject is 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, livestock animals (eg, cows, pigs), pets (eg, dogs, cats), and rodents (eg, mice and rats).
  • mammals such as humans, primates, livestock animals (eg, cows, pigs), pets (eg, dogs, cats), and rodents (eg, mice and rats).
  • the main experimental reagents (sources) of the present disclosure Fmoc-Thr (tBu)-Wang resin (Gill Biochemical), HCTU (Haofan Biology), Fmoc-Aib-OH (Gill Biochemical), nitrogen, nitrogen-dimethylformamide ( Sinopharm), dichloromethane (Sinopharm), trifluoroacetic acid (TCI Chemicals), triisopropylsilane (TCI Chemicals), acetonitrile (Merck-Millipore), diisopropylethylamine (Sigma), 4-methylpiperene Pyridine (TCI Chemicals), methyl tert-butyl ether (TCI Chemicals), 4-methylmorpholine (TCI Chemicals), Fmoc-Nle-OH (Gill Biochemical).
  • 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-type base plate (PS) (Axygen WIPP02280), 96-well plate (Cisbio 66PL96100), Cell Counting Chamber Slides (Invitrogen C10228), Cisbio-cAMP-Gs Dynamic kit (Cisbio 62AM4PEC), 0.25% trypsin-EDTA and Phenol Red (ThermoFisher 25200-114), fetal bovine serum (GibcoTM, ThermoFisher 10099-141), Human glucagon (Abmole M9312).
  • the main experimental instrument (source) 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 multi-channel peptide solid-phase synthesizer (Protein Technologies), microplate reader (BioTek H1MFD; Tecan-Infinite F Plex).
  • the deprotection of the Fmoc group was carried out using nitrogen, nitrogen-dimethylformamide solution containing 20% 4-methylpiperidine for 2 reactions at room temperature, 10 minutes each time.
  • nitrogen, nitrogen-dimethylformamide solution containing 20% 4-methylpiperidine for the condensation of the N-terminal amino acid next to Aib, two or even three condensations are required at room temperature, and the condensation time is 30-60 minutes each time, which is very important to improve the purity of the crude peptide.
  • the crude peptide was dissolved in a mixed solvent containing 0.1% (v/v) trifluoroacetic acid, 20% (v/v) acetonitrile/water, and filtered through a 0.22 ⁇ m membrane with a WATERS Prep150 LC reversed-phase high-performance liquid chromatography system. Separated, the mobile phase is A (0.1% trifluoroacetic acid, 10% acetonitrile, aqueous solution, v/v) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution, v/v).
  • the chromatographic column is an X-SELECT OBD C-18 (WATERS) reversed-phase chromatographic column.
  • the detection wavelength of the chromatograph is set at 220nm, and the flow rate is 20mL/min.
  • the product-related fractions were collected and freeze-dried to obtain the pure polypeptide of Compound No. 1, with a yield of 20%.
  • the purity and compound identity of the pure peptide were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry, wherein the purity was 96.09%, and the molecular weight was correct.
  • the polypeptide compound of compound 2-49 of the present disclosure was synthesized using the experimental scheme of compound 1, and the purity and molecular weight of the compound were determined by analytical ultra-high performance liquid chromatography and liquid chromatography/mass spectrometry. The molecular weight of the compound is correct, and the purity is shown in Table 2.
  • GCGR glucagon receptor
  • GLP-1R glucagon-like peptide-1 receptor
  • GIPR glucose-dependent insulinotropic polypeptide receptor
  • Glucagon Analogues of the Disclosure for Human Glucagon Receptor (GCGR), Glucagon-Like Peptide-1 Receptor (GLP-1R) and Glucose-Dependent Insulin-Stimulating Polypeptide Receptor (GIPR) agonist activity Activation of GCGR by related compounds will stimulate the formation of cellular cyclic adenosine monophosphate (cAMP). Accordingly, cAMP production in appropriate receptor-expressing cells can be used to monitor receptor activity.
  • cAMP cyclic adenosine monophosphate
  • Detection of agonist activity on GCGR Take out the frozen CHO-K1/GCGR/CRE-Luc stably transfected cell line from the liquid nitrogen tank, put it in a 37°C water bath to thaw quickly, and use DMEM/F12 medium (Sigma Cat #D8437) was resuspended, centrifuged, and the cells were inoculated into T75 culture flasks, and the cells were passaged at least once before being used for subsequent tests.
  • DMEM/F12 medium Sigma Cat #D8437
  • Detection of agonist activity on GLP-1R The difference from the above method for detection of agonist activity on GCGR is that a CHO-GLP-1R stably transfected cell line is used.
  • Detection of agonist activity on GIPR The difference from the detection method of agonist activity on GCGR above is that a CHO-K1/GIPR stably transfected cell line is used, and the cell density is 3,000 cells/5 ⁇ L/well.
  • Tecan-Infinite F Plex microplate reader to read the signal of HTRF, the excitation wavelength is 320nm, and the emission wavelength is 620nm and 665nm.
  • the signal ratio (665nm/620nm*10,000) was calculated, and it was fitted nonlinearly with the sample concentration using a four-parameter equation to obtain the EC 50 value, see Table 3 and Table 4.
  • the results in Table 3 show that the agonist activity of the glucagon analogs of the present disclosure on human GCGR is significantly better than that of Dasiglucagon.
  • the results in Table 4 show that, compared with natural glucagon, the glucagon analogs of the present disclosure have extremely low agonist activity on GLP-1R and GIPR, which can be ignored, that is, the glucagon analogs of the present disclosure have GCGR has good specificity.
  • native glucagon as a direct therapeutic agent is limited because its solubility at neutral pH 7.4 is well below 1 mg/mL.
  • the purpose of this example was to evaluate the solubility of glucagon analogs of the present disclosure in neutral pH buffers, with higher solubility expected compared to native glucagon.
  • glucagon analogs of the present disclosure have significantly improved solubility compared to native glucagon, see Table 5. Comparing compounds 2 to 5, it was found that compound 3 containing Aib at position 17 had improved solubility compared to compound 4 containing Aib at position 18; Compound 2 and Compound 4 based on Ser-Ser have enhanced solubility. It can be seen that the inclusion of Aib at the 17th position of the glucagon analog plays an important role in improving the solubility of the polypeptide.
  • Native glucagon has very poor physical and chemical stability in solution.
  • the natural glucagon aqueous solution will form a gel or fibrous precipitate within hours to days, and it is easier to gel under the conditions of high peptide concentration, external disturbance and salt content.
  • the chemical instability of natural glucagon is mainly due to its sequence isomerization (aspartic acid residues), deamidation (asparagine, glutamine residues) and oxidation (methionine residues). acid residues) and so on.
  • the purpose of this example was to evaluate the physical and chemical stability of the disclosed glucagon analogs in solution.
  • ThT fluorescence assay fibril formation was monitored by Thioflavin-T (ThT) fluorescence assay.
  • the lyophilized glucagon analog was dissolved in 0.1N HCl aqueous solution at a concentration of 1 mg/mL.
  • the polypeptide solution was incubated on a constant temperature shaker for 120h (37°C, 300rmp), and the fibril content of the solution was detected at 24h, 48h and 120h, respectively.
  • ThT fluorescence test accurately weigh 8 mg of ThT powder and dissolve it in 50 mL of PBS buffer (1 ⁇ ), filter through a 0.22 ⁇ m filter membrane and store in the dark at 4°C for later use. When in use, dilute 1:500 with PBS buffer (1 ⁇ ) to prepare a working solution.
  • glucagon glucagon analogs of the present disclosure exhibit excellent physical and chemical stability; wherein the chemical stability of compound 40 in phosphate buffer is higher than Dasiglucagon is superior.
  • Example 5 Drug efficacy evaluation of glucagon analogues in normoglycemic rat models
  • This example is to compare and evaluate the blood sugar-raising efficacy of the glucagon analog 40 in the present disclosure, natural glucagon (Glucagon) and Dasiglucagon in a normal rat model.
  • mice Male SD rats (Sprague-Dawley rats) aged 7-8 weeks were fed adaptively for one week. Group according to body weight, ensure that the average body weight and standard deviation of rats in each group are similar after grouping. 4 hours before the experiment and during the experiment fasting, normal water supply. After fasting for 4 hours, rats in the experimental group were subcutaneously given 0.5nmol/kg, 2nmol/kg, 6nmol/kg of glucagon analog 40 or 2nmol/kg of natural glucagon or 2nmol/kg Dasiglucagon, the rats in the control group were subcutaneously administered the same phosphate buffer as the glucagon analog. Before the administration, the tail blood was collected to measure the blood glucose at T0 point, and then the blood glucose of the rats were measured at 15min, 30min, 45min, 60min, 75min, 90min, 105min, and 120min respectively.
  • Glucagon analogue 40 elevates the blood glucose level of rats in a dose-dependent manner, and both natural glucagon and Dasiglucagon have a short-term onset of effect, but the glucagon analogue 40 at the same dose has a higher blood glucose level. High, the specific results are shown in Figure 2 and Table 7.
  • glucagon analog 40 after administration by subcutaneous injection, glucagon analog 40, natural glucagon (Glucagon) and Dasiglucagon could all rapidly increase the blood glucose level in rats.
  • Glucagon analog 40 at a dose of 0.5nmol/kg and Dasiglucagon at a dose of 2nmol/kg have similar effects on raising blood sugar, and at the same dose (2nmol/kg), the drug effect of glucagon analog 40 is significantly better than Dasiglucagon.
  • the glucagon analog 40 in the present disclosure has a higher glucose level and better drug efficacy under the same dosage condition.
  • Example 6 Drug efficacy evaluation of glucagon analogs in hypoglycemia rat model
  • hypoglycemia symptoms were induced in rats by subcutaneous injection of insulin, simulating the hypoglycemia scene in humans, and then treated with glucagon analogues subcutaneously, so that blood sugar quickly returned to normal levels.
  • glucagon analog 40 of the present disclosure was evaluated in vivo in a hypoglycemic rat model compared to Dasiglucagon.
  • mice Male SD rats (Sprague-Dawley rats) aged 7-8 weeks were fed adaptively for one week. According to blood glucose grouping, after grouping, ensure that the average blood glucose and standard deviation of rats in each group are similar. 4 hours before the experiment and during the experiment fasting, normal water supply. After fasting for 4 hours, blood was collected from the tail to measure blood glucose at point T0. Rats in the model group were injected subcutaneously with 0.65 IU/kg insulin immediately, and rats in the non-model group were injected subcutaneously with the same volume of normal saline. Blood glucose was then measured at 15min, 30min, and 45min.
  • the rats in the model group were subcutaneously given 6 nmol/kg, 12 nmol/kg, and 20 nmol/kg of glucagon analogs 40 or 12 nmol/kg of Dasiglucagon, respectively.
  • the mice were subcutaneously administered the same phosphate-buffered saline as in the preparation of glucagon analog 40. Then continue to measure blood glucose in rats at 60min, 75min, 90min, 105min, 120min, 150min and 180min.
  • glucagon analog 40 in the rat model of insulin-induced hypoglycemia, glucagon analog 40, natural glucagon (Glucagon) and Dasiglucagon could rapidly recover and increase the blood glucose level within 15 minutes after subcutaneous injection.
  • glucagon analogue 40 At the same dose (12nmol/kg), glucagon analogue 40 has a significantly better effect on raising blood sugar than Dasiglucagon, and the duration is longer.
  • the glucagon analogue 40 has better efficacy than Dasiglucagon.
  • Hemolysis refers to the phenomenon that the red blood cell membrane is destroyed, and the transparency increases and turns dark red; some pharmaceutical ingredients and excipients contain hemolytic ingredients, which can cause hemolytic reactions in the human body, causing local swelling, blood circulation dysfunction and other adverse reactions.
  • the solution of the test compound was added to the red blood cell suspension of rats, and after incubation, the absorbance value was measured with a microplate reader to evaluate the degree of hemolysis.
  • Preparation of erythrocyte suspension Take 100 ⁇ L fresh rat whole blood, then add 900 ⁇ L 1 ⁇ PBS solution, place on a plate shaker, shake at 30 rpm for 5 min, then centrifuge at 1000 g for 5 min, discard the supernatant; repeat the above washing steps, Until the supernatant does not appear red, it is used for testing.
  • Preparation of the test solution add an appropriate amount of 1 ⁇ PBS solution to the solid of the test product, dissolve to obtain a standard stock solution, and then dilute with 1 ⁇ PBS to obtain a concentration of 1 ⁇ g/mL, 3 ⁇ g/mL, 10 ⁇ g/mL, 30 ⁇ g/mL mL, 100 ⁇ g/mL and 300 ⁇ g/mL of the test solution.
  • the blank 1 ⁇ PBS was used as a negative control
  • the 1 ⁇ PBS solution containing 0.1% Triton X-100 (1 ⁇ g/mL) was used as a positive control.
  • Incubation process Add 500 ⁇ L of the test solution to the erythrocyte suspension, shake on a plate shaker at 30 rpm for 5 minutes to make it fully mixed, then incubate the suspension at 37°C for 1 hour, and then centrifuge at 1000g for 5 minutes. Take 100 ⁇ L of the supernatant to a single well of the microtiter plate, and measure the absorbance at 540 nm.
  • the calculation formula of hemolysis rate (%) is (absorbance of test product-absorbance of negative control)/(absorbance of positive control-absorbance of negative control) ⁇ 100%.
  • the hemolytic test results of the glucagon analogs of the present disclosure are shown in FIG. 4A , FIG. 4B and Table 9.
  • the results showed that the glucagon analogue 40 and Dasiglucagon of the present disclosure, within the concentration range of 1 to 300 ⁇ g/mL, the hemolytic tests all met the specified requirements, and no significant risk of hemolytic side effects was found.
  • liver and kidney are the most important metabolic organs of the human body. They are rich in first-phase and second-phase metabolic enzymes, which will affect the metabolic rate of drugs, play an important role in the degradation and clearance of drugs, and affect blood drug concentration and half-life. Commonly used models of liver and kidney metabolism include liver and kidney microsomes, S9 fractions, hepatocytes, tissue homogenate, etc.
  • the metabolic stability of liver and kidney in vitro is one of the main indicators to predict the pharmacokinetic and pharmacodynamic properties in vivo.
  • liver-kidney microsome model uses microsomes extracted from the liver or kidney, adds a reduced coenzyme (NADPH) regeneration system, simulates metabolic reactions in a physiological environment, and evaluates the in vitro metabolic stability of test compounds, which can be used to predict drug in vivo degradation and clearance rate.
  • NADPH reduced coenzyme
  • glucagon analogues need to be quickly absorbed and cleared, and have a relatively fast degradation rate in the body to rescue hypoglycemia while avoiding the toxic side effects of high blood sugar.
  • Solution configuration Weigh an appropriate amount of pure compound, add appropriate amount of DMSO to dissolve to obtain 1 mM test solutions of Dasiglucagon and 40, respectively.
  • MgCl 2 -PBS buffer solution preparation Take 200 ⁇ L of 200 mM PBS buffer solution, add 106 ⁇ L of H2O and 40 ⁇ L of MgCl 2 solution (50 mM) in sequence, vortex and mix to obtain MgCl 2 -PBS buffer solution.
  • test compound solution and positive control compound (Verapamil for human liver microsomes, and benzydamine hydrochloride for human kidney microsomes) to the incubation reaction system, and the final concentration of the incubation system is 1 ⁇ M.
  • Incubation temperature is 37 °C;
  • verapamil and benzydamine hydrochloride are the substrate of known hepatic metabolic enzyme and renal metabolic enzyme respectively (referring to Pauli-Magnus C etc., J Pharmacol Exp Ther.2000 May; 293 (2 ): 376-82.), used to confirm the enzyme activity of the microsomal incubation system;
  • the metabolism rate of Dasiglucagon and glucagon analog 40 in liver microsomes is slower than that in kidney microsomes, and the metabolism has nothing to do with NADPH.
  • the degradation of glucagon analog 40 is slightly faster than that of Dasiglucagon (in vitro T1/2: 40 is 68.76min, Dasiglucagon is 161.74min).
  • Dasiglucagon and glucagon analog 40 are metabolized in renal microsomes, but the metabolism has nothing to do with NADPH; second, glucagon analog 40 is degraded faster than Dasiglucagon (in vitro T1/2 : 40 is 35.73min, Dasiglucagon is 51.27min).
  • the glucagon analogue 40 of the present disclosure has a faster metabolism rate than Dasiglucagon, so it is easier to be cleared in the body, and the risk of hyperglycemia side effects is lower, which is more beneficial Blood sugar control in diabetics.
  • the general method of animal pharmacokinetic research is to use the route of administration intended for clinical use, give animals a certain dose of test compound, and then use HPLC, GC, HPLC-MS and other analytical instruments to measure the drug concentration in biological matrices such as blood and urine. Changes over time, so as to understand the dynamic changes of the compound in the body, obtain the blood drug concentration-time curve measurement, and then obtain the pharmacokinetic parameters of the drug, and clarify the absorption, distribution, metabolism and excretion process and characteristics of the compound .
  • Sampling time 0min before administration (predose), 2min, 4min, 7min, 10min, 20min, 40min, 1h and 2h after administration. Blood was collected from rat jugular vein cannula.
  • Plasma samples were stored at -80°C for cryopreservation.
  • Sample pretreatment method take 100 ⁇ L of plasma sample, add 300 ⁇ L methanol solution containing 1 ng/mL internal standard (verapamil), vortex for 5 minutes, centrifuge at low temperature for 10 minutes, take 100 ⁇ L of supernatant, add 100 ⁇ L of 0.1% formic acid aqueous solution, mix Uniform, into the instrument analysis;
  • Liquid chromatography and mass spectrometry analysis LC-30A liquid chromatography system (Shimadzu Corporation, Japan); API 5500 liquid chromatography-triple quadrupole mass spectrometer (Analyst Software data processing system, American AB Company).
  • the chromatographic column is a nanomicro Unisil C18aq (4.6mm ⁇ 150mm, 5 ⁇ m) chromatographic column; the column temperature is 40°C; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile solution; the flow rate is 0.5mL/min; 20 ⁇ L; the gradient conditions are shown in Table 13 below.
  • the mass spectrometer adopts electrospray ionization source (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning method.
  • ESI electrospray ionization source
  • MRM multiple reaction monitoring
  • T1/2 of glucagon analog 40 was significantly shorter than that of Dasiglucagon (T 1/2 of 40 was 0.41h, and T 1/2 of Dasiglucagon was 0.51h), indicating that pancreatic hypertrophy Glucagon analog 40 was cleared faster; glucagon analog 40 also had a shorter Tmax than Dasiglucagon (0.117h for 40 vs. 0.167h for Dasiglucagon), indicating that glucagon analogues 40 is absorbed significantly faster than Dasiglucagon.
  • glucagon analog 40 has faster absorption onset and faster clearance, and has better pharmacokinetic properties.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Emergency Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

一种胰高血糖素类似物及其医药用途。具体地，所述胰高血糖素类似物具有显著提高的体外活性、优异的物理/化学稳定性和较高的溶解度，可用于治疗低血糖、肥胖症、糖尿病等代谢性疾病。

Description

胰高血糖素类似物及其医药用途
本申请要求2021年06月18日提交的申请号为202110676681.3的中国专利申请的优先权。
技术领域
本公开涉及胰高血糖素类似物及其医药用途。本公开的胰高血糖素类似物可用于治疗低血糖、肥胖症、糖尿病等代谢性疾病或缓解病症。
背景技术
控制体内血糖的平衡对机体代谢极其重要。正常情况下，人体血糖水平在多种不同多肽类激素(以胰岛素和胰高血糖素为主)调节下保持动态平衡。血糖失衡可能导致并发症。当血糖浓度低于正常水平，就会出现低血糖(Hypoglycemia)现象。胰岛中胰腺产生胰高血糖素，使血糖水平增加到正常范围。非严重的低血糖通常产生自主性的或神经性的低血糖症状，患者能够识别低血糖的发病，因此无需他人辅助治疗。而当血液中葡萄糖浓度因其它药物、或疾病、或者激素或酶的缺陷而下降(如治疗糖尿病、肾功能衰竭、特定肿瘤、肝疾病、甲状腺机能低下症、遗传性代谢缺陷、败血症与反应性低血糖所引起的一系列临床的严重低血糖症状)，体内的胰高血糖素就不足以使血糖恢复，甚至可危及到生命，通常表现为认知或身体功能受损，自我治疗能力受限，所以需要他人的帮助治疗才能恢复正常血糖。
目前，在低血糖防治中，完全清醒的低血糖患者的治疗方法首选口服糖类食物；对于意识障碍者，需要在非医护人员或医护人员的帮助下接受静脉注射50％葡萄糖液或肌肉注射0.5至2mg胰高血糖素的治疗。非医护人员较难开展50％葡萄糖液的静脉注射。注射剂量过高则会引起反弹性高血糖，如果葡萄糖液渗漏出血管，会造成局部组织损伤。因此，在非医院环境下，意识障碍的严重低血糖患者的最佳治疗策略是通过皮下或肌肉注射胰高血糖素。胰高血糖素作为严重低血糖的急救药物也已应用于临床；并且基于胰高血糖素的降脂及促进能量消耗作用，其与肠促胰素的双重受体激动剂也使肥胖的糖尿病患者进一步获益。
胰高血糖素为由胰腺α-细胞分泌的29个氨基酸组成的直链多肽，控制肝脏生产葡萄糖和酮体。胰高血糖素在夜间及两餐之间分泌，通过增加源自氨基酸前体的葡萄糖生成(糖异生)和促进糖原降解成葡萄糖(糖酵解)，导致肝脏的葡萄糖外流增加，从而提高血糖浓度。胰高血糖素与胰腺β细胞分泌的胰岛素共同精确控制体内血糖的平衡。胰高血糖素除了有低血糖的治疗效果，还抑制食欲、 并活化脂肪细胞的激素敏感性脂肪酶以促进脂肪分解，从而显示出抗肥胖效果。
胰高血糖素是抵抗低血糖防御机制的重要组成部分，低剂量的胰高血糖素可防止胰岛素诱导性的低血糖，并改善从低血糖恢复的能力。低剂量的胰高血糖素还可引起饱腹感抑制食欲，同时可活化脂肪细胞的激素敏感性脂肪酶，促进脂肪分解，从而有潜在的抗肥胖效果，可用于治疗许多患有II型糖尿病患者的体重超重或者肥胖。
然而，天然胰高血糖素在中性pH的水相溶液中溶解度极低。此外，胰高血糖素含有可产生氨基酸残基侧链脱酰氨基化、氧化、环状亚胺中间物形成的氨基酸或氨基酸序列，以及含有可发生异构化、肽链切割等的氨基酸或氨基酸序列，无法保持长时间稳定，并且在几小时至几天内就会形成凝胶或者原纤维(fibril)，化学及物理稳定性极差。因此，需要开发溶解度优异，物理/化学稳定性良好的新型胰高血糖素类似物以满足注射制剂开发的要求。
第一代胰高血糖素注射制剂，如胰高血糖素应急试剂盒(
Figure PCTCN2022099357-appb-000001
Eli Lilly)、胰高血糖素试剂盒(
Figure PCTCN2022099357-appb-000002
Novo Nordisk A/S)，受限于胰高血糖素不理想的成药性质，因而以冻干粉末的形式储存，使用时需要利用溶剂重溶解后再进行皮下/肌肉注射，不便于患者及非医护照料人员使用。
第二代胰高血糖素注射制剂，如人胰高血糖素鼻喷粉末(
Figure PCTCN2022099357-appb-000003
Eli Lilly)、预充的人胰高血糖素注射液(GVOKE
Figure PCTCN2022099357-appb-000004
XERIS)，对制剂做了针对性的改良，保证治疗效果的同时显著提高了患者及非医护人员操作的便利性。但是，
Figure PCTCN2022099357-appb-000005
采用鼻腔喷雾给药，起效较慢，相比肌肉注射需三倍剂量，有不良反应。GVOKE
Figure PCTCN2022099357-appb-000006
以DMSO为制剂，虽然可直接皮下注射，但有明显的注射部位不适及其它副作用。此外，Dasiglucagon(开发公司Zealand Pharma)是在天然人胰高血糖素的基础上进行了7个氨基酸位点的改造，在不影响活性的基础上，对溶解度和物理/化学稳定性进行了改善，可以注射液剂型直接皮下注射。Dasiglucagon已于近期获得美国食品药品管理局的批准上市。
本领域仍然亟需高活性、溶解度优异、物理/化学稳定性良好的胰高血糖素类似物。本公开提供了一类高活性(相比天然胰高血糖素，活性提高)、水性溶液中溶解度高(尤其在生理pH下)和稳定性(包含物理稳定性和化学稳定性)改善的胰高血糖素类似物。与本领域内的胰高血糖素类似物相比，本公开的胰高血糖素类似物具有更强的体外胰高血糖素受体激动剂活性，对降低皮下给药的体积和次数、提高低血糖患者的使用便利性、减轻患者经济负担都有很大帮助。本公开的胰高血糖素类似物可单独或与其它治疗性药物联合用于治疗低血糖、肥胖症、糖尿病等代谢性疾病或病症方法中。
发明内容
本公开提供胰高血糖素类似物或其可药用盐和/或溶剂化物，包含其的药物组合物，编码所述胰高血糖素类似物的多核苷酸、含有所述多核苷酸的载体、含有所述多核苷酸或载体的宿主细胞。以及，所述胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物用于治疗疾病、缓解病症(例如低血糖、肥胖症、糖尿病等代谢性疾病或病症)的方法和相关制药用途。
胰高血糖素类似物或其可药用盐和/或溶剂化物
本公开提供了如式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物：
R 1-His-Ser-X 3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X 15-X 16-X 17-Arg-Ala-X 20-X 21-Phe-Val-X 24-Trp-Leu-X 27-X 28-Thr-R 2(SEQ ID NO：50)
(I)
本公开还提供了包含式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中，R 1和/或R 2不存在；
其中：
R 1是氢、C 1-4烷基、乙酰基、甲酰基、苯甲酰基、三氟乙酰基或pGlu；
R 2是-OH或-NH 2；
X 3、X 15、X 16、X 20、X 21、X 24、X 27和X 28相互独立地选自任意天然氨基酸残基或非天然氨基酸残基；X 17是Aib。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20选自Ala、Gln、Glu，Ser，Thr或Lys；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20选自Ala、Gln、Glu,Ser,Thr或Lys；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、 Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20选自Ala、Gln、Glu,Ser,Thr或Lys；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21是Glu；X 24选自Gln或Glu；X 27是Glu； X 28是Ser。
在一些实施方案中，R 1是氢；R 2是-OH或-NH 2。在一些实施方案中，R 1是氢；R 2是-OH。
本公开的可药用盐和/或溶剂化物选自：无机盐或有机盐。
本公开的胰高血糖素类似物可与酸性化合物或碱性化合物反应而生成相应的盐。
“与酸性化合物反应而生成相应的盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于：盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于：甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。可通过本领域已知的方法制备这些盐。
“与碱性化合物反应而生成相应的盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于：钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。衍生自有机碱的盐包括但不限于：伯胺、仲胺、叔胺、取代的胺、环状胺；例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、乙二胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。可通过本领域已知的方法制备这些盐。
术语“溶剂化物”表示本公开的胰高血糖素类似物或其可药用盐与适宜的溶剂形成的复合物。溶剂的非限制性实例包括：水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。在具体的实施方案中，所述溶剂化物为水合物。
在一些实施方案中，本公开提供胰高血糖素类似物或其可药用盐和/或溶剂化物，所述胰高血糖素类似物是选自SEQ ID NO：1-49中任一所示的化合物。
在一些具体的实施方案中，本公开提供胰高血糖素类似物或其可药用盐和/或溶剂化物，所述胰高血糖素类似物是选自以下的化合物：
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)。
在本公开的每一个胰高血糖素类似物的序列中，C端“-OH”部分(moiety)可以替换成C端的“-NH 2”。
在一些具体的实施方案中，本公开提供胰高血糖素类似物或其可药用盐和/或溶剂化物，所述胰高血糖素类似物包含选自SEQ ID NO：39、40、41、42、43、47任一的结构，其中，所述SEQ ID NO：39、40、41、42、43、47的N端的H和/或C端的OH是不存在的。
本公开提供了如式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物：
R 1-His-Ser-X 3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X 15-X 16-X 17-Arg-Ala-X 20-X 21-Phe-Val-X 24-Trp-Leu-X 27-X 28-Thr-R 2(SEQ ID NO：50)
(I)
本公开还提供了包含式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中，R 1和/或R 2不存在；
其中：
R 1是氢、C 1-4烷基、乙酰基、甲酰基、苯甲酰基、三氟乙酰基或pGlu；
R 2是-OH或-NH 2；
X 3、X 17、X 20、X 21、X 24、X 27和X 28相互独立地选自任意天然氨基酸残基或非天然氨基酸残基；X 15是Asp；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser，例如选自Thr、Leu、Val或Ile。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17选自Aib或Ala；X 20不是Glu；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17选自Aib或Ala；X 20不是Glu；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys； X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20不是Glu；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20不是Glu；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20不是Glu；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20选自Ala、Gln、Glu、Ser、Thr或Lys；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21是Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，R 1是氢；R 2是-OH或-NH 2。在一些实施方案中，R 1是氢；R 2是-OH。
本公开提供了一种胰高血糖素类似物或其可药用盐和/或溶剂化物，所述胰高血糖素类似物包含如式(I)所示的结构：
R 1-His-Ser-X 3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X 15-X 16-X 17-Arg-Ala-X 20-X 21-Phe-Val-X 24-Trp-Leu-X 27-X 28-Thr-R 2(SEQ ID NO：50)
(I)
本公开还提供了包含式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中，R 1和/或R 2不存在；
其中：
R 1是氢、C 1-4烷基、乙酰基、甲酰基、苯甲酰基、三氟乙酰基或pGlu；
R 2是-OH或-NH 2；
X 3、X 15、X 16、X 17、X 21、X 24、X 27和X 28相互独立地选自任意天然氨基酸残基或非天然氨基酸残基；X 20是Gln。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17选自Aib或Ala；X 20是Gln；X 21选自Asp或Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21是Glu；X 24选自Gln或Glu；X 27是Glu； X 28是Ser。
在一些实施方案中，R 1是氢；R 2是-OH或-NH 2。在一些实施方案中，R 1是氢；R 2是-OH。
本公开提供式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物：
R 1-His-Ser-X 3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X 15-X 16-X 17-Arg-Ala-X 20-X 21-Phe-Val-X 24-Trp-Leu-X 27-X 28-Thr-R 2(SEQ ID NO：50)
(I)
本公开还提供了包含式(I)所示结构的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中，R 1和/或R 2不存在；
其中：
R 1是氢、C 1-4烷基、乙酰基、甲酰基、苯甲酰基或三氟乙酰基或pGlu；
R 2是-OH或-NH 2；
X 3、X 15、X 16、X 17、X 21、X 27和X 28相互独立地选自任意天然氨基酸残基或非天然氨基酸残基；X 20是Glu，X 24是Arg。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20是Glu；X 21选自Asp或Glu；X 24是Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Glu；X 21选自Asp或Glu；X 24是Arg；X 27选自Met、Glu、Nle、Leu或Ser；X 28选自Asn、Glu或Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15选自Asp或Glu；X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；X 17是Aib；X 20是Glu；X 21选自Asp或Glu；X 24是Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His、Dap(Ac)或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Glu；X 21选自Asp或Glu；X 24是Arg；X 27是Glu；X 28是Ser。
在一些实施方案中，X 3选自His或Gln；X 15是Asp；X 16选自Thr、Leu、Val或Ile；X 17是Aib；X 20是Gln；X 21是Glu；X 24选自Gln或Glu；X 27是Glu；X 28是Ser。
在一些实施方案中，R 1是氢；R 2是-OH或-NH 2。在一些实施方案中，R 1是氢；R 2是-OH。
本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物具有胰高血糖素受 体(GCGR)激动剂活性，其与GCGR的结合可用作激动剂活性的指示。在一些可选实施方案中，也可测量由化合物与受体结合时引起的细胞内信号转导。例如，胰高血糖素受体激动剂对GCGR的活化，将刺激细胞环磷酸腺苷(cAMP)的形成。因此，可使用合适的表达受体的细胞中cAMP的产生来监测受体活性。例如，可使用实施例2中给出的材料(例如CHO-K1/GCGR/CRE-Luc稳转细胞株和Cisbio-cAMP-Gs Dynamic kit)并且在所述条件下进行测定。可使用EC 50值作为对GCGR的激动剂效力的数值量度。EC 50值是：在特定测定中，对于所讨论的受体实现化合物最大活性的一半时，所需的该化合物的浓度。
在一些实施方案中，胰高血糖素受体是哺乳动物的胰高血糖素受体，例如人胰高血糖素受体。
在一些实施方案中，本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物对人GCGR的激动剂活性是天然胰高血糖素的至少1.5倍、至少1.6倍、至少1.7倍、至少1.8倍、至少1.9倍、至少2.0倍、至少2.1倍、至少2.2倍、至少2.3倍、至少2.4倍、至少2.5倍、至少2.6倍、至少2.7倍、至少2.8倍、至少2.9倍、至少3.0倍、或至少3.1倍。
在一些实施方案中，本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物对人GLP-1R和/或GIPR无激动剂活性或可以忽略不计。
在一些实施方案中，与天然胰高血糖素相比，本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物具有提高的溶解度和/或稳定性。
在一些实施方案中，提高的溶解度可以包括或者是指：在pH 4(例如，在pH 4的醋酸盐缓冲液中)、pH 5(例如，在pH 5的醋酸盐缓冲液中)、pH 6(例如，在pH 6的磷酸盐缓冲液中)、pH 7(例如，在pH7的磷酸盐缓冲液中)和/或pH 7.5(例如，在pH 7.5的磷酸盐缓冲液中)下，与天然胰高血糖素相比，具有提高的溶解度。作为一个示例，可以在实施例5中给出的条件下进行测定。可以期望溶解度≥1mg/mL。
在一些实施方案中，例如通过测量在280nm的吸光度，并依据比尔定律计算的，本公开的胰高血糖素类似物在pH7.4的磷酸盐缓冲液中的溶解度为≥1.00mg/mL、≥1.10mg/mL、≥1.20mg/mL、≥1.30mg/mL、≥1.40mg/mL、≥1.50mg/mL、≥1.60mg/mL、≥1.70mg/mL、≥1.80mg/mL、≥1.90mg/mL、≥2.00mg/mL、≥2.10mg/mL、≥2.20mg/mL、≥2.30mg/mL、≥2.40mg/mL、≥2.50mg/mL、≥2.60mg/mL、≥2.70mg/mL、≥2.80mg/mL、≥2.90mg/mL、≥3.00mg/mL、≥3.10mg/mL、≥3.20mg/mL、≥3.30mg/mL、≥3.40mg/mL、≥3.50mg/mL、≥3.60mg/mL、≥3.70mg/mL、≥3.80mg/mL、≥3.90mg/mL、≥4.00mg/mL、≥4.10mg/mL、≥4.20mg/mL、≥4.30mg/mL、≥4.40mg/mL、≥4.50mg/mL、≥4.60mg/mL、≥4.70mg/mL、≥4.80mg/mL、≥4.90mg/mL、或≥5.00mg/mL。
在一些实施方案中，提高的稳定性可以包括或者是指：与天然人胰高血糖素相比提高的物理稳定性和/或提高的化学稳定性。
在一些实施方案中，提高的物理稳定性可以包括或者是指：聚集(例如形成可溶性或不溶性聚集体(例如，原纤维))的趋势降低。可以以0.1N HCl溶解胰高血糖素类似物，以1mg/mL的浓度37℃下测定聚集(例如，原纤维形成)。可使用任何合适的时间段，例如，24小时、48小时、96小时或120小时。可以适用ThT荧光测试测定聚集。可以在实施例4给出的条件下测定聚集。
在一些实施方案中，提高的化学稳定性可以包括或者是指：在水性缓冲液(通常不存在污染性蛋白酶或肽酶活性)中肽被切割或降解的趋势降低。可以以例如pH 7.4的磷酸盐缓冲液，溶解胰高血糖素类似物，以1mg/mL的浓度4℃、25℃或40℃下测定稳定性。评估可包括在孵育合适的时间段例如1天、7天、14天、21天、或28天后测定稳定性。这可包括按照实施例4中所限定，来测定化合物纯度，即各色谱图中主峰相对于所有整合峰的总面积的面积百分比。可以在实施例4给出的条件下测定稳定性。
多核苷酸
本公开提供编码本公开胰高血糖素类似物前体肽的多核苷酸，可以为RNA、DNA或cDNA。根据本公开的一些实施方案，本公开的多核苷酸是基本上分离的多核苷酸。
所述前体肽是经修饰后能够获得本公开胰高血糖素类似物的肽。例如，前体肽的X 17为Arg或其他天然氨基酸，经修饰后获得X 17为Aib的本公开胰高血糖素类似物。
本公开的多核苷酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供胰高血糖素类似物在体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达，对所述元件及其序列进行选择为本领域技术人员的常识。
在一些实施方案中，载体包含5’→3’方向上的且可操作地连接的以下元件(element)：用于驱动核酸片段表达的启动子，编码使得能够进行分泌(分泌至胞外相或适当时分泌到周质(periplasma)中)的前导肽的任选的多核苷酸，编码前体肽的多核苷酸，以及编码终止子的任选的多核苷酸。
本公开的多核苷酸可基于本公开胰高血糖素类似物前体肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得。
宿主细胞
本公开提供表达本公开的胰高血糖素类似物前体肽或含有本公开的多核苷酸或载体的重组宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的细胞不能发育成完成的植株或动物个体。
生产或制备方法
本公开提供制备本公开的胰高血糖素类似物的方法，包括例如以下的方法：
(a)通过化学合成(例如，固相或液相合成)方法合成胰高血糖素类似物，并回收由此获得的所合成的胰高血糖素类似物；或者
(b)由编码前体肽的多核苷酸或载体表达前体肽，回收表达产物，以及修饰所述前体肽，以产生本公开的胰高血糖素类似物，例如，可通过引入一种或多种非天然氨基酸(例如Aib)来修饰前体肽。
在一个实施方案中，包含以下步骤：
-在允许表达前体肽的条件下，培养本公开的宿主细胞；及
-从培养物中回收前体肽；及
-修饰所述前体肽，获得本公开的胰高血糖素类似物。可选地，包含纯化前体肽的步骤。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造前体肽的方法中的多肽分离及纯化技术为本领域技术人员所公知。固 相或液相合的方法在本领域中是已知的，例如，可参考WO98/11125，尤其是Fields，GB et al.，2002，“Principles and practice of solid-phase peptide synthesis”.In：Synthetic Peptides(第二版)，以及本公开中的实施例。
在一些实施方案中，还提供胰高血糖素类似物形成其可药用盐和/或溶剂化物的方法。例如，本公开胰高血糖素类似物可以与药学上可接受的酸性或碱性化合物反应成盐。
在一些实施方案中，本公开的胰高血糖素类似物可以采用微生物重组表达的方法来制备，也可以采用Fmoc固相合成的方法来制备(例如SPPS方法，包括氨基酸脱保护的方法、从树脂上切割肽的方法及其纯化的方法)。
在一些实施方案中，采用Fmoc固相合成的方法时，合成载体为Fmoc-L-Thr(tBu)-Wang树脂(吉尔生化，载量0.533mmol/g)。在一些实施方案中，合成过程中使用的氨基酸衍生物包括：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-OH，Fmoc-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-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等。在一些实施方案中，合成过程包括：洗涤固相合成载体后，首先用含20％4-甲基哌啶的氮,氮-二甲基甲酰胺(DMF)溶液脱去固相合成载体α-氨基上的Fmoc保护基团，接着该固相载体与序列中下一个氨基酸衍生物在过量情况下使用HCTU/4-甲基吗啉活化后进行缩合形成酰胺键以延长肽链；重复缩合→洗涤→去保护→洗涤→下一轮氨基酸缩合的操作以达到所要合成的多肽链长度，最后用三氟乙酸：水：三异丙基硅烷(90：5：5，v：v：v)的混合溶液与树脂反应将多肽从固相载体上裂解下来，再由冷冻甲基叔丁基醚沉降后得到多肽衍生物的固体粗品；多肽固体粗品由含0.1％三氟乙酸的乙腈/水的混合溶液溶解后由C-18反相制备色谱柱纯化分离后得到多肽及其衍生物的纯品。
药物组合物
本公开提供药物组合物，其含有预防、治疗或缓解有效量的本公开的胰高血糖素类似物或可药用盐和/或溶剂化物，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
在一些实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的本公开的胰高血糖素类似物或可药用盐和/或溶剂化物。在另一些实施方案中，药物组合物单位剂量中含本公开的胰高血糖素类似物或可药用盐和/或溶剂化物的量为0.1-2000mg；一些具体实施方案中为1-1000mg。
在一些实施方案中，药物组合物进一步包括至少一种具有代谢性疾病治疗活性的化合物或物质。
在一些实施方案中，所述具有代谢性疾病治疗活性的化合物或物质选自以下中的一种或多种：葡萄糖依赖性促胰岛素肽(GIP)、胰高血糖素样肽-1(GLP-1)受体激动剂、胰岛素、肠高血糖素、神经肽Y5受体拮抗剂、乙酰辅酶A羧化酶抑制剂、Leptin受体激动剂、格列奈、α-葡萄糖苷酶抑制剂(AGi)、噻唑烷二酮(TZD)、二肽基肽酶-4抑制剂类(DPP-IV)、钠葡萄糖协同转运蛋白2抑制剂(SGLT-2)、法尼醇X受体(FXR)激动剂和肥胖抑制素(Obestatin)。
本公开的药物组合物可以通过胃肠外给药，用注射器(任选笔型注射器)，通过皮下、肌内、腹膜内或静脉内注射进行。或者，胃肠外给药可以用输注泵进行。另外的选择是组合物可以是经鼻或肺喷雾的形式给予胰高血糖素肽的溶液剂或混悬剂；或者，其可以是透皮给药例，如通过无针注射或由贴剂、任选离子导入贴剂；或者，其可以是经粘膜(例如口腔)给药。
试剂盒(或药盒)
本公开还提供了一种药盒，所述药盒包括本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物和施用装置。
在一些实施方案中，所述装置包含注射器和针。在一些具体实施方案中，所述装置是预充针。
在一些实施方案中，本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物于预充针中。
预防、治疗疾病或病症的方法和制药用途
本公开还提供了上述胰高血糖素类似物或其可药用盐和/或溶剂化物，和/或上述药物组合物在制备用于预防、治疗疾病或病症的药物中的用途。
本公开还提供上述胰高血糖素类似物或其可药用盐和/或溶剂化物，和/或上述药物组合物，其用于预防、治疗疾病或病症。
本公开还提供了预防、治疗疾病或病症方法，其包括向有其需要的对象施用治疗有效量的本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物，和/或施用治疗有效量的本公开的药物组合物。
在一些实施方案中，所述疾病或病症选自低血糖、高血糖症、2型糖尿病、1型糖尿病、冠心病、动脉粥样硬化、β-受体阻滞剂中毒、胰岛素瘤和冯吉尔克病；所述病症或病症选自葡萄糖耐量减低、血脂异常、高血压、体重超重、暴食症、肝脂肪变性、肥胖。
在一些实施方案中，所述低血糖选自病理性低血糖或非病理性低血糖。
在一些实施方案中，所述低血糖选自糖尿病性低血糖、非糖尿病性低血糖、 空腹低血糖、药物诱发性低血糖、急性胰岛素诱发低血糖、胃旁路术诱发性低血糖、酒精诱发性低血糖、反应性低血糖和妊娠期低血糖。
在一些实施方案中，提供了一种迅速增加葡萄糖水平、使血糖水平正常、使血糖水平稳定、或者预防或治疗低血糖的方法，其包括向有需要的对象施用有效量的本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物。
在一些实施方案中，提供了一种减少体重增加或引起体重下降的方法，其包括向有需要的对象施用有效量的本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物。减少体重增加或引起体重下降的方法预期可用于治疗各种起因的肥胖，包括药物诱导的肥胖，并减少与肥胖相关的并发症，包括血管疾病(例如冠状动脉疾病、中风、周围血管疾病、缺血性再灌注等)、高血压、II型糖尿病、高脂血症和骨骼肌疾病。本公开提供相应胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物用于制备预防、治疗、缓解肥胖的药物的制药用途。
在一些实施方案中，提供了一种治疗高血糖症或糖尿病的方法，其包括联合施用胰岛素与本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物。联合施用胰岛素(例如基础、速效或长效胰岛素)和本公开的胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物，能够减少夜间低血糖和/或缓冲胰岛素的使血糖过低的效应。本公开提供相应胰高血糖素类似物或其可药用盐和/或溶剂化物或包含其的药物组合物用于制备预防、治疗、缓解高血糖症或糖尿病的制药用途。
在一些实施方案中，上述方法是用于人受试者的，上述制药用途是用于人受试者的药物的制备用途。
附图说明
图1为通过ThT原纤维形成测定法评价胰高血糖素类似物的物理稳定性的结果。
图2为正常血糖大鼠模型中胰高血糖素类似物40和Dasiglucagon的药效结果。
图3为低血糖大鼠模型中胰高血糖素类似物40和Dasiglucagon的药效结果。
图4A为Dasiglucagon的溶血性检查结果；图4B为胰高血糖素类似物40的溶血性检查结果。
图5A和图5B分别为胰高血糖素类似物40和Dasiglucagon在人肝微粒体中、人肾微粒体中的稳定性结果。
图6为胰高血糖素类似物40和Dasiglucagon经皮下或静脉注射给药后的药时曲线结果。
具体实施方式
定义
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本公开中的它处另有明确定义，否则本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
术语“胰高血糖素类似物”是指具有天然胰高血糖素对胰高血糖素受体的至少一部分、全部、或更高的激动剂活性的化合物。例如胰高血糖素类似物包括但不限于：在SEQ ID NO：51所示的氨基酸序列基础上包含任意氨基酸替换、添加或删除，或包含翻译后修饰或其他化学修饰的多肽，前提是所述类似物具有刺激胰高血糖素受体的活性，例如使用实施例2描述的测定法通过cAMP的产生来测定的。
本公开中，胰高血糖素类似物也包括具有修饰的氨基末端和/或羧基末端的肽。例如，包含以酰胺基团取代末端羧酸的肽。例如，本公开的胰高血糖素类似物可以在序列的氨基端(N-端)引入“H-”部分，在序列的羧基端(C-端)引入“-OH”部分或“-NH 2”部分。在这种情况下，除非另有说明，否则所讨论序列N端的“H-”部分表示氢原子[即，在式(I)中，R 1＝氢＝H；相当于在N端存在游离伯氨基或仲氨基]，而序列C端的“-OH”或“-NH 2”部分分别表示羟基[例如，在式(I)中，R 2＝OH；相当于在C端存在羧基(COOH)]或氨基[例如，在式(I)中，R 2＝NH 2；相当于在C端存在酰胺基(CONH 2)]。在本公开的每一个序列中，C端“-OH”部分可以替换成C端的“-NH 2”，反之亦然。
“天然胰高血糖素”是指具有以下序列的天然人胰高血糖素：
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)。
“Dasiglucagon”特指由西兰制药公司(Zealand Pharma)开发的胰高血糖素类似物，其氨基酸序列为：H-HSQGTFTSDYSKYLDAibARAEEFVKWLEST-OH(SEQ ID NO：52)。
本公开中，如果没有进一步解释，术语“GLP-1”是指人GLP-1(7-36或7-37)；术语“GIP”是指人GIP(1-42)。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。除非明确说明，否则本公开中所有氨基酸残基的构型为L-型。另外，Aib是2-氨基异丁酸(又称α-氨基异丁酸)，α-甲基-Ser(α-甲基丝氨酸)是α位 H被甲基取代的丝氨酸，pGlu是焦谷氨酸。
“天然氨基酸”是指20种常规氨基酸(即丙氨酸(A)，半胱氨酸(C)，天冬氨酸(D)，谷氨酸(E)，苯丙氨酸(F)，甘氨酸(G)，组氨酸(H)，异亮氨酸(I)，赖氨酸(K)，亮氨酸(L)，甲硫氨酸(M)，天冬酰胺(N)，脯氨酸(P)，谷氨酰胺(Q)，精氨酸(R)，丝氨酸(S)，苏氨酸(T)，缬氨酸(V)，色氨酸(W)和酪氨酸(Y)。
“非天然氨基酸”是指不是天然编码的或不在任何生物体的遗传密码中发现的氨基酸。它们可以是例如纯化学合成的化合物。非天然氨基酸的实例包括但不限于：羟基脯氨酸，γ-羧基谷氨酸，O-磷酸丝氨酸，氮杂环丁烷羧酸，2-氨基己二酸，3-氨基己二酸，β-丙氨酸，氨基丙酸，2-氨基丁酸，4-氨基丁酸，6-氨基己酸，2-氨基庚酸，2-氨基异丁酸(Aib)，3-氨基异丁酸，2-氨基庚二酸，叔丁基甘氨酸，2,4-二氨基异丁酸(Dap)，锁链素(desmosine)，2，2′-二氨基庚二酸，2,3-二氨基丙酸(Dab)，N-乙基甘氨酸，N-甲基甘氨酸，N-乙基天冬酰胺，高脯氨酸，羟基赖氨酸，别羟基赖氨酸(allo-hydroxylysine)，3-羟基脯氨酸，4-羟基脯氨酸，异锁链素(isodesmosine)，别异亮氨酸，N-甲基丙氨酸，N-甲基甘氨酸，N-甲基异亮氨酸，N-甲基戊基甘氨酸，N-甲基缬氨酸，萘基丙氨酸(naphthalanine)，正缬氨酸，正亮氨酸(Nle)，鸟氨酸(Orn)，D-鸟氨酸，D-精氨酸，对氨基苯丙氨酸，戊基甘氨酸，呱啶酸(pipecolic acid)和硫代脯氨酸。此外，“非天然氨基酸”还包括在天然氨基酸或非天然氨基酸的C-末端羧基，N-末端氨基和/或其侧链官能团上经化学修饰所得的化合物。
术语“激动剂”定义为激活所讨论的受体类型的物质或配体。例如，本公开上下文中使用的术语GLP-1或GIP或胰高血糖素(受体)激动剂指可以激活GLP-1受体或GIP受体或胰高血糖素受体的物质或配体。
术语“可药用盐”是指对待治疗的患者或对象无害的盐。这样的盐通常是酸加成盐或碱式盐。
术语“溶剂化物”是指由限定化学计量的溶质(在这种情况下为本公开的胰高血糖素类似物或者其可药用盐)和溶剂形成的复合物。
术语“药物组合物”表示含有一种或多种本公开的胰高血糖素类似物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。
术语“可药用载体”包括任何标准药用载体或稀释剂，例如用于适于经口、肺、直肠、鼻、局部、皮下、肌内、静脉内、腹膜内、皮内、透皮或阴道内施用的组合物或制剂中使用的那些。
术语“治疗”是指用于获得有益的或期望的临床结果的方法。就本公开目的 而言，有益的或期望的临床结果包括但不限于缓解病症、减轻疾病程度、稳定(即，不恶化)疾病状态、延迟或减缓疾病发展、改善或缓和疾病状态以及(部分或全部地)消除(remission)，无论是可检出的还是不可检出的。“治疗”还可以指与未接受治疗时的预期存活相比延长存活期。“治疗”是以防止疾病发生或改变疾病病理状况为目的而进行的介入。因此，“治疗”是指治疗性处理和预防性或防护性手段二者。如在预防性或防护性手段的上下文中使用的，化合物未必完全防止疾病或病症发生。需要治疗的对象包括已经患病或需要预防疾病发生的对象。“治疗”也指与不进行治疗的情况相比抑制或减少病理状况或症状(例如，体重增加或低血糖)的增加，并且不暗示相关病症的完全终止。
术语“有效量”或“治疗有效量”是指足以治愈、缓解、或部分阻止、或者以其他方式促进给定病症(紊乱、疾病)或损伤以及优选地由它们引起的并发症的治愈或康复的量或剂量。对于特定目的有效的量或剂量取决于疾病或病症的严重程度以及待治疗对象或患者的体重和总体状况。合适的量或剂量的确定在经过训练的一般技术的医生(或兽医)的能力范围之内。
术语“对象”可与“患者”、“个体”、“受试者”等互换使用，并且均指人或非人动物，包括但不限于哺乳动物，例如人、灵长类动物、家畜动物(例如，牛、猪)、宠物(例如，狗、猫)和啮齿类动物(例如，小鼠和大鼠)。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如常规多肽Fmoc固相合成法手册，冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
本公开主要实验试剂(来源)：Fmoc-Thr(tBu)-Wang树脂(吉尔生化)，HCTU(昊帆生物)，Fmoc-Aib-OH(吉尔生化)，氮，氮-二甲基甲酰胺(国药)，二氯甲烷(国药)，三氟乙酸(TCI Chemicals)，三异丙基硅烷(TCI Chemicals)，乙腈(Merck-Millipore)，二异丙基乙胺(Sigma)，4-甲基哌啶(TCI Chemicals)，甲基叔丁基醚(TCI Chemicals)，4-甲基吗啉(TCI Chemicals)，Fmoc-Nle-OH(吉尔生化)。DMEM/F12(Gibco 11330032)，酪蛋白(Casein)，3-异丁基-1-甲基黄嘌呤(Sigma I7018-250MG)，cAMP-Gs Dynamic kit(Cisbio 62AM4PEC，20000个测试)，384孔板(Sigma CLS4514-50EA)，96V型底板(PS)(Axygen WIPP02280)，96孔板(Cisbio 66PL96100)，
Figure PCTCN2022099357-appb-000007
Cell Counting Chamber Slides(Invitrogen C10228)，Cisbio-cAMP-Gs Dynamic试剂盒(Cisbio 62AM4PEC)， 0.25％胰蛋白酶-EDTA和Phenol Red(ThermoFisher 25200-114)，胎牛血清(GibcoTM,ThermoFisher 10099-141)，人胰高血糖素(Abmole M9312)。
本公开主要实验仪器(来源)：H-CLASS分析型超高效液相色谱(WATERS)，Agilent 1290-6530超高效液相色谱-质谱仪(Agilent Technologies)，Prep-150制备型高效液相色谱(WATERS)，Prelude-X多通道多肽固相合成仪(Protein Technologies)，酶标仪(BioTek H1MFD；Tecan-Infinite F Plex)。
实施例1.胰高血糖素类似物的合成
本实施例设计并按照以下方法合成了表1的化合物。
表1.胰高血糖素类似物(又称化合物或本公开化合物)
Figure PCTCN2022099357-appb-000008
Figure PCTCN2022099357-appb-000009
1.化合物1的化学合成
1.1多肽骨架的合成
称取0.1mmol固相合成载体Fmoc-Thr(tBu)-Wang树脂置于一次性聚丙烯多肽合成固相反应管中，加入DMF(10mL)溶胀树脂10分钟，真空抽掉DMF，加入DMF(10mL)洗涤树脂，重复洗涤2次。使用Prelude-X全自动多肽合成仪完成多肽骨架的合成，其中酰胺键缩合使用10倍过量，相应的Fmoc-保护氨基酸，用HCTU/4-甲基吗啉活化后在室温下反应30分钟进行氨基酸的缩合。Fmoc基团脱保护使用含20％4-甲基哌啶的氮，氮-二甲基甲酰胺溶液在室温下反应2次，每次10分钟。此外，对于Aib旁N-端氨基酸的缩合，需要在室温下进行二次乃至三次缩合，缩合时间每次30-60分钟，这对改善粗品肽的纯度至关重要。
1.2树脂肽的裂解
上述步骤得到的树脂肽依次用DMF，DCM洗涤3次后进行真空干燥，后加入新鲜配制的裂解液10mL(三氟乙酸：三异丙基硅烷：水＝90：5：5，v：v：v)在室温下振荡反应3小时。反应结束后过滤，用三氟乙酸洗涤树脂2次，合并滤液后加入大量冷冻甲基叔丁基醚析出固体，离心后除去上清液并得到化合物1的多肽粗品。
1.3粗品肽的反相液相色谱纯化
将粗品肽溶于含有0.1％(v/v)三氟乙酸，20％(v/v)乙腈/水的混合溶剂中， 经过0.22μm膜过滤后用WATERS Prep150 LC反相高效液相色谱系统进行分离，流动相为A(0.1％三氟乙酸，10％乙腈，水溶液，v/v)和B(0.1％三氟乙酸，90％乙腈，水溶液，v/v)。其中，色谱柱为X-SELECT OBD C-18(WATERS)反相色谱柱，纯化过程中色谱仪检测波长设定为220nm，流速为20mL/min。收集产物相关馏分冻干后得到化合物编号1的多肽纯品，收率20％。多肽纯品通过分析性高效液相色谱和液相色谱/质谱联用确定纯度及化合物身份，其中纯度＝96.09％，且分子量正确。
2.化合物2-49的化学合成
采用化合物1的实验方案合成本公开化合物2-49的多肽化合物，并用分析型超高效液相色谱和液相色谱/质谱联用确定纯度和化合物分子量，其中化合物分子量正确，纯度如表2。
表2.化合物纯度
Figure PCTCN2022099357-appb-000010
以下实施例对实施例1合成的胰高血糖素类似物进行了生物学测试评价。
实施例2.胰高血糖素类似物对胰高血糖素受体(GCGR)、胰高血糖素样肽-1受体(GLP-1R)和葡萄糖依赖性促胰岛素分泌多肽受体(GIPR)的激动剂活性评估
1.实验目的
测定本公开的胰高血糖素类似物对人胰高血糖素受体(GCGR)、胰高血糖素样肽-1受体(GLP-1R)和葡萄糖依赖性促胰岛素分泌多肽受体(GIPR)的激动剂活性。相关化合物对GCGR的活化将刺激细胞环磷酸腺苷(cAMP)的形成。因此，可使用合适的表达受体的细胞中cAMP的产生来监测受体活性。
2.实验方法
对GCGR的激动剂活性检测：从液氮罐中取出冻存的CHO-K1/GCGR/CRE-Luc稳转细胞株，放于37℃水浴锅中快速融化，用DMEM/F12培养基(Sigma Cat#D8437)重悬，离心后将细胞接种至T75培养瓶中，细胞至少传代一次之后，再用于后续测试。测试前，胰酶消化细胞，离心后清洗一遍细胞，使用不含血清DMEM/F12培养基重悬细胞，调整细胞密度，以2,500个细胞/5μL/孔的密度铺于96孔板；配制Cisbio-cAMP-Gs Dynamic试剂盒中的1×缓冲液，添加IBMX至终浓度为0.5mM；每孔加入5μL缓冲液梯度稀释的多肽，轻震混匀，37℃静置孵育30分钟；将cAMP-d2和Anti-cAMP-Eu 3+-Cryptate分别用cAMP Lysis&Detection buffer稀释20倍混匀，每孔加入5μL稀释后的cAMP-d2溶液，再加入5μL稀释后的Anti-cAMP-Eu 3+-Cryptate溶液，轻震混匀，室温避光静置孵育1小时。
对GLP-1R的激动剂活性检测：与上述对GCGR的激动剂活性检测方法不同在于，使用的是CHO-GLP-1R稳转细胞株。
对GIPR的激动剂活性检测：与上述对GCGR的激动剂活性检测方法不同在于，使用的是CHO-K1/GIPR稳转细胞株，细胞密度是3,000个细胞/5μL/孔。
3.实验结果
使用Tecan-Infinite F Plex酶标仪读取HTRF的信号，激发波长为320nm，发射波长为620nm和665nm。计算信号比值(665nm/620nm*10,000)，将其与样品浓度使用四参数方程非线性拟合，得出EC 50值，见表3和表4。
表3.化合物对人GCGR的激动剂活性
化合物编号 人GCGR受体EC 50(nM) 相对活性(％)
Dasiglucagon 0.14 142
39 0.072 268
40 0.060 318
41 0.058 333
42 0.093 207
43 0.052 371
47 0.076 253
(相对活性是以天然胰高血糖素的活性为100％，进行归一化。)
表4.化合物对人GLP-1R和GIPR的激动剂活性
Figure PCTCN2022099357-appb-000011
(n.d.表示未检出。)
表3结果显示，本公开的胰高血糖素类似物对人GCGR的激动剂活性显著优于Dasiglucagon。表4结果显示，与天然胰高血糖素相比，本公开的胰高血糖素类似物对GLP-1R和GIPR的激动剂活性极低，可以忽略，即本公开的胰高血糖素类似物对GCGR有良好的特异性。
实施例3.胰高血糖素类似物在生理条件缓冲液中的溶解度评估
1.实验目的
天然胰高血糖素直接作为治疗剂的使用受到限制，是因为其在中性pH 7.4条件下溶解度远低于1mg/mL。本实施例的目的是评估本公开胰高血糖素类似物在中性pH缓冲液中的溶解度，期望与天然胰高血糖素相比具有更高的溶解度。
2.实验方法
在pH 7.4的磷酸盐缓冲液中制备部分本公开胰高血糖素类似物的饱和溶液，室温超声5min后，离心(10000g，5min)，吸取上清液并确保无可见沉淀。测量溶液在280nm的吸光度(Abs λ＝280nm)，基于胰高血糖素类似物序列中色氨酸和酪氨酸的含量计算多肽在280nm处的摩尔吸光系数(ε＝8250M -1cm -1)，依据比尔定律计算各测试胰高血糖素类似物在中性pH缓冲液中的最大溶解度。
3.实验结果
本公开的多种胰高血糖素类似物相比天然的胰高血糖素具有显著提升的溶解度，见表5。比较化合物2至化合物5发现，在第17位包含Aib的化合物3比在18位包含Aib的化合物4具有提升的溶解度；在第17位包含Aib的化合物3比在16或19位包含α-甲基-Ser的化合物2和化合物4具有提升的溶解度。可见胰高血糖素类似物在第17位包含Aib对提升多肽溶解度有重要作用。
表5.化合物的溶解度数据
化合物编号 最大溶解度(mg/mL)
Dasiglucagon >3.75
2 1.27
3 >2.98
4 1.40
5 0.872
38 >16.4
39 >4.79
40 5.19
41 2.56
42 >2.49
43 1.62
47 >2.07
实施例4.胰高血糖素类似物的物理稳定性和化学稳定性评估
天然的胰高血糖素在溶液中的物理和化学稳定性极差。天然胰高血糖素水溶液会在数小时至数天内形成凝胶或者纤维状沉淀，并且在多肽浓度较高、外界扰动及含盐情况下更容易凝胶化。此外，天然胰高血糖素的化学不稳定性主要是由于其序列发生异构化(天冬氨酸残基)、脱酰胺化(天冬酰胺、谷氨酰胺残基)及氧化(甲硫氨酸残基)等导致。
1.实验目的
本实施例的目的是评估本公开胰高血糖素类似物在溶液中的物理稳定性和化学稳定性。
2.物理稳定性评估实验方法
本实验通过Thioflavin-T(ThT)荧光测试方法监测原纤维形成。将冻干后的胰高血糖素类似物溶解于0.1N HCl水溶液，浓度1mg/mL。多肽溶液在恒温振荡器上孵育120h(37℃，300rmp)并分别于24h、48h及120h检测溶液原纤维含量。ThT荧光测试中，准确称取8mg ThT粉末溶于50mL PBS缓冲液(1×)，经过0.22μm滤膜过滤后避光保存于4℃备用。使用时用PBS缓冲液(1×)进行1：500的比例稀释配制成工作溶液，在96孔板中每个孔中分别加入100μL工作溶液及5μL待测多肽溶液，混匀后室温孵育20-30min，使用Tecan-Infinite F Plex酶标仪读取ThT荧光信号，激发波长为450nm，发射波长为482nm，每个多肽的测试包含四次生物学重复。
3.化学稳定性评估实验方法
将胰高血糖素类似物冻干粉末溶于磷酸盐缓冲液(10mM磷酸二氢钠，15 mg/mL丙二醇，0.01％吐温-80，pH 7.4)至终浓度1mg/mL(通过测量溶液在280nm的吸光度确定)，将样品置于玻璃瓶中并在4℃和25℃下孵育30天。在第0天、7天、14天、21天和28天取样后利用超高效液相色谱检测样品纯度和降解产物，色谱使用C-18反相色谱柱，UV＝214nm。化合物纯度定义为各色谱图中主峰相对于所有整合峰的总面积的面积百分比，以给定多肽不同时间的纯度参照第0天样品的纯度进行归一化。
4.实验结果
通过以上实验方法，部分本公开胰高血糖素类似物在水溶液中的物理稳定性如图1所示，在磷酸盐缓冲液中的化学稳定性如表6所示。
表6.化合物在磷酸盐缓冲液中的化学稳定性
Figure PCTCN2022099357-appb-000012
(n.d.表示多肽沉淀，纯度未测出。)
通过研究发现，本公开的胰高血糖素类似物与天然胰高血糖素(Glucagon)相比展现出优异的物理稳定性和化学稳定性；其中化合物40在磷酸盐缓冲液中的化学稳定性比Dasiglucagon更优。
实施例5.胰高血糖素类似物在正常血糖大鼠模型体内的药效评估
1.实验目的
本实施例是为对比评估本公开中的胰高血糖素类似物40、天然胰高血糖素(Glucagon)和Dasiglucagon在正常大鼠模型体内的升血糖药效。
2.实验方法
7-8周龄的雄性SD大鼠(Sprague-Dawley rats)适应性饲养一周。根据体重分组，分组后确保每组大鼠的平均体重和标准差相似。实验前4小时及实验过程中禁食，正常给水。完成禁食4小时后，实验组大鼠分别单次皮下给予0.5nmol/kg、 2nmol/kg、6nmol/kg的胰高血糖素类似物40或2nmol/kg的天然胰高血糖素或2nmol/kg的Dasiglucagon，对照组大鼠皮下给予与配置胰高血糖素类似物相同的磷酸盐缓冲液。给药前尾部采血测定T0点血糖，之后分别在15min，30min，45min，60min，75min，90min，105min，120min测定大鼠血糖。
3.实验结果
胰高血糖素类似物40提升大鼠血糖水平呈现剂量依赖性，与天然胰高血糖素、Dasiglucagon都呈现短时起效的特点，但相同剂量下的胰高血糖素类似物40升糖水平更高，具体结果见图2和表7。
表7.化合物在正常大鼠中的升糖数据
Figure PCTCN2022099357-appb-000013
4.实验结论
结果显示，经皮下注射给药后，胰高血糖素类似物40、天然胰高血糖素(Glucagon)和Dasiglucagon均能快速地提高大鼠的血糖水平。0.5nmol/kg剂量的胰高血糖素类似物40与2nmol/kg剂量的Dasiglucagon对血糖提升的效果相近，且同等剂量下(2nmol/kg)，胰高血糖素类似物40的药效明显好于Dasiglucagon。
在正常血糖大鼠模型中，与Dasiglucagon相比，同等剂量条件下本公开中的胰高血糖素类似物40升糖水平更高，药效更好。
实施例6.胰高血糖素类似物在低血糖大鼠模型体内的药效评估
通过皮下注射胰岛素诱导大鼠出现低血糖症状，模拟人发生低血糖场景，再皮下给予胰高血糖素类似物治疗，使血糖快速恢复至正常水平。
1.实验目的
对比Dasiglucagon，评估本公开中的胰高血糖素类似物40在低血糖大鼠模型体内的药效。
2.实验方法
7-8周龄的雄性SD大鼠(Sprague-Dawley rats)适应性饲养一周。根据血糖分组，分组后确保每组大鼠的平均血糖和标准差相似。实验前4小时及实验过程中禁食，正常给水。完成禁食4小时后，尾部采血测定T0点血糖，造模组大鼠立即皮下注射0.65IU/kg胰岛素，非造模组大鼠皮下注射相同体积的生理盐水。 之后在15min，30min，45min测定血糖。在45min时，造模组大鼠分别单次皮下给予6nmol/kg、12nmol/kg、20nmol/kg的胰高血糖素类似物40或12nmol/kg的Dasiglucagon，对照组大鼠和非造模组大鼠皮下给予与配置胰高血糖素类似物40相同的磷酸盐缓冲液。之后继续在60min，75min，90min，105min，120min，150min，180min测定大鼠血糖。
3.实验结果
胰岛素处理后，在大鼠血糖最低点给予本公开中的胰高血糖素类似物40治疗，能迅速恢复大鼠血糖且呈现剂量依赖性，虽然本公开的胰高血糖素类似物40与Dasiglucagon都呈现短时起效的特点，但在相同剂量下本公开中的胰高血糖素类似物40升糖水平更高，具体结果见图3和表8。
表8.化合物在低血糖大鼠模型中的升糖结果
Figure PCTCN2022099357-appb-000014
4.实验结论
结果显示，在胰岛素诱导低血糖大鼠模型中，经皮下注射给药后，胰高血糖素类似物40、天然胰高血糖素(Glucagon)和Dasiglucagon均能在15分钟内快速地恢复并提高大鼠的血糖水平。同等剂量下(12nmol/kg)，胰高血糖素类似物40对血糖的提升效果明显优于Dasiglucagon，并且持续时间更长。
在胰岛素诱导低血糖大鼠模型中，同等剂量条件下，胰高血糖素类似物40相比Dasiglucagon药效更好。
实施例7.胰高血糖素类似物的溶血性实验评估
溶血指红细胞膜被破坏，并且透明度增加呈深红色的现象；某些药物成分，辅料等含有溶血成分，可导致人体的溶血反应，引起局部肿胀，血液循环功能障碍等不良反应。根据红细胞破裂释放出来的血红蛋白在可见光波段具有吸收的原理，将供试化合物溶液加入大鼠的红细胞混悬液，孵育后用酶标仪测定吸光度值，评价溶血程度。
1.实验目的
观察胰高血糖素类似物40是否会引起溶血反应。
2.溶血性实验方法
红细胞混悬液的制备：取100μL新鲜大鼠全血，然后加入900μL 1×PBS溶液，置于平板摇床上，30rpm震摇5min，然后1000g离心5min，弃去上清液；重复以上清洗步骤，直到上清液不显红色为止，供测试用。
供试品溶液的制备：向供试品固体中加入适量1×PBS溶液，溶解得到标准储备液，然后用1×PBS稀释，得到浓度为1μg/mL，3μg/mL，10μg/mL，30μg/mL，100μg/mL和300μg/mL的供试品溶液。同时将空白1×PBS作为阴性对照，将含有0.1％Triton X-100(1μg/mL)的1×PBS溶液作为阳性对照。
孵育过程：加入500μL供试品溶液到红细胞混悬液，平板摇床上，30rpm震摇5min，使其充分混匀，然后将混悬液在37℃下温育1小时，然后1000g速度离心5min，取上清液100μL至酶标板单孔中，测定540nm处的吸光度。
3.实验结果
溶血率(％)计算公式为(供试品吸光度-阴性对照吸光度)/(阳性对照吸光度-阴性对照吸光度)×100％。当结果小于5％，判断无溶血性；当结果大于5％，判断待测物有溶血性。
本公开的胰高血糖素类似物的溶血性检查结果如图4A、图4B和表9所示。结果显示本公开的胰高血糖素类似物40与Dasiglucagon，在1至300μg/mL浓度范围内，溶血性检查均符合规定要求，未发现显著溶血副作用风险。
表9.化合物在人全血中的溶血率％
Figure PCTCN2022099357-appb-000015
实施例8.胰高血糖素类似物的人体肝、肾微粒体稳定性评估
肝脏和肾脏是人体最主要的代谢器官，富含一相和二相代谢酶，会影响药物的代谢速度，对药物的降解清除起重要作用，影响血药浓度和半衰期。肝肾代谢常用的模型包括肝肾微粒体，S9组分，肝细胞，组织匀浆等。体外肝肾代谢稳定性是预测体内药动和药效学性质的主要指标之一。就肝肾微粒体模型而言，其采用从肝脏或肾脏提取的微粒体，加入还原型辅酶(NADPH)再生系统，模拟生理环境下代谢反应，评价测试化合物的体外代谢稳定性，可用于预测药物的体内降解和清除速度。理想条件下，胰高血糖素类似物需要快速吸收和清除，在体内有相对快的降解速度，解救低血糖的同时，避免高血糖的毒副反应。
1.实验目的
比较和评价胰高血糖素类似物在人的肝微粒体和肾微粒体体外代谢系统中的代谢稳定性。
2.人肝微粒、肾微粒体检测实验方法
溶液配置：称取适量化合物纯品，加入适量DMSO溶解分别得到Dasiglucagon和40的1mM供试溶液。
MgCl 2-PBS缓冲溶液配置：取200μL 200mM的PBS缓冲溶液，依次加入106μL H2O和40μL MgCl 2溶液(50mM)，涡旋混匀，得到MgCl 2-PBS缓冲溶液。
稳定性实验过程：样本分别按照以下方式进行处理，
(1)孵育系统准备
a)加入NADPH：取10μL 20mg/mL的肝微粒体和40μL 10mM NADPH溶液加入到孵育系统中，肝微粒体和NADPH的终浓度分别为0.5mg/mL和1mM；
b)不加NADPH：取10μL 20mg/mL的肝微粒体和40μL H 2O加入到孵育系统中。
(2)孵育反应系统中加入4μL 100μM的测试化合物溶液和阳性对照化合物(人肝微粒体采用维拉帕米(Verapamil)，人肾微粒体采用盐酸苄达明)，孵育体系终浓度为1μM，孵育温度为37℃；其中，维拉帕米和盐酸苄达明分别为已知的肝代谢酶和肾代谢酶的底物(参见Pauli-Magnus C等，J Pharmacol Exp Ther.2000May；293(2)：376-82.)，用于确证微粒体孵育系统的酶反应活性；
(3)分别在0min，15min，30min，60min和120min取样50μL，加入4倍体积的含有内标和3％甲酸的乙腈溶液终止反应。样本进一步经过3220g速度离心40min，取100μL上清液，加入等体积的H 2O混合均匀，进HPLC-MS/MS分析。
3.实验结果：
本公开中的胰高血糖素类似物人肝微粒体和肾微粒体的稳定性结果如图5A、图5B和表10、表11所示。
表10.人肝微粒体孵育体系的稳定性结果
Figure PCTCN2022099357-appb-000016
表11.人肾微粒体孵育体系的稳定性结果
Figure PCTCN2022099357-appb-000017
4.实验结论
肝代谢方面，其一，Dasiglucagon和胰高血糖素类似物40在肝微粒体中代谢速度比肾微粒体中慢，代谢与NADPH无关。其二，胰高血糖素类似物40降解比Dasiglucagon稍快(体外T1/2：40为68.76min，Dasiglucagon为161.74min)。
肾代谢方面，其一，Dasiglucagon和胰高血糖素类似物40在肾微粒体中被代谢，但代谢与NADPH无关；其二，胰高血糖素类似物40降解比Dasiglucagon更快(体外T1/2：40为35.73min，Dasiglucagon为51.27min)。
总之，在肝肾微粒体孵育过程中，本公开中的胰高血糖素类似物40比Dasiglucagon的代谢速度更快，因此体内更容易被清除，高血糖副反应的发生风险更低，更有利于糖尿病患者的血糖控制。
实施例9.胰高血糖素类似物的大鼠体内药代动力学评估
动物药代动力学研究一般方法是采用临床拟用的给药途径，给予动物一定剂量的测试化合物，然后采用HPLC，GC，HPLC-MS等分析仪器方法测定血液，尿液等生物基质中药物浓度随着时间的变化情况，从而了解该化合物在体内的动态变化规律，获得血药浓度-时间曲线测定，进而得到药物的药代动力学参数，阐明化合物的吸收，分布，代谢和排泄过程和特点。
1.实验目的
以雄性SD大鼠为受试动物，单次皮下注射给予Dasiglucagon和胰高血糖素类似物40，研究Dasiglucagon和胰高血糖素类似物40在SD大鼠体内(血浆)的药代动力学特征。
2.实验方法
(1)按照表12对动物分组，给药前大鼠自由饮水和进食。
表12.动物实验分组
Figure PCTCN2022099357-appb-000018
(2)采血
采样时间：给药前0min(predose)，给药后2min，4min，7min，10min，20min，40min，1h和2h。从大鼠颈静脉插管采血。
(3)样本采集
1)每个时间点取0.2mL全血，转移至含有EDTA-K2抗凝的离心管中；
2)血样本在4℃下4000g速度离心5min，分离获得血浆；
3)血浆样本储存在-80℃冷冻保存。
(4)生物样本分析
样本前处理方法：取100μL血浆样本，加入300μL含有1ng/mL内标(维拉帕米)甲醇溶液，涡旋5min，低温离心10min，取上清液100μL，加入100μL0.1％甲酸水溶液，混合均匀，进仪器分析；
液相色谱和质谱分析：使用LC-30A液相色谱系统(日本岛津公司)；API 5500型液相色谱-三重四级杆质谱联用仪(Analyst Software数据处理系统，美国AB公司)。色谱柱为nanomicro Unisil C18aq(4.6mm×150mm，5μm)色谱柱；柱温40℃；流动相A为0.1％甲酸水溶液，流动相B为0.1％甲酸乙腈溶液；流速0.5mL/min；进样量20μL；梯度条件如下表13所示。质谱采用电喷雾离子源(ESI)，正离子分析模式，多反应监测(MRM)扫描方式。
表13.液相梯度程序
时间(min) A相(％) B相(％)
0.5 95 5
5.5 5 95
8.5 5 95
8.51 95 5
10.0 95 5
3.实验结果
采用以上HPLC-MS/MS方法测定血浆中各待测物的血药浓度，绘制血药浓度时间曲线，并用PKSolver软件计算药动学参数。通过以上实验方法，本公开的胰高血糖素类似物的药代动力学(PK)参数如表14和图6所示。
表14.皮下注射(SC)给药后Dasiglucagon和40的药动学参数比较
Figure PCTCN2022099357-appb-000019
4.实验结论
结果显示，经皮下注射给药后，胰高血糖素类似物40的T1/2明显短于Dasiglucagon(40的T 1/2为0.41h，Dasiglucagon的T 1/2为0.51h)，说明胰高血糖素类似物40的清除速度更快；胰高血糖素类似物40的T max也短于Dasiglucagon(40的T max为0.117h，Dasiglucagon的T max为0.167h)，说明胰高血糖素类似物40的吸收速度要明显比Dasiglucagon更快。
总之，与Dasiglucagon相比，胰高血糖素类似物40能够更快地吸收起效和更迅速的清除，具有更优的药代动力学特性。

Claims (16)

  1. 胰高血糖素类似物或其可药用盐和/或溶剂化物，其是式(I)结构所示：
    R 1-His-Ser-X 3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-X 15-X 16-X 17-Arg-Ala-X 20-X 21-Phe-Val-X 24-Trp-Leu-X 27-X 28-Thr-R 2
    (I)
    其中：
    R 1是氢、C 1-4烷基、乙酰基、甲酰基、苯甲酰基、三氟乙酰基或pGlu；
    R 2是-OH或-NH 2；
    X 3、X 15、X 16、X 20、X 21、X 24、X 27和X 28相互独立地选自任意天然氨基酸残基或非天然氨基酸残基；X 17是Aib。
  2. 根据权利要求1所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：
    X 3选自His、Dap(Ac)或Gln；
    X 15选自Asp或Glu；
    X 16选自Ser、Thr、Leu、Val、Ile或α-甲基-Ser；
    X 20选自Ala、Gln、Glu、Ser、Thr或Lys；
    X 21选自Asp或Glu；
    X 24选自Ala、Gln、Ser、Glu、α-甲基-Ser或Arg；
    X 27选自Met、Glu、Nle、Leu或Ser；
    X 28选自Asn、Glu或Ser。
  3. 根据权利要求1或2所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：
    X 15是Asp；
    X 16选自Thr、Leu、Val或Ile。
  4. 根据权利要求1-3中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：X 20是Gln。
  5. 根据权利要求1-4中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：X 24选自Gln或Glu。
  6. 根据权利要求1-4中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：X 27是Glu；X 28是Ser。
  7. 根据权利要求1-6中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：
    X 3选自His或Gln；
    X 15是Asp；
    X 16选自Thr、Leu、Val或Ile；
    X 17是Aib；
    X 20是Gln；
    X 21是Glu；
    X 24选自Gln或Glu；
    X 27是Glu；
    X 28是Ser。
  8. 根据权利要求7所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，其中：
    R 1是氢；R 2是-OH或-NH 2，优选-OH。
  9. 根据权利要求1-8中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，所述胰高血糖素类似物选自以下任一项的化合物：
    H-HSQGTFTSDYSKYLDLAibRAQEFVQWLEST-OH(SEQ ID NO：40)；
    H-HSQGTFTSDYSKYLDTAibRAQEFVQWLEST-OH(SEQ ID NO：39)；
    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)。
  10. 药物组合物，其包含：
    根据权利要求1-9中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物，以及
    一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  11. 根据权利要求10所述的药物组合物，其进一步包括至少一种具有代谢性疾病治疗活性的化合物。
  12. 根据权利要求11所述的药物组合物，其中所述具有代谢性疾病治疗活性的化合物选自以下中的一种或多种：葡萄糖依赖性促胰岛素肽(GIP)、胰高血糖素样肽-1(GLP-1)受体激动剂、胰岛素、肠高血糖素、神经肽Y5受体拮抗剂、乙酰辅酶A羧化酶抑制剂、Leptin受体激动剂、格列奈、α-葡萄糖苷酶抑制剂(AGi)、噻唑烷二酮(TZD)、二肽基肽酶-4抑制剂类(DPP-IV)、钠葡萄糖协同转运蛋白2抑制剂(SGLT-2)、法尼醇X受体(FXR)激动剂和肥胖抑制素(Obestatin)。
  13. 权利要求1-9中任一项所述的胰高血糖素类似物或其可药用盐和/或溶剂化物或权利要求10-12中任一项所述的药物组合物在制备用于治疗疾病或病症的药物中的用途；
    优选地，所述疾病或病症选自低血糖、高血糖症、2型糖尿病、1型糖尿病、冠心病、动脉粥样硬化、β-受体阻滞剂中毒、胰岛素瘤、冯吉尔克病、葡萄糖耐量减低、血脂异常、高血压、体重超重、暴食症和肝脂肪变性。
  14. 根据权利要求13所述的用途，其中所述低血糖是病理性低血糖或非病理性低血糖。
  15. 根据权利要求13所述的用途，其中所述低血糖选自糖尿病性低血糖、非糖尿病性低血糖、空腹低血糖、药物诱发性低血糖、急性胰岛素诱发低血糖、胃旁路术诱发性低血糖、酒精诱发性低血糖、反应性低血糖和妊娠期低血糖。
  16. 制备权利要求1-9任一项胰高血糖素类似物或其可药用盐和/或溶剂化物的方法，其包括采用固相合成、液相合成或细胞重组表达的方法制备所述胰高血糖素类似物或其可药用盐和/或溶剂化物。
PCT/CN2022/099357 2021-06-18 2022-06-17 胰高血糖素类似物及其医药用途 Ceased WO2022262837A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
KR1020247000856A KR20240022551A (ko) 2021-06-18 2022-06-17 글루카곤 유사체 및 이의 의학적 용도
EP22824309.3A EP4357358A4 (en) 2021-06-18 2022-06-17 GLUCAGONE ANALOGUE AND MEDICAL USE THEREOF
CA3221829A CA3221829A1 (en) 2021-06-18 2022-06-17 Glucagon analog and medical use thereof
CN202280032659.8A CN117242087A (zh) 2021-06-18 2022-06-17 胰高血糖素类似物及其医药用途
MX2023015068A MX2023015068A (es) 2021-06-18 2022-06-17 Analogo de glucagon y uso medico del mismo.
JP2023576365A JP2024525144A (ja) 2021-06-18 2022-06-17 グルカゴン類似体及びその医薬用途
US18/570,551 US20240279298A1 (en) 2021-06-18 2022-06-17 Glucagon analog and medical use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110676681.3 2021-06-18
CN202110676681 2021-06-18

Publications (1)

Publication Number Publication Date
WO2022262837A1 true WO2022262837A1 (zh) 2022-12-22

Family

ID=84526889

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/099357 Ceased WO2022262837A1 (zh) 2021-06-18 2022-06-17 胰高血糖素类似物及其医药用途

Country Status (9)

Country Link
US (1) US20240279298A1 (zh)
EP (1) EP4357358A4 (zh)
JP (1) JP2024525144A (zh)
KR (1) KR20240022551A (zh)
CN (1) CN117242087A (zh)
CA (1) CA3221829A1 (zh)
MX (1) MX2023015068A (zh)
TW (1) TW202317606A (zh)
WO (1) WO2022262837A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119954932A (zh) * 2025-04-10 2025-05-09 广州呼吸药物工程技术有限公司 一种含β-氨基酸的胰高血糖素类似物或其药学上可接受的盐及其应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011125A1 (en) 1996-09-09 1998-03-19 Zealand Pharmaceuticals A/S Improved solid-phase peptide synthesis and agent for use in such synthesis
WO2012153196A2 (en) * 2011-05-10 2012-11-15 Zealand Pharma A/S Glu-glp-1 dual agonist signaling-selective compounds
WO2013098408A1 (en) * 2011-12-30 2013-07-04 Zealand Pharma A/S Glucagon and cck receptor agonist peptide conjugates
WO2013192129A1 (en) * 2012-06-21 2013-12-27 Indiana University Research And Technology Corporation Glucagon analogs exhibiting gip receptor activity
WO2014016300A1 (en) * 2012-07-23 2014-01-30 Zealand Pharma A/S Glucagon analogues
WO2015185640A1 (en) * 2014-06-04 2015-12-10 Novo Nordisk A/S Glp-1/glucagon receptor co-agonists for medical use
WO2019101035A1 (zh) * 2017-11-24 2019-05-31 浙江道尔生物科技有限公司 一种治疗代谢疾病的胰高血糖素类似物
WO2020163125A1 (en) * 2019-02-05 2020-08-13 Eli Lilly And Company Glucagon analog agonists and methods of using the same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011163473A1 (en) * 2010-06-25 2011-12-29 Indiana University Research And Technology Corporation Glucagon analogs exhibiting enhanced solubility and stability in physiological ph buffers
HK1211232A1 (zh) * 2012-12-21 2016-05-20 Sanofi 作为glp1/gip双重激动剂或glp1/gip/胰高血糖素三重激动剂的毒蜥外泌肽-4衍生物

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011125A1 (en) 1996-09-09 1998-03-19 Zealand Pharmaceuticals A/S Improved solid-phase peptide synthesis and agent for use in such synthesis
WO2012153196A2 (en) * 2011-05-10 2012-11-15 Zealand Pharma A/S Glu-glp-1 dual agonist signaling-selective compounds
WO2013098408A1 (en) * 2011-12-30 2013-07-04 Zealand Pharma A/S Glucagon and cck receptor agonist peptide conjugates
WO2013192129A1 (en) * 2012-06-21 2013-12-27 Indiana University Research And Technology Corporation Glucagon analogs exhibiting gip receptor activity
WO2014016300A1 (en) * 2012-07-23 2014-01-30 Zealand Pharma A/S Glucagon analogues
WO2015185640A1 (en) * 2014-06-04 2015-12-10 Novo Nordisk A/S Glp-1/glucagon receptor co-agonists for medical use
WO2019101035A1 (zh) * 2017-11-24 2019-05-31 浙江道尔生物科技有限公司 一种治疗代谢疾病的胰高血糖素类似物
WO2020163125A1 (en) * 2019-02-05 2020-08-13 Eli Lilly And Company Glucagon analog agonists and methods of using the same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., vol. 243, 1968, pages 3558
PAULI-MAGNUS C ET AL., J PHARMACOL EXP THEN, vol. 293, no. 2, May 2000 (2000-05-01), pages 376 - 82
See also references of EP4357358A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119954932A (zh) * 2025-04-10 2025-05-09 广州呼吸药物工程技术有限公司 一种含β-氨基酸的胰高血糖素类似物或其药学上可接受的盐及其应用
CN119954932B (zh) * 2025-04-10 2025-07-22 广州呼吸药物工程技术有限公司 一种含β-氨基酸的胰高血糖素类似物或其药学上可接受的盐及其应用

Also Published As

Publication number Publication date
TW202317606A (zh) 2023-05-01
CA3221829A1 (en) 2022-12-22
EP4357358A4 (en) 2024-11-06
JP2024525144A (ja) 2024-07-10
EP4357358A1 (en) 2024-04-24
US20240279298A1 (en) 2024-08-22
CN117242087A (zh) 2023-12-15
KR20240022551A (ko) 2024-02-20
MX2023015068A (es) 2024-01-17

Similar Documents

Publication Publication Date Title
KR102129235B1 (ko) 글루카곤 유사체
US9764004B2 (en) Glucagon receptor agonists
EP3398961B1 (en) Triple activator activating glucagon, glp-1 and gip receptor
JP6612251B2 (ja) エキセンジン−４に由来するペプチド二重ｇｌｐ−１／グルカゴン受容体アゴニスト
CN102197049B (zh) 胰岛淀粉样多肽衍生物
WO2022111370A1 (zh) 一类glp-1/胰高血糖素受体双重激动剂及其应用
TW201609797A (zh) 雙重ｇｌｐ－１／升糖素受體促效劑
JP2017519718A (ja) ペプチド二重ｇｌｐ−１／グルカゴン受容体アゴニストとしてのエキセンジン−４誘導体
TW201609800A (zh) 做為雙重ｇｌｐ－１／升糖素受體促效劑之艾塞那肽－４胜肽類似物
JP2016503770A (ja) 官能基化されたエキセンディン−４誘導体
JP2017517483A (ja) エキセンジン−４に由来する二重ｇｌｐ−１／グルカゴン受容体アゴニスト
KR20210024081A (ko) 당뇨병을 위한 gip/glp1 공효능제의 사용 방법
EP4345105A1 (en) Polypeptide derivative having effect of dual targeted activation of glp-1r and gipr, preparation method therefor, and use thereof
EA038073B1 (ru) Ацилированное инсулиновое соединение
EP2631244A1 (en) Amylin family polypeptide-6 (AFP-6) analogs and methods of making and using them
TW202409070A (zh) 芳族含硼化合物及相關胰島素類似物
WO2022262837A1 (zh) 胰高血糖素类似物及其医药用途
IL322205A (en) GIP/GLP1/GCG trireceptor agonists and their uses
KR20200080331A (ko) 글루카곤 유사체
HK1210787B (zh) 胰高血糖素类似物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22824309

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202280032659.8

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 3221829

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2023576365

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 202317085167

Country of ref document: IN

Ref document number: MX/A/2023/015068

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 18570551

Country of ref document: US

ENP Entry into the national phase

Ref document number: 20247000856

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020247000856

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2022824309

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022824309

Country of ref document: EP

Effective date: 20240118