WO2021136303A1 - 长效glp-1化合物 - Google Patents

长效glp-1化合物 Download PDF

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Publication number
WO2021136303A1
WO2021136303A1 PCT/CN2020/141057 CN2020141057W WO2021136303A1 WO 2021136303 A1 WO2021136303 A1 WO 2021136303A1 CN 2020141057 W CN2020141057 W CN 2020141057W WO 2021136303 A1 WO2021136303 A1 WO 2021136303A1
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Prior art keywords
human insulin
γglu
desb30 human
diacyl
ethoxy
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PCT/CN2020/141057
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English (en)
French (fr)
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甘忠如
陈伟
张一宁
薛方凯
蔡玲玉
牛江红
穆彬
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甘李药业股份有限公司
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Priority to CA3166496A priority Critical patent/CA3166496A1/en
Priority to BR112022013042A priority patent/BR112022013042A2/pt
Priority to AU2020418207A priority patent/AU2020418207A1/en
Priority to MX2022008139A priority patent/MX2022008139A/es
Priority to KR1020227026310A priority patent/KR20220119731A/ko
Priority to EP20909624.7A priority patent/EP4086277A4/en
Priority to JP2022540870A priority patent/JP2023510218A/ja
Priority to CN202080091240.0A priority patent/CN114901680B/zh
Priority to US17/758,113 priority patent/US20240239859A1/en
Publication of WO2021136303A1 publication Critical patent/WO2021136303A1/zh

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    • 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
    • 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
    • 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
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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

Definitions

  • the present invention relates to the field of therapeutic peptides, in particular to a new long-acting GLP-1 compound, its pharmaceutical preparation, its pharmaceutical composition with long-acting insulin, and the medical use of the compound, pharmaceutical preparation and pharmaceutical composition.
  • Glucagon-like peptide 1 (GLP-1) and its analogs and derivatives are very effective in the treatment of type 1 and 2 diabetes, but the high clearance rate limits the effectiveness of these compounds.
  • GLP-1 glucagon-like peptide 1
  • WO99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives with lipophilic substitutions attached to the C-terminal amino acid residue.
  • WO00/34331 discloses acylated GLP-1 analogs.
  • WO00/69911 discloses activated insulinotropic peptides for injection into a patient.
  • the currently marketed GLP-1 drugs include, for example, the natural GLP-1 analog Exenatide (Exenatide), which is administered twice a day; Liraglutide and Lisna, which are administered once a day.
  • Peptide (Lixisenatide) the former is a GLP-1 compound modified with hexadecanoic acid, and the latter is a new molecule obtained by structural modification of exenatide; and Semaglutide (Semaglutide) administered once a week , Exenatide LAR, Abiglutide, Dulaglutide (also known as dulaglutide) and polyethylene glycol losenatide.
  • exenatide microspheres are prepared by encapsulating exenatide into a polylactic acid glycolic acid copolymer matrix by a microencapsulation method, and abiglutide is prepared by dividing two modified GLP-1 peptide chains into two
  • the polymer form is fused with human albumin to become a recombinant fusion protein;
  • Dulaglutide is obtained by fusing the modified GLP-1 chain to the Fc fragment of recombinant G4 immunoglobulin through disulfide bonds;
  • polyethylene glycol loxena Peptide is modified by amino acid modification based on the chemical structure of Exenatide and modified by polyethylene glycol;
  • Somaglutide is mainly replaced by non-protein amino acid Aib on GLP-1(7-37) peptide Ala at position 8 is achieved once a week, but the presence of non-protein amino acids in somaglutide may have unknown and various potential side effects in the human body compared to natural amino acids.
  • a compound preparation containing two active ingredients of insulin and GLP-1 peptide may be a very effective therapeutic agent. Therefore, there is still a need for synergistic realization of compound preparations with better physical and chemical stability, longer action time, and better efficacy.
  • the first aspect of the present invention provides a new GLP-1 compound (also called a GLP-1 derivative).
  • a new GLP-1 compound also called a GLP-1 derivative.
  • the new GLP-1 compound has better potency, pharmacodynamics or efficacy, and smaller potential The risk of side effects, better weight loss effects, longer duration of action or half-life in vivo, better or equivalent GLP-1 receptor binding affinity, and better or equivalent DPP-IV stability.
  • the long-acting GLP-1 compound of the present invention and the pharmaceutical composition or combined preparation of the long-acting insulin provided by the present invention will not weaken the physical stability of the GLP-1 compound and the insulin compound, and the combined preparation has Better physical stability than single-drug formulations.
  • the physical stability of the combination preparation of the present invention is unexpected.
  • the combined preparation also increases the chemical stability of the GLP-1 compound and the acylated insulin compared to the single-drug preparation.
  • Both the GLP-1 compound of the present invention and the combined preparations containing the GLP-1 compound and the pancreatic islet compound provided by the present invention can achieve long pharmacokinetics (hereinafter also referred to as PK) characteristics, making it difficult for diabetic patients Subcutaneous treatments twice a week, once a week, once fortnightly, or less frequently are possible.
  • PK pharmacokinetics
  • the GLP-1 compound provided in the first aspect of the present invention is a compound of formula B, or a pharmaceutically acceptable salt, amide or ester thereof:
  • G1 is a GLP-1 analogue with Arg at position 34 corresponding to GLP-1(7-37) (SEQ ID NO:1) and Ala or Gly at position 8
  • [Acy-(L1) r -(L2) q ] is a substituent attached to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog, wherein
  • r is an integer of 1-10
  • q is an integer of 0 or 1-10
  • Acy is an aliphatic diacid containing 20-24 carbon atoms, in which the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid;
  • L1 is an amino acid residue selected from: ⁇ Glu, ⁇ Glu, ⁇ Asp, ⁇ Asp, ⁇ -D-Glu, ⁇ -D-Glu, ⁇ -D-Asp or ⁇ -D-Asp;
  • L2 is a neutral amino acid residue containing alkylene glycol
  • G1 is [Gly8,Arg34]GLP-1-(7-37) peptide or [Arg34]GLP-1-(7-37) peptide, preferably [Gly8,Arg34]GLP-1-( 7-37) Peptides.
  • r is 1, 2, 3, 4, 5 or 6, preferably r is 1, 2, 3 or 4, preferably r is 1 or 2, preferably r is 1.
  • q is 0, 1, 2, 3, 4, 5, 6, 7, or 8, preferably, q is 0, 1, 2, 3, or 4, more preferably, q is 0, 1. , Or 2.
  • Acy is an aliphatic diacid containing 20-23 carbon atoms, preferably Acy is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein the hydroxyl group has been formally removed from the fat One of the carboxyl groups of the diacid is removed.
  • L2 is: -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-CH 2 -CO-, -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-, -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-, -HN-(CH 2 ) 2 -O-( CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-, -HN-(CH 2 )
  • L1 is selected from ⁇ Glu or ⁇ Asp, preferably L1 is ⁇ Glu.
  • Acy is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 19 -CO-, HOOC-(CH 2 ) 20 -CO-, HOOC-(CH 2 ) 21 -CO -Or HOOC-(CH 2 ) 22 -CO-, Acy is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 20 -CO- or HOOC-(CH 2 ) 22 -CO-.
  • Acy, L1, and L2 in formula (B) are sequentially connected by an amide bond, and the C-terminus of L2 is connected to the epsilon amino group of the Lys residue at position 26 of the GLP-1 analog.
  • the compound of the first aspect of the present invention is selected from the following compounds:
  • the compound of the first aspect of the present invention is selected from the following compounds:
  • the compound of the first aspect of the present invention is selected from the following compounds:
  • the second aspect of the present invention provides a pharmaceutical preparation comprising the compound described in the first aspect of the present invention and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient is selected from one or more of buffering agents, preservatives, isotonic agents, stabilizers and chelating agents. In another embodiment, the pharmaceutically acceptable excipients are buffers, preservatives and isotonic agents.
  • the pharmaceutical preparation comprises: the compound according to the first aspect of the present invention, an isotonic agent, a preservative and a buffering agent.
  • the compound according to the first aspect of the present invention is N- ⁇ 26 -[2-(2-[2-(4-[19-carboxynonadenoylamino]-4( S)-Carboxybutyrylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide, or N- ⁇ 26 -[2-(2-[2- (2-[2-(2-[4-(21-carboxyeicosananoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy] Ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide.
  • the isotonic agent is selected from one or more of sodium chloride, propylene glycol, mannitol, sorbitol, glycerol, glucose and xylitol, preferably propylene glycol, mannitol or sodium chloride.
  • the preservative is selected from phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2 -One or more of phenethyl alcohol and benzyl alcohol, preferably phenol or m-cresol.
  • the buffer is selected from sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, dihydrogen phosphate
  • sodium, sodium phosphate, and tris(hydroxymethyl)-aminomethane preferably sodium acetate, citrate, sodium dihydrogen phosphate, or disodium hydrogen phosphate.
  • the pH of the formulation is from about 6.0 to about 10.0, preferably from about 6.5 to about 10.0, preferably from about 6.5 to about 9.5, preferably from about 6.5 to about 8.5, more preferably from about 7.0 to about 8.5 , More preferably about 7.0 to about 8.1, still more preferably about 7.3 to about 8.1.
  • the pharmaceutical formulation contains the following ingredients:
  • the Isotonic agent is selected from one or more of propylene glycol, glycerin, mannitol or sodium chloride;
  • the agent is selected from one or more of phenol or m-cresol;
  • the pH of the pharmaceutical preparation is from about 6.0 to about 10.0, preferably from about 6.5 to about 9.5, preferably from about 6.5 to about 8.5, more preferably from about 7.0 to about 8.5, more preferably from about 7.0 to about 8.1, further preferably About 7.3 to about 8.1.
  • the pharmaceutical preparation contains: about 0.3-0.7 mM, more preferably about 0.48-0.6 mM N- ⁇ 26 -[2-(2-[2-(2-[2-(2 -[4-(21-Carboxydocanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][ Gly8,Arg34]GLP-1-(7-37) peptide or N- ⁇ 26 -[2-(2-[2-(4-[19-carboxynonadenoylamino]-4(S)-carboxybutane Acylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7-37) peptide; about 180-200mM, more preferably about 183-195mM propylene glycol; about 45-60mM, more Preferably about 50-60 mM phenol; about 5
  • the pharmaceutical preparation contains: about 0.5 mM N- ⁇ 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxyl twenty-one Alkanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-(7- 37) Peptide or N- ⁇ 26 -[2-(2-[2-(4-[19-carboxynonadenoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy) Acetyl] [Gly8, Arg34] GLP-1-(7-37) peptide; about 184 mM propylene glycol; about 58.5 mM phenol; about 10 mM disodium hydrogen phosphate; and the pH of the pharmaceutical preparation is about 6.5 to about 8.5, more preferably about 7.0 to
  • the pharmaceutical preparation contains: about 2.0 mg/ml of N- ⁇ 26 -[2-(2-[2-(2-[2-(2-[4-(21-carboxydi Undecanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][Gly8,Arg34]GLP-1-( 7-37) peptide or N- ⁇ 26 -[2-(2-[2-(4-[19-carboxynonadenoylamino]-4(S)-carboxybutyrylamino)ethoxy]ethoxy Group) acetyl] [Gly8, Arg34] GLP-1-(7-37) peptide; about 14 mg/ml propylene glycol; about 5.5 mg/ml phenol; about 1.42 mg/ml disodium hydrogen phosphate; and
  • the pH of the pharmaceutical preparation is from about 6.5 to about 8.5, more preferably from about 7.0 to about 8.5, more preferably from about 7.0 to about 8.1, further preferably from about 7.3 to about 8.1.
  • the third aspect of the present invention provides a pharmaceutical composition comprising the GLP-1 compound described in the first aspect of the present invention and acylated insulin.
  • the acylated insulin is B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-OEG), desB30 human insulin; A14E, B16H, B25H, B29K(N( ⁇ )-20 Alkanedioyl- ⁇ Glu-2xOEG), desB30 human insulin; or B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-12xPEG), desB30 human insulin.
  • the acylated insulin is an insulin: the insulin parent of the acylated insulin is a naturally-occurring insulin or an insulin analog and contains at least one lysine residue, and the acylated insulin
  • the acyl moiety of is connected to the lysine residue of the insulin parent or the amino group of the N-terminal amino acid residue, and the acyl moiety is as shown in formula (A):
  • I is a neutral amino acid containing alkylene glycol Residue;
  • II is an acidic amino acid residue;
  • III is an aliphatic diacid containing 20-24 carbon atoms, in which formally the hydroxyl group has been removed from one of the carboxyl groups of the aliphatic diacid; III, II, And I are connected by an amide bond; and the order in which II and I appear in formula (A) can be independently interchanged.
  • n is 5-15, preferably n is 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, preferably n is 5, 6, 7, 8, 9, 10, 11 or 12, preferably n is 5, 6, 7, 8, 9, or 10, preferably n is 5, 6, 7, 8 or 9, preferably n is 5, 6, 7, or 8.
  • n is 1-6, preferably m is 1, 2, 3, or 4, preferably m is 1 or 2, preferably m is 1.
  • III is an aliphatic diacid containing 20-23 carbon atoms, preferably III is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein the hydroxyl group has been formally removed from the Remove one of the carboxyl groups of aliphatic diacids.
  • the insulin parent contains a lysine residue.
  • I is: -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-CH 2 -CO-, -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-, -HN-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-, -HN-(CH 2 ) 2 -O-( CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -CO-,
  • II is an amino acid residue selected from: ⁇ Glu, ⁇ Glu, ⁇ Asp, ⁇ Asp, ⁇ -D-Glu, ⁇ -D-Glu, ⁇ -D-Asp or ⁇ -D-Asp; Preferably, II is selected from ⁇ Glu or ⁇ Asp.
  • III is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 19 -CO-, HOOC-(CH 2 ) 20 -CO-, HOOC-(CH 2 ) 21- CO- or HOOC-(CH 2 ) 22 -CO-, preferably III is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 20 -CO- or HOOC-(CH 2 ) 22 -CO- .
  • formula (A) is linked to the amino group of the lysine residue or the N-terminal amino acid residue of the insulin parent through the C-terminus of I.
  • the acyl moiety is linked to the epsilon amino group of the lysine residue of the insulin parent.
  • the lysine residue of the insulin parent is located at position B29.
  • the insulin parent is selected from the following insulins or insulin analogues: desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 5, respectively representing the A chain and the B chain); A14E, B16H, B25H ,desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 7, respectively representing the A chain and B chain); A14E, B16E, B25H, desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 9, respectively, representing A chain and B chain); human insulin (SEQ ID NO: 10 and SEQ ID NO: 11, representing the A chain and B chain, respectively); A21G human insulin (SEQ ID NO: 12 and SEQ ID NO: 13, respectively, representing A Chain and B chain); A21G, desB30 human insulin (SEQ ID NO: 14 and SEQ ID NO: 15, representing the A chain and B chain respectively); or B28D human insulin (SEQ ID NO: 16 and SEQ ID NO: 17, (Respectively represent A chain and B chain).
  • desB30 human insulin SEQ ID
  • the acylated insulin is selected from the following insulins: B29K(N( ⁇ )-eicosandioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosandi Acyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl-5xOEG- ⁇ Glu), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl-6xOEG- ⁇ Glu), desB30 Human insulin; B29K (N( ⁇ )-eicosane diacyl-6xOEG- ⁇ Glu), desB30 Human insulin; B29K (N( ⁇ )-eicos
  • the inventor unexpectedly discovered that the pharmaceutical composition of the compound and acylated insulin according to the first aspect of the present invention not only does not impair the physical stability of the compound, but also that the combined preparation has better physical properties than the single-drug preparation. stability. Compared with other long-acting insulin derivatives (for example, insulin deglubber and liraglutide), the physical stability of the combined preparation of the present invention is unexpected. In addition, the combined preparation also increases the chemical stability of the acylated insulin compared to the single-drug preparation.
  • the fourth aspect of the present invention provides the use of the compound according to the first aspect of the present invention, the pharmaceutical preparation according to the second aspect of the present invention, or the pharmaceutical composition according to the third aspect of the present invention as a medicine.
  • the compound according to the first aspect of the present invention, the pharmaceutical preparation according to the second aspect of the present invention, or the pharmaceutical composition according to the third aspect of the present invention are used to treat or prevent hyperglycemia, diabetes, and / Or obesity.
  • the fifth aspect of the present invention provides that the compound according to the first aspect of the present invention, the pharmaceutical preparation according to the second aspect of the present invention, or the pharmaceutical composition according to the third aspect of the present invention are prepared for the treatment or prevention of hyperglycemia, Use in medicine for diabetes and/or obesity.
  • the sixth aspect of the present invention provides a method for treating or preventing hyperglycemia, diabetes, and/or obesity, the method comprising administering an effective amount of the compound according to the first aspect of the present invention, the second aspect of the present invention
  • the diseases include, but are not limited to, for example, hyperglycemia, diabetes, and obesity.
  • Figure 1a shows the hypoglycemic effect and time of action of the title compound, liraglutide, and vehicle of Examples 1-3 of the present invention on db/db mice, where the percentage of the ordinate refers to The baseline blood glucose before the drug is the benchmark, and the blood glucose at each monitoring point after administration is compared with the blood glucose percentage at the corresponding time point (the same below).
  • Figure 1b corresponds to Figure 1a showing the AUC of the hypoglycemic effect of the title compound, liraglutide, and vehicle of Examples 1-3 of the present invention on db/db mice.
  • Figure 2a shows the hypoglycemic effect and action time of the title compound, somaglutide, and vehicle of Example 2 of the present invention on db/db mice.
  • Figure 2b corresponds to Figure 2a showing the AUC of the hypoglycemic effect of the title compound, somaglutide, and vehicle of Example 2 of the present invention on db/db mice.
  • Figure 3a shows the hypoglycemic effect and action time of the title compound, liraglutide, and vehicle of Example 3-4 of the present invention on db/db mice.
  • Figure 3b corresponds to Figure 3a showing the AUC of the hypoglycemic effect of the title compound, liraglutide, and vehicle of Example 3-4 of the present invention on db/db mice.
  • Figure 4a shows the hypoglycemic effect and time of action of the title compound of Example 1-3, the title compound of Comparative Example 3-4, and the vehicle on db/db mice of the present invention.
  • Figure 4b corresponds to Figure 4a showing the AUC of the hypoglycemic effect of the title compound of Example 1-3 of the present invention, the title compound of Comparative Example 3-4, and the vehicle on db/db mice.
  • Figure 5a shows the effect of the title compound of Example 11 of the present invention at 100 ⁇ g/kg and 300 ⁇ g/kg, the title compound of Comparative Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal small mice. Rat (normal control) hypoglycemic effect and duration of action.
  • Figure 5b corresponding to Figure 5a shows the effect of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal control ) AUC of hypoglycemic effect.
  • Figure 5c shows the weight loss effects of the title compound of Example 11, the title compound of Comparative Example 2, and the vehicle (model control group) on obese C57BL mice or normal mice (normal control) induced by a high-fat diet.
  • Figure 6a shows the title compound of Example 11 of the present invention, the title compound of Comparative Example 2, and the vehicle (model control group) to obese C57BL mice or normal mice (normal control) induced by a high-fat diet after the first administration. Hypoglycemic effect when ipGTT is performed in hours.
  • Figure 6b corresponds to Figure 6a showing the effects of the title compound of Example 11 of the present invention, the title compound of Control Example 2, and the vehicle (model control group) on high-fat diet-induced obese C57BL mice or normal mice (normal Control) ⁇ AUC of the hypoglycemic effect when ipGTT was administered 48 hours after the first administration.
  • Figure 7a shows the hypoglycemic effect of the title compound of Example 2 of the present invention, the title compound of Comparative Example 2 and the vehicle on db/db mice.
  • Fig. 7b shows the ⁇ AUC of the hypoglycemic effect of the title compound of Example 2 of the present invention, the title compound of Comparative Example 2 and the vehicle on db/db mice corresponding to Fig. 7a.
  • Figure 7c shows the control effects of the title compound of Example 2 of the present invention, the title compound of Comparative Example 2 and the vehicle on the food intake of db/db mice.
  • Figure 7d shows the control effect of the title compound of Example 2 of the present invention, the title compound of Comparative Example 2 and the solvent on the drinking water volume of db/db mice
  • Figure 8a shows the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2 and the vehicle on db/db mice.
  • Figure 8b corresponds to Figure 8a showing the AUC of the long-term hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2 and the vehicle on db/db mice.
  • Figure 8c shows the long-term weight loss effects of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2 and the vehicle on db/db mice.
  • Figure 8d shows the control effect of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2 and the vehicle on the long-term food intake of db/db mice.
  • Figure 8e shows the control effect of the title compound of Example 11 of the present invention, the title compound of Comparative Example 2 and the vehicle on the long-term water intake of db/db mice.
  • Figure 9a shows the hypoglycemic effect of the title compound of Example 11 of the present invention, the title compound of Example 2, dulaglutide and vehicle on Kkay mice.
  • Figure 9b corresponds to Figure 9a showing the AUC of the hypoglycemic effect of the title compound of Example 11, the title compound of Example 2, dulaglutide and vehicle on Kkay mice.
  • Figure 9c shows the HbA1c-reducing effect of the title compound of Example 11, the title compound of Example 2, dulaglutide and vehicle on Kkay mice of the present invention.
  • Figure 10a shows the long-term hypoglycemic effect of the title compound, dulaglutide, and vehicle (model control group) of Example 11 of the present invention on db/db mice or normal mice (normal control).
  • Figure 10b corresponds to Figure 10a showing the long-term hypoglycemic effect of the title compound, dulaglutide, and vehicle (model control group) in db/db mice or normal mice (normal control) of Example 11 of the present invention The delta AUC of the effect.
  • Figure 10c shows the administration of the title compound of Example 11 of the present invention, dulaglutide, and vehicle (model control group) before, third and fifth injections in db/db mice or normal mice (normal control). Random blood glucose values after the first and eleventh injections.
  • Figure 10d shows the title compound, dulaglutide, and vehicle (model control group) of Example 11 of the present invention when ipGTT was performed 48 hours after the first administration of db/db mice or normal mice (normal control group) Hypoglycemic effect.
  • Figure 10e corresponds to Figure 10d showing the title compound, dulaglutide, and vehicle (model control group) of Example 11 of the present invention after the first administration to db/db mice or normal mice (normal control group) AUC of hypoglycemic effect when ipGTT was performed for 48 hours.
  • Figure 11a shows the long-term weight loss effect of the title compound, dulaglutide, and vehicle (model control group) of Example 11 of the present invention on obese C57BL mice or normal mice (normal control group) induced by a high-fat diet.
  • Figure 11b shows the control effect of the title compound, dulaglutide and vehicle (model control group) of Example 11 of the present invention on the long-term food intake of obese C57BL mice induced by a high-fat diet.
  • Figure 11c shows the effect of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) on the reduction of periovarian fat in obese C57BL female mice induced by high-fat diet.
  • Figure 11d shows the effect of the title compound of Example 11 of the present invention, dulaglutide and vehicle (model control group) on epididymal fat reduction in obese C57BL male mice induced by a high-fat diet.
  • GLP-1 analog refers to a peptide or compound that is a variant of human glucagon-like peptide-1 (GLP-1(7-37)), wherein One or more amino acid residues of GLP-1(7-37) are replaced, and/or one or more amino acid residues are deleted, and/or one or more amino acid residues are added.
  • GLP-1 (7-37) is shown in SEQ ID NO:1 in the sequence list.
  • the peptide having the sequence shown in SEQ ID NO: 1 can also be referred to as "natural" GLP-1 or "natural” GLP-1 (7-37).
  • the amino acid residue numbering or position numbering of the GLP-1(7-37) sequence referred to herein is the sequence of His starting at position 7 and Gly ending at position 37.
  • [Gly8, Arg34] GLP-1-(7-37) peptide is a GLP with Gly and Arg at positions 8 and 34 corresponding to GLP-1(7-37) (SEQ ID NO: 1) -1 analog, [Arg34]GLP-1-(7-37) peptide, is similar to GLP-1 with Arg at position 34 corresponding to GLP-1(7-37) (SEQ ID NO:1) Things. Specifically, the amino acid sequences of [Gly8,Arg34]GLP-1-(7-37) peptide and [Arg34]GLP-1-(7-37) peptide are as shown in SEQ ID NO: 2 and SEQ ID NO in the sequence list, respectively. :3 shown.
  • the term "derivative" as used herein refers to a chemically modified GLP-1 peptide or analogue in which one or more substituents have been covalently attached to the peptide .
  • Substituents can also be referred to as side chains.
  • the GLP-1 derivatives of formula (B) of the present invention may exist in different stereoisomeric forms, which have the same molecular formula and connected atomic sequence, but differ only in the three-dimensional direction of their atomic space. Unless otherwise stated, the present invention relates to all stereoisomeric forms of the claimed derivatives.
  • peptide when used, for example, in the GLP-1 analog of the present invention, refers to a compound comprising a series of amino acids connected to each other by amide (or peptide) bonds.
  • the peptide is largely or predominantly composed of amino acids interconnected by amide bonds (e.g., at least 50%, 60%, 70%, 80%, or at least 90% of the molar mass). In another specific embodiment, the peptide is composed of amino acids connected to each other by peptide bonds.
  • Amino acids are molecules containing amino and carboxylic acid groups, optionally containing one or more additional groups, commonly referred to as side chains.
  • amino acid includes proteinaceous amino acids (encoded by the genetic code, including natural amino acids and standard amino acids), as well as non-proteinaceous (not found in proteins, and/or not encoded in the standard genetic code), and synthetic amino acids .
  • Non-protein-derived amino acids are parts that can be integrated into peptides through peptide bonds, but are not protein-derived amino acids.
  • Synthetic non-protein-derived amino acids include amino acids produced by chemical synthesis, that is, D-isomers of amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid) , Abu ( ⁇ -aminobutyric acid), 3-aminomethyl benzoic acid, anthranilic acid, deamino-histidine, ⁇ -analogs of amino acids such as ⁇ -alanine, D-histidine, Amino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, etc.
  • D-isomers of amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid) , Abu ( ⁇ -aminobutyric acid), 3-aminomethyl benzoic acid, anthranilic acid, deamino-histidine, ⁇ -analogs
  • Non-limiting examples of amino acids not encoded by the genetic code are ⁇ -carboxyglutamate, ornithine, D-alanine, D-glutamine, and phosphoserine.
  • Non-limiting examples of synthetic amino acids are D-isomers of amino acids such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid), ⁇ -alanine and des-amino-group Acid (desH, alternative name imidazopropionic acid, abbreviation Imp).
  • the GLP-1 derivatives, analogs and intermediates of the present invention may be in the form of pharmaceutically acceptable salts, amides or esters.
  • the salt can be a basic salt, an acid salt, or a neutral salt.
  • Basic salts in water produce hydroxide ions, and acid salts produce hydronium ions.
  • the salt of the derivative of the present invention can be formed with an added cation or anion that reacts with an anionic group or a cationic group, respectively. These groups may be located in the peptide moiety and/or in the side chain of the derivative of the invention.
  • Non-limiting examples of anionic groups of the derivatives of the invention include side chains (if any) and free carboxyl groups in the peptide moiety.
  • the peptide moiety usually includes a free carboxylic acid at the C-terminus, and it may also include free carboxyl groups on internal acidic amino acid residues such as Asp and Glu.
  • Non-limiting examples of cationic groups of the peptide moiety include the N-terminal free amino group (if any) and any free amino groups on internal basic amino acid residues such as His, Arg, and Lys.
  • esters of the derivatives of the present invention can be formed, for example, by the reaction of a free carboxylic acid group with an alcohol or phenol, which results in the substitution of at least one hydroxyl group by an alkoxy or aryloxy group.
  • the formation of the ester may involve the free carboxyl group at the C-terminus of the peptide, and/or any free carboxyl group on the side chain.
  • the amide of the derivative of the present invention can be formed, for example, by reacting a free carboxylic acid group with an amine or substituted amine, or by reacting a free or substituted amino group with a carboxylic acid.
  • the formation of amides may involve the free carboxyl group at the C-terminus of the peptide, any free carboxyl group at the side chain, the free amino group at the N-terminus of the peptide, and/or any free or substituted peptide amino group in the peptide and/or side chain.
  • the GLP-1 compound or GLP-1 derivative of the present invention is in the form of a pharmaceutically acceptable salt. In another specific embodiment, it is in the form of a pharmaceutically acceptable amide, preferably having an amide group at the C-terminus of the peptide. In a further specific embodiment, the peptide or derivative is in the form of a pharmaceutically acceptable ester.
  • GLP-1(7-37) peptides and GLP-1 analogs of the present invention are well known in the art.
  • classical peptide synthesis such as solid-phase peptide synthesis using t-Boc or Fmoc chemistry or other sophisticated techniques, can be used to produce the GLP-1 peptide part (or fragment thereof) of the derivative of the present invention and the peptide of the present invention.
  • GLP-1 analogs see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and by WCChan and "Fmoc Solid Phase Peptide Synthesis” edited by PD White, Oxford University Press, 2000.
  • the intact GLP-1 analog of the present invention such as [Gly8,Arg34]GLP-1-(7-37) peptide can be produced by recombinant methods, that is, by culturing a host cell that contains the analog
  • the DNA sequence of the substance can be used to express the peptide in a suitable nutrient medium under conditions that allow the expression of the peptide.
  • host cells suitable for expressing these peptides are: Escherichia coli, Saccharomyce scerevisiae, and mammalian BHK or CHO cell lines. In some embodiments, this complete recombination fermentation step of the production process is satisfactory, for example due to production economic considerations.
  • the fusion protein inclusion body containing the main chain of the GPL-1 compound is denatured and renatured to obtain the fusion protein with the correct conformation. After a series of treatments such as enzyme digestion, sedimentation, and centrifugation, a higher content of GLP-1 compound is obtained. Main chain. Purified by ion exchange chromatography, the main chain of GLP-1 compound with higher purity is obtained after treatment.
  • excipient broadly refers to any ingredient other than the active therapeutic ingredient.
  • the excipient may be an inert substance, an inactive substance, and/or a non-pharmacologically active substance.
  • Excipients can be used for various purposes, such as as carriers, vehicles, diluents, tablet aids, and/or to improve administration and/or absorption of active substances.
  • Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, isotonic agents, chelating agents and stabilizers.
  • the GLP-1 derivatives and analogs of the present invention have GLP-1 activity. Having GLP-1 activity refers to the ability to bind to GLP-1 receptors and initiate signal transduction pathways to produce insulin-promoting effects or other physiological effects.
  • potency, efficacy and/or activity refer to in vitro efficacy, that is, performance in a functional GLP-1 receptor assay, more particularly in cell lines expressing cloned human GLP-1 receptor The ability to stimulate the formation of cAMP.
  • the derivatives of the present invention are potent in vivo, which can be determined in any suitable animal model and in clinical trials in a manner known in the art.
  • diabetic db/db mice are an example of a suitable animal model, in which the blood glucose lowering effect can be measured in such mice, for example, as described in the Examples section of the present invention.
  • insulin includes naturally-occurring insulin, such as human insulin, as well as its insulin analogs and insulin derivatives.
  • insulin analogue encompasses a polypeptide having one or more amino acid residues and/or addition of at least one amino acid residue that can be present in natural insulin by deletion and/or substitution (replacement) in form, and A molecular structure derived from the structure of a naturally occurring insulin, such as human insulin.
  • the substituted amino acid residue is a codable amino acid residue.
  • insulin derivative refers to a naturally-occurring insulin or insulin analogue that has been chemically modified.
  • the modification can be, for example, the introduction of a side chain or oxidation or reduction at one or more positions of the insulin backbone.
  • the groups of amino acid residues on insulin either convert free carboxyl groups into ester groups or acylate free amino groups or hydroxyl groups.
  • the acylated insulin of the present invention belongs to insulin derivatives.
  • insulin parent refers to an insulin derivative or an insulin portion of an acylated insulin (also referred to herein as parent insulin), for example, in the present invention, it refers to a portion of an acylated insulin without an additional acyl group.
  • the parent insulin may be a naturally occurring insulin, such as human insulin or porcine insulin.
  • the parent insulin may be an insulin analogue.
  • amino acid residue includes an amino acid from which a hydrogen atom has been removed from an amino group and/or a hydroxyl group has been removed from a carboxyl group and/or a hydrogen atom has been removed from a sulfhydryl group.
  • amino acid residues can be called amino acids.
  • amino acids mentioned herein are L-amino acids.
  • alkylene glycol encompasses oligo- and polyalkylene glycol moieties as well as monoalkylene glycol moieties.
  • Monoalkylene glycols and polyalkylene glycols include, for example, chains based on mono-polyethylene glycol, mono-polypropylene glycol, and mono-polybutylene glycol, that is, based on the repeating unit -CH 2 CH 2 O- , -CH 2 CH 2 CH 2 O- or -CH 2 CH 2 CH 2 CH 2 O- chain.
  • the alkylene glycol moiety can be monodisperse (having a well-defined length/molecular weight) as well as polydisperse (having a less well-defined length/average molecular weight).
  • the monoalkylene glycol moiety includes -OCH 2 CH 2 O-, -OCH 2 CH 2 CH 2 O-, or -OCH 2 CH 2 CH 2 CH 2 O- containing different groups at each end.
  • fatty acid includes straight or branched chain aliphatic carboxylic acids, which have at least two carbon atoms and are saturated or unsaturated.
  • Non-limiting examples of fatty acids are, for example, myristic acid, palmitic acid, stearic acid, and arachidic acid.
  • aliphatic diacid includes linear or branched aliphatic dicarboxylic acids, which have at least two carbon atoms and are saturated or unsaturated.
  • Non-limiting examples of aliphatic diacids are adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid , Eicosandioic acid, Docosanedioic acid and tetracosanedioic acid.
  • the naming of insulin or GLP-1 compounds is based on the following principles: according to mutations and modifications (such as acylation) relative to human insulin, or mutations and modifications (such as acylation) of natural GLP-1 (7-37) ) Give a name.
  • mutations and modifications such as acylation
  • mutations and modifications such as acylation of natural GLP-1 (7-37)
  • OEG represents the group -NH(CH 2 ) 2 O(CH 2 ) 2 OCH 2 CO- (ie, 2-[ The abbreviation for 2-(2-aminoethoxy)ethoxy]acetyl), ⁇ Glu (and gGlu) is the abbreviation for the amino acid ⁇ glutamic acid in the L configuration.
  • the acyl moiety can be named according to the IUPAC nomenclature (OpenEye, IUPAC format). According to this nomenclature, the above-mentioned acyl moiety of the present invention is called the following name: "[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19- Carboxynonadenoylamino)butyryl]-amino]-ethoxy]-ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]" or "[2-(2-[2 -(2-[2-(2-[4-(19-carboxynonadenoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy] Ethoxy)acetyl]".
  • the insulin of Example 6 of the present invention (having the sequence/structure given below) is called "B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin", “B29K(N ⁇ ) -Eicosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin” or “B29K (N ⁇ -eicosane diacyl-gGlu-5xOEG), desB30 human insulin” to indicate that the amino acid K at position B29 in human insulin has passed
  • the ⁇ nitrogen of the lysine residue of B29 (called N ⁇ or (N( ⁇ )) is modified by acylation with the residue eicosane diacyl-gGlu-2xOEG, and the amino acid T at position B30 in human insulin has
  • the insulin of Comparative Example 5 (having the sequence/structure given below) is called "A14E, B16H, B25H, B29K (N ⁇ eicosane diacyl-gGlu-2
  • NxPEG herein means -NH(CH 2 CH 2 O) n CH 2 CO-, where n is an integer.
  • 12xPEG represents the group -NH(CH 2 CH 2 O) 12 CH 2 CO-.
  • Insulin is a polypeptide hormone secreted by ⁇ -cells in the pancreas. It is composed of two polypeptide chains, A and B, which are connected by two interchain disulfide bonds. In addition, the A chain is characterized by an intra-chain disulfide bond.
  • the prior art discloses many methods for expressing insulin precursors in Escherichia coli or Saccharomyces cerevisiae. For example, refer to US Patent No. 5,962,267, WO95/16708, EP0055945, EP0163529, EP0347845 and EP0741188.
  • the construction, expression, processing, and purification of the insulin analog vector can be carried out using techniques known to those skilled in the art.
  • the insulin analog can be prepared by a well-known technique disclosed in US Patent No. 6,500645 by expressing a DNA sequence encoding the target insulin analog in a suitable host cell.
  • insulin analogues can also be prepared by the method reported in the following literature: Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain ⁇ -Helix for Receptor Binding; Biochemistry 2008 47 4743-4751. This document uses overlap extension PCR to introduce mutations into the insulin encoding vector.
  • Insulin analogs are expressed in Saccharomyces cerevisiae strain MT663 as a pre-insulin-like fusion protein with Ala-Ala-Lys small C-peptide.
  • A. lyticus endoprotease the single-chain precursor is enzymatically converted into a double-chain desB30 analog.
  • the isolated insulin analogs can be acylated at desired positions by acylation methods known in the art. Examples of such insulin analogs have been described in, for example, Chinese patent applications with publication numbers CN1029977C, CN1043719A and CN1148984A.
  • each insulin analog polypeptide encoded can be synthesized by established standard methods, such as the method described in Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or Matthes et al. (1984) EMBO Journal 3 : The method described in 801-805.
  • cAMP is cyclic adenosine monophosphate
  • BHK is a baby hamster kidney cell
  • DNA is deoxyribonucleic acid
  • Na 2 HPO 4 is disodium hydrogen phosphate
  • NaOH sodium hydroxide
  • OEG is the amino acid residue -NH(CH 2 ) 2 O(CH 2 ) 2 OCH 2 CO-;
  • OSu is succinimidyl-1-yloxy-2,5-dioxo-pyrrolidin-1-yloxy
  • OtBu is oxy tert-butyl
  • HCl is hydrogen chloride
  • ⁇ Glu or gGlu is ⁇ L-glutamyl
  • NHS is N-hydroxysuccinimide
  • DCC is dicyclohexylcarbodiimide
  • AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid
  • Gly is glycine
  • Arg is arginine
  • TFA is trifluoroacetic acid
  • HbA1c is glycosylated hemoglobin.
  • [Gly8, Arg34]GLP-1-(7-37) peptide is prepared by general protein recombinant expression method (for specific method, please refer to Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Cold Spring Harbor Press, 2012) .
  • [Gly8,Arg34]GLP-1-(7-37) peptide (5g, 1.48mmol) was dissolved in 100mM Na 2 HPO 4 aqueous solution (150mL), and acetonitrile (100mL) was added, and the pH was adjusted to pH with 1N NaOH 10-12.5.
  • the mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), the precipitated product was separated by filtration, and washed twice with n-heptane.
  • the product was purified by ion exchange chromatography (Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA) , The purified fractions were combined, the pH was adjusted to 5.2 with 1N HCl, the precipitate was separated, and lyophilized to obtain the title compound.
  • the reference compound liraglutide was prepared according to Example 37 of patent CN1232470A.
  • control compound Semaglutide was prepared according to Example 4 of the patent CN101133082A.
  • the purpose of this study is to confirm the regulatory effect of the GLP-1 derivatives of the present invention on hyperglycemia (BG) in the case of diabetes.
  • BG hyperglycemia
  • mice aged 8-9 weeks are kept in a barrier environment in a breeding box of suitable specifications, free access to standard food and purified water, and the environmental conditions are controlled at a relative humidity of 40%-60%.
  • the temperature is 22°C-24°C. After an adaptation period of 1-2 weeks, start to be used in experiments.
  • mice were weighed. According to random blood glucose and body weight, the mice were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of GLP-1 derivative 100 ⁇ g/kg, where the vehicle contained: propylene glycol 14mg/ml, phenol 5.5mg/ ml, disodium hydrogen phosphate 1.133 mg/ml, the pH of the solvent is 8.12.
  • the GLP-1 derivative is dissolved in a solvent to an administration concentration of 20 ⁇ g/ml, and the administration volume is 5 ml/kg (that is, 50 ⁇ l/10 g body weight). It is administered subcutaneously, once subcutaneously on the back of the neck.
  • the corresponding GLP-1 derivative was given at approximately 10:30 in the morning (time 0).
  • the animals were free to eat and drink, and at 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours after administration Assess blood glucose in mice. Clean the tail of the rat with an alcohol cotton ball, collect blood drops from the tail with a disposable lancet, and measure it with a blood glucose meter and matching test paper (Roche). The food intake and body weight of each mouse were measured at 24, 48 and 72 hours after administration.
  • the blood glucose at each monitoring point after administration is compared with the blood glucose percentage at the corresponding time point.
  • a dose-response curve of blood glucose percentage versus time is drawn.
  • AUC is the area under the curve of the time-glucose percentage curve. The smaller the AUC value, the better the hypoglycemic effect and the better the drug effect.
  • Figures 1a-4b show that the GLP-1 derivatives of the present invention have unexpectedly increased pharmacodynamics.
  • the title compound of Examples 1-4 has significantly better hypoglycemic effect on db/db mice than liraglutide , And the compound of Comparative Example 3-4.
  • the hypoglycemic effect of the compound of Example 2 of the present invention is better than that of somaglutide.
  • the GLP-1 derivatives of the present invention such as the compounds of Examples 1-4, have a significantly longer effective time of action in db/db mice compared to liraglutide and the compounds of Comparative Examples 3-4, especially The effective time of hypoglycemic effect of the compound of Example 2 in db/db mice was longer than that of somaglutide.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-5xOEG
  • desB30 human insulin compound 5
  • DesB30 human insulin (5 g, 0.876 mmol) was dissolved in 100 mM Na 2 HPO 4 aqueous solution (150 mL), and acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1 N NaOH.
  • Dissolve tert-butyl eicosane diacyl- ⁇ Glu-(5xOEG-OSu)-OtBu (1.36 g, 0.964 mmol) in acetonitrile (50 mL), and slowly add to the insulin solution. Maintain the pH at 10-12.5. After 120 minutes, the reaction mixture was added to water (150 mL), and the pH was adjusted to 5.0 with 1N aqueous HCl.
  • the precipitate was separated by centrifugation and lyophilized.
  • the crude product was added to a mixed solution of trifluoroacetic acid (60 mL) and dichloromethane (60 ml), and stirred at room temperature for 30 minutes.
  • the mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), the precipitated product was separated by filtration, and washed twice with n-heptane.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-6xOEG
  • desB30 human insulin compound 6
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-8xOEG), desB30 human insulin (compound 7)
  • Compound 7 was prepared in a procedure similar to that of Example 6, Part 2.
  • B29K N( ⁇ )-docosanediacyl- ⁇ Glu-6xOEG
  • desB30 human insulin compound 8
  • Compound 8 was prepared in a procedure similar to that of Example 6, Part 2.
  • B29K N( ⁇ )-docosanediacyl- ⁇ Glu-8xOEG
  • desB30 human insulin compound 9
  • the purpose of this example is to test the in vitro efficacy or activity of the GLP-1 derivatives of the present invention
  • the cells expressing GLP-1R were resuscitated, and the cells were inoculated into 25 mL cell culture flasks using ham's-F12 medium, and cultured overnight at 37° C., 5% CO 2.
  • the title compound (compound 10) and liraglutide of Example 11 of the present invention were separately prepared to 150 ⁇ g/mL, and then the samples were serially diluted to 750 ng/ml, 150 ng/ml, 30 ng/ml, 6 ng/ml, 1.2 ng /ml, 0.24ng/ml, 0.048ng/ml, 0.0096ng/ml and 0.00192ng/ml.
  • test subject EC 50 (nM) Liraglutide 0.439 Compound 10 0.677
  • the GLP-1 derivative of the present invention has satisfactory in vitro efficacy, and its in vitro activity is close to that of liraglutide, which confirms that it has GLP-1 receptor agonistic activity.
  • Example 13 Pharmacodynamic experiment in obese C57BL mice induced by high-fat diet
  • the purpose of this study is to verify the blood glucose regulation and weight loss effects of the GLP-1 derivatives of the present invention in high-fat diet-induced obese C57BL mice.
  • mice C57BL mice aged 5 weeks and weighing 17-22g (male and female) were raised in a barrier environment in a suitable feeding box (3-5 mice/box).
  • the high-fat diet induction group was free to get high-fat feed And purified water.
  • the normal control group has free access to standard food and purified water.
  • the environmental conditions are controlled at a relative humidity of 40%-60%, a temperature of 22°C-24°C, and reared for 10 weeks.
  • the body weight is selected to exceed 30 of the body weight of the normal control group. %-50% of mice were evaluated for efficacy.
  • the basal blood glucose was evaluated at time -1/1h (9:30 am), and the mice were weighed. According to random blood glucose and body weight, the mice in the high-fat diet induction group were matched and assigned to the vehicle group (ie, the model control group) or the treatment group, and received the following treatment: subcutaneous injection of the vehicle, or subcutaneous injection of the control compound somaglutide 100 ⁇ g/kg, or The title compound of Example 11 of the present invention was injected subcutaneously at 100 ⁇ g/kg and 300 ⁇ g/kg.
  • the solvent includes: propylene glycol 14 mg/ml, phenol 5.5 mg/ml, disodium hydrogen phosphate 1.133 mg/ml, and the pH of the solvent is 7.4.
  • GLP-1 derivatives were administered subcutaneously, once subcutaneously on the back of the neck (5 ⁇ l/g body weight).
  • GLP-1 derivatives were administered at approximately 10:30 in the morning (time 0), and the blood glucose of the mice was assessed at 3, 6, 24, 48, and 72 hours after administration. At the same time, the body weight of the mice was monitored daily.
  • a ⁇ blood glucose-time curve is drawn for each single dose of GLP-1 derivative.
  • ⁇ AUC area under the blood glucose-time curve
  • ipGTT intraperitoneal glucose tolerance test
  • a dose-response curve of blood glucose versus time and a dose-response curve of daily weight change versus time were drawn.
  • ⁇ AUC blood glucose-time difference area under the curve
  • Figures 5a-6b show that the GLP-1 derivatives of the present invention have unexpectedly increased pharmacodynamics.
  • the hypoglycemic effect of compound 10 of Example 11 on obese C57BL mice induced by a high-fat diet is at the same dose as There is no significant difference in the marketed reference compound semaglutide.
  • the hypoglycemic effect of the GLP-1 derivative of the present invention is slightly better than that of semaglutide.
  • the average blood glucose of the same dose of compound 10 group was lower than the average blood glucose of the same dose of somaglutide group.
  • the hypoglycemic effect of the GLP-1 derivative of the present invention is dose-dependent, and as the dose of the GLP-1 of the present invention increases, the hypoglycemic effect is also significantly improved.
  • Figures 6a-6b show that in the ipGTT experiment, the compound 10 of Example 11, compared with the vehicle, had a significant effect on blood glucose after the ipGTT experiment was carried out 48 hours after the first administration of obese C57BL mice induced by a high-fat diet.
  • the inhibitory effect is slightly better than the hypoglycemic effect of the same dose of semaglutide.
  • FIG. 5c shows that the GLP-1 derivatives of the present invention, such as compound 10 of Example 11, have an excellent weight loss effect, and its weight loss effect is better than that of semaglutide.
  • the purpose of this study is to verify the blood glucose regulation effect of the GLP-1 derivatives of the present invention in the case of diabetes.
  • Example 11 In db/db mice, the title compound of Example 11 and the control compound liraglutide were tested for their blood glucose-lowering effects at different doses of 0.3, 1, 3, 10, 30, and 100 nmol/kg, and the ED 50 was calculated.
  • mice aged 8-9 weeks are kept in a barrier environment in a breeding box of suitable specifications, free access to standard food and purified water, and the environmental conditions are controlled at a relative humidity of 40%-60%.
  • the temperature is 22°C-24°C. After an adaptation period of 1-2 weeks, start to be used in experiments.
  • mice were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of the vehicle, or subcutaneous injection of the compound of Example 11 or the control compound liraglutide 0.3, 1, 3, 10, 30 and 100 nmol/kg, where the solvent contains: propylene glycol 14 mg/ml, phenol 5.5 mg/ml, disodium hydrogen phosphate 1.133 mg/ml, and the pH of the solvent is 7.4.
  • Subcutaneous administration method 50 ⁇ l/10g body weight
  • the compound of Example 11 was administered at approximately 10:00 in the morning (time 0), and the blood glucose of the mice was evaluated at 1, 2, 3, 6, 12, 24, 48, and 72 hours after the administration.
  • ⁇ refers to the actual blood glucose at a given time minus the baseline, where baseline is the blood glucose at time 0.
  • ⁇ AUC the area under the curve of ⁇ blood glucose from 0 to 72 hours
  • ED 50 the effective dose 50%
  • the purpose of this study is to verify the control of GLP-1 derivatives of the present invention on blood sugar, food intake and water consumption.
  • Example 2 On type II diabetic db/db mice, the title compound of Example 2 and the control compound semaglutide were tested in a single dose study.
  • mice aged 8-9 weeks are kept in a barrier environment in a breeding box of suitable specifications, free access to standard food and purified water, and the environmental conditions are controlled at a relative humidity of 40%-60%.
  • the temperature is 22°C-24°C. After an adaptation period of 1-2 weeks, start to be used in experiments.
  • mice were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of the vehicle, or subcutaneous injection of the compound of Example 2 or the control compound semaglutide 100 ⁇ g/kg, where the vehicle contains propylene glycol 14mg/ml, phenol 5.5mg/ml, and disodium hydrogen phosphate 1.133mg/ml, pH 7.4.
  • the GLP-1 derivative is dissolved in a solvent to an administration concentration of 20 ⁇ g/ml, and administered subcutaneously (50 ⁇ l/10 g body weight), and administered once by subcutaneous injection on the back of the neck.
  • the compound of Example 2 was administered at approximately 10:00 in the morning (time 0), and the blood glucose of the mice was evaluated at 1, 2, 3, 6, 12, 24, 48, and 72 hours after the administration. Clean the tail of the rat with an alcohol cotton ball, collect blood drops from the tail with a disposable lancet, and measure it with a blood glucose meter (Roche) and matching test paper. At the same time, the food intake and water intake of the mice were measured every day.
  • a dose response curve of blood glucose versus time For each single dose of GLP-1 derivative, a dose response curve of blood glucose versus time, a dose response curve of food intake versus time, and a dose response curve of water consumption versus time were drawn.
  • the difference in area under the blood glucose-time curve from 0 to the monitoring endpoint ( ⁇ AUC) was calculated. Among them, the smaller the ⁇ AUC value, the better the hypoglycemic effect and the better the drug effect.
  • Figures 7a-7d show that the GLP-1 derivative of the present invention has an unexpectedly increased hypoglycemic effect and an inhibitory effect on the increase in food intake and drinking water after administration. This further proves that the title compound of Example 2 has better hypoglycemic effect on db/db mice after administration than semaglutide at the same dose. In addition, the title compound of Example 2 can effectively control food intake and drinking water, and its effect is better than semaglutide, suggesting that the GLP-1 derivative of the present invention has a better weight loss effect.
  • the purpose of this study is to verify the long-term hypoglycemic effect, weight loss, and diet control effect of the GLP-1 derivative of the present invention on type II diabetic db/db mice.
  • the GLP-1 derivative of Example 11 and the control compound somaglutide were tested on type II diabetic db/db mice. GLP-1 derivatives were administered to mice at different doses of 100 and 300 ⁇ g/kg and somaglutide was administered at a dose of 100 ⁇ g/kg, and it was determined that the GLP-1 derivatives and the control compound somaglutide reduced blood sugar, reduced body weight, The effect of reducing food intake and drinking water.
  • mice aged 8-9 weeks are kept in a barrier environment in a breeding box of suitable specifications, free access to standard food and purified water, and the environmental conditions are controlled at a relative humidity of 40%-60%.
  • the temperature is 22°C-24°C. After an adaptation period of 1-2 weeks, start to be used in experiments.
  • mice were matched and assigned to the vehicle group or treatment group, and received the following treatments: subcutaneous injection of vehicle, or subcutaneous injection of GLP-1 derivatives 100 and 300 ⁇ g/kg, or subcutaneous injection of control compound somaglutide 100 ⁇ g /kg.
  • the solvent includes: propylene glycol 14 mg/ml, phenol 5.5 mg/ml, disodium hydrogen phosphate 1.133 mg/ml, and the pH of the solvent is 7.4.
  • Subcutaneous administration 50 ⁇ l/10g body weight
  • subcutaneous injection on the back of the neck administration of GLP-1 derivatives at approximately 10:00 in the morning (time 0), respectively on the 0th, 3rd, 7th, 10th, 13th, and 16th , 19, 22, 25, and 28 days of administration.
  • the blood glucose of the mice was evaluated before each administration and 72 hours after the last administration.
  • the weight, food intake and water consumption of the mice were measured every day from day 0-17, and the weight, food intake and water consumption of the mice were monitored every 3 days after the 17th day.
  • Figures 8a-8f show that the GLP-1 derivative of the present invention has unexpectedly increased hypoglycemic efficacy, increased weight loss, and suppressed food and water intake after long-term administration.
  • compound 10 of Example 11 has a better hypoglycemic effect on db/db mice after long-term administration.
  • Figures 8c-8d show that, compared with the same dose of semaglutide, the GLP-1 derivative of the present invention, such as the title compound of Example 11, has a better weight loss effect and an effect of suppressing food intake and water consumption.
  • the purpose of this study is to verify the hypoglycemic effect of GLP-1 derivatives of the present invention on type II diabetic Kkay mice
  • the compound 10 of Example 11, the compound 2 of Example 2 and the control compound dulaglutide (also known as dulaglutide) were tested on Kkay mice with type II diabetes.
  • Compound 10 and compound 2 were administered to mice at different doses of 100 and 300 ⁇ g/kg, and dulaglutide was administered to mice at a dose of 600 ⁇ g/kg.
  • the GLP-1 derivatives of the present invention and the control compound dulaglutide were tested for their blood sugar lowering effect and HbA1c. Effect.
  • mice Male Kkay mice aged 12-14 weeks are reared in a barrier environment in a suitable feeding box, free access to standard food and purified water, and the environmental conditions are controlled at a relative humidity of 40%-60% and a temperature of 22°C-24°C . After an adaptation period of 1-2 weeks, start to be used in experiments.
  • mice were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of the vehicle, or subcutaneous injection of the GLP-1 derivatives of the present invention 100 and 300 ⁇ g/kg, or subcutaneous injection of the control compound Dura Glycopeptide 600 ⁇ g/kg.
  • the solvent contains: propylene glycol 14mg/ml, phenol 5.5mg/ml, disodium hydrogen phosphate 1.133mg/ml, pH 7.4.
  • Subcutaneous administration 50 ⁇ l/10g body weight
  • subcutaneous injection on the back of the neck administration of the GLP-1 derivative, dulaglutide or vehicle of the present invention at approximately 10:00 am (time 0), every 2 days
  • the drug was administered once for 16 consecutive times.
  • the blood glucose of the mice was evaluated at 3h, 6h, 1 day and 2 days after the first administration. 48h after the last administration, EDTA was taken to detect HbA1c.
  • Figures 9a-9b show that the GLP-1 derivative of the present invention has an unexpectedly increased hypoglycemic effect after administration.
  • the title compound of Example 11 and Example 2 has a significantly better hypoglycemic effect on Kkay mice.
  • Dulaglutide shows that the GLP-1 derivative of the present invention has a significantly better effect on reducing HbA1c in type II diabetic Kkay mice than dulaglutide.
  • C max maximum measured plasma concentration
  • T max time corresponding to maximum measured plasma concentration
  • T 1/2 terminal elimination half-life
  • AUC 0-t 0-t time-area under the blood glucose concentration time curve
  • MRT average residence time
  • 24 cynomolgus monkeys each group of 6 (male and female), divided into compound 10 low-dose group, compound 10 medium-dose group, compound 10 high-dose group, subcutaneously injected 10, 60, 360 ⁇ g/kg, and compound In the 10 intravenous injection group, 60 ⁇ g/kg of compound 10 was intravenously injected.
  • the compound 10 low, medium and high dose groups were taken before administration (0 min), 1, 3, 6, 8, 10, 12, 16, 24, 48, 72, 120, 168, and 240 hours to determine the blood concentration of the compound.
  • blood was collected before administration (0min), 1min, 10min, 1, 3, 6, 8, 10, 12, 24, 48, 72, 120, 168, 240 h after administration to determine the blood drug concentration.
  • the non-compartmental model of WinNonLin v6.4 software was used to calculate the pharmacokinetic parameters, C max , T max , T 1/2 , AUC 0-t , and MRT. The test results are shown in Table 4.
  • the GLP-1 derivative compound 10 of the present invention exhibits a longer half-life, a larger AUC 0-t and a longer MRT in rats and cynomolgus monkeys.
  • the GLP-1 derivatives of the present invention are all dose-dependent, and their efficacy increases with increasing dose.
  • the purpose of this experiment is to measure the chemical stability of the GLP-1 derivative preparation of the present invention.
  • the chemical stability of the formulation in this example can be shown by the change of high molecular weight protein (HMWP) relative to day 0 after storage at 37°C for 27 days, and the amount of related substances measured after storage at 37°C for 28 days can also be used. Change to express.
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • Protein content of high molecular weight (of HMWP) was determined by high performance liquid chromatography (HPLC), in Waters TSKgel G2000SWXL (7.8 * 300mm) , 5 ⁇ m column, at a column temperature of 30 °C, the temperature of the sample cell 5 °C, with The mobile phase was tested at a flow rate of 0.5 ml/min, where the mobile phase contained 300 ml isopropanol, 400 ml glacial acetic acid and 300 ml purified water. The detection wavelength is 276nm, and the injection volume is 25 ⁇ l. Table 5 shows the increase in HMWP when stored at 37°C for 27 days relative to the 0th day.
  • Phase A contains 90 mM potassium dihydrogen phosphate and 10% acetonitrile (v/v), pH 2.4
  • Phase B is 75% (v/v) acetonitrile.
  • the detection wavelength is 214nm
  • the flow rate is 1.0ml/min
  • the injection volume is 15 ⁇ l.
  • Table 5 shows the increased amount of related substances stored at 37°C for 28 days relative to that on day 0.
  • the formulation has good chemical stability when the pH is between 6.5 and 8.4, and when the pH is between 7.0 and 8.0, the chemical stability of the formulation is the best.
  • the purpose of this experiment is to measure the chemical stability of the GLP-1 derivative preparation of the present invention.
  • A14E, B16H, B25H, desB30 human insulin by conventional methods of preparing insulin analogues (see Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain ⁇ -Helix for Receptor Binding; Biochemistry 2008 47 4743-4751).
  • A14E, B16H, B25H, desB30 human insulin (5 g, 0.888 mmol) was dissolved in 100 mM Na 2 HPO 4 aqueous solution (150 mL), and acetonitrile (100 mL) was added, and the pH was adjusted to pH 10-12.5 with 1N NaOH.
  • the mixture was concentrated to about 30 ml, poured into ice-cold n-heptane (300 mL), and the precipitated product was separated by filtration and washed twice with n-heptane. After vacuum drying, it was purified by ion exchange chromatography ((Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), and the purified The fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was separated and lyophilized to obtain control compound 5.
  • ion exchange chromatography (Ressource Q, 0.25%-1.25% ammonium acetate gradient in 42.5% ethanol, pH 7.5), reverse phase chromatography (acetonitrile, water, TFA), and the purified
  • the fractions were combined, the pH was adjusted to 5.2 with 1N HCl, and the precipitate was separated and lyophilized to
  • A14E, B16H, B25H, B29K N( ⁇ )-eicosandioyl- ⁇ Glu-6xOEG), desB30 human insulin (compound 11)
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin (compound 12)
  • A14E, B16H, B25H, B29K N( ⁇ )-eicosandioyl- ⁇ Glu-12xOEG), desB30 human insulin (compound 13)
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin (compound 14)
  • the purpose of this example is to test the in vitro receptor binding affinity of the GLP-1 derivatives of the present invention and how the presence of albumin potentially affects binding.
  • Receptor binding is a measure of the affinity of the GLP-1 derivative for the human GLP-1 receptor.
  • the binding affinity of the GLP-1 derivatives and control compounds of the present invention to the human GLP-1 receptor is determined as follows: namely, by measuring their ability to replace 125 I-GLP-1 from the receptor. In order to determine the binding of GLP-1 derivatives to albumin (HSA), low concentration albumin (0.005% (w/v)) and high concentration albumin (2% (w/v)) were used for the determination. Change in binding affinity IC 50 indicates that GLP-1 derivatives bound to albumin, thereby predicting the kinetics of potentially prolonged drug derivative of GLP-1 in an animal model.
  • test compound was prepared with 10 mM Na 2 HPO 4 of pH 7.3, and the reference standard GLP-1 (7-37) was used to prepare a 1 mM stock solution with ultrapure water. Under the condition of 0.005% HSA, all test compounds and reference standards were diluted to 2 ⁇ M with assay buffer, and then a 4-fold serial dilution was performed, with a total of 10 concentration gradients.
  • the reference standard GLP-1(7-37) was diluted to 2 ⁇ M, liraglutide was diluted to 20 ⁇ M, compound 10 and somaglutide were diluted to 800 ⁇ M, and then all samples were serially diluted by 4 times. , A total of 10 concentration gradients.
  • IC 50 value is a measure of the influence of albumin GLP-1 derivatives and GLP-1 receptor binding pair.
  • GLP-1 derivatives also bind to albumin, which is usually the desired effect, which will extend their plasma lifespan.
  • IC 50 values generally higher than the albumin IC 50 values at low albumin, corresponding to reduced binding to the GLP-1 receptor, which is bound by a binding competition with the GLP-1 receptor albumin Caused by.
  • a high ratio (IC 50 value (high albumin)/IC 50 value (low albumin)) can be used, as the target derivative binds well to albumin (it can be determined that it has a long half-life), and that it has a long half-life with itself and GLP 1 receptor binding is also good (IC 50 value (high albumin) high, IC 50 values (low albumin) low) instructions.
  • the ratio of the GLP-1 derivatives of the present invention is higher than the control compounds Semaglutide, Liraglutide and GLP-1 (7-37), which indicates that the compound of the present invention has a longer half-life And it also binds well to the GLP-1 receptor.
  • Example 16 With reference to the similar experimental procedure in Example 16, a long-term pharmacodynamic study was carried out in type II diabetic db/db mice. The difference is that the control compound used is dulaglutide, and the dosage of dulaglutide is 300 ⁇ g/kg. Subcutaneous administration (50 ⁇ l/10g body weight), subcutaneous injection on the back of the neck, and GLP-1 derivatives were administered at approximately 10:00 in the morning (time 0), at 0, 3, 6, 9, 12, and 15 , 18, 21, 24, 27, 30 days of administration. The blood glucose of the mice was evaluated at 3, 6, 9, 12, 24, 48, and 72 hours after the first administration, and the area under the blood glucose-time curve ( ⁇ AUC) was calculated.
  • ⁇ AUC blood glucose-time curve
  • ipGTT intraperitoneal glucose tolerance test
  • Figures 10a-10e show that the GLP-1 derivative of the present invention still has an unexpectedly increased hypoglycemic effect after long-term administration.
  • the compound 10 of Example 11 has a better hypoglycemic effect on db/db mice after administration than dulaglutide.
  • compound 10 has a better hypoglycemic effect on db/db mice after long-term administration.
  • the GLP-1 derivative of the present invention has a more obvious inhibitory effect on blood sugar, which is better than the hypoglycemic effect of dulaglutide.
  • Example 13 With reference to the similar experimental procedure of Example 13, the pharmacodynamic experiment was carried out in obese C57BL mice induced by a high-fat diet. The difference is that the control compound used is dulaglutide, and the dose of dulaglutide is 300 ⁇ g. /kg.
  • Subcutaneous administration is adopted, once subcutaneously on the back of the neck (5 ⁇ l/g body weight), once every 3 days, a total of 11 times.
  • the GLP-1 derivative was administered at approximately 10:30 in the morning (time 0), and the blood glucose of the mice was evaluated at 3, 6, 9, 12, 24, 48, and 72 hours after the administration. At the same time, the weight and food intake of the mice were monitored every 3 days.
  • the subcutaneous fat, perirenal fat, and genital fat were weighed.
  • FIGS 11a-11d show that the GLP-1 derivatives of the present invention have unexpectedly improved weight loss effects, diet control effects, and lipid-lowering effects.
  • B29K N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG
  • desB30 human insulin compound 15
  • the compound B29K (N( ⁇ )-docosanediacyl- ⁇ Glu-12xOEG), desB30 human insulin was prepared in a procedure similar to the second part of Example 6.
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-18xOEG), desB30 human insulin (compound 16)
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-24xOEG), desB30 human insulin (compound 17)
  • B29K N( ⁇ )-docosanedioyl- ⁇ Glu-OEG
  • desB30 human insulin compound 18
  • the compound B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-OEG), desB30 human insulin was prepared by the similar procedure as in Example 6 Part 2.
  • the compound B29K (N( ⁇ )-docosanediacyl- ⁇ Glu-12xPEG), desB30 human insulin was prepared in a procedure similar to the second part of Example 6.

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Abstract

一种新的GLP-1衍生物，其相比于利拉鲁肽、索玛鲁肽等已上市的GLP-1衍生物，具有相当或更好的效力、药效或功效、更长或相当的体内持续作用时间或体内半衰期、具有更好或相当的GLP-1受体结合亲和力、具有更好或相当的DPP-IV稳定性。

Description

长效GLP-1化合物 技术领域
本发明涉及治疗性肽领域，具体涉及新的长效GLP-1化合物、其药物制剂、其与长效胰岛素的药物组合物、及所述化合物、药物制剂和药物组合物的医药用途。
背景技术
胰高血糖素样肽1(GLP-1)及其类似物和衍生物在治疗1和2型糖尿病中非常有效，然而高清除率限制了这些化合物的有效性。为了提供在体内作用持续时间更长的GLP-1化合物，已经将一系列不同方法用于修饰胰高血糖素样肽1(GLP-1)的结构。例如WO99/43708公开了具有连接至C-端氨基酸残基的亲脂性取代物的GLP-1(7-35)和GLP-1(7-36)衍生物。WO00/34331公开了酰化的GLP-1类似物。WO00/69911公开了用于注射进患者体内的活化的促胰岛素肽。
目前已上市的GLP-1类药物中有例如：每天给药两次的天然的GLP-1类似物艾塞那肽(Exenatide)；每天给药一次的利拉鲁肽(Liraglutide)和利司那肽(Lixisenatide)，前者为十六烷酸修饰的GLP-1化合物，后者为对艾塞那肽进行结构改造修饰而得到的新分子；和每周给药一次的索玛鲁肽(Semaglutide)、艾塞那肽微球(ExenatideLAR)、阿必鲁肽(Abiglutide)、度拉糖肽(Dulaglutide)(也称杜拉鲁肽)和聚乙二醇洛塞那肽。其中，艾塞那肽微球是通过微囊化方法将艾塞那肽包裹至聚乳酸乙醇酸共聚物基质制备得到，阿必鲁肽是通过将两条修饰后的GLP-1肽链以二聚体形式与人白蛋白融合成为重组融合蛋白；度拉糖肽是将修饰后的GLP-1链通过二硫键融合到重组G4免疫球蛋白的Fc片段上得到；聚乙二醇洛塞那肽是在艾塞那肽的化学结构式基础上进行氨基酸改造和经聚乙二醇修饰而成；索玛鲁肽主要是通过在GLP-1(7-37)肽上使用非蛋白质性氨基酸Aib替换了第8位的Ala而实现了一周一次，但索玛鲁肽中非蛋白质性氨基酸的存在，相对于天然的氨基酸，在人体内可能存在未知的、各种潜在的副作用风险。
一方面，目前仍然需要开发能够相对于已上市的同类药物例如利拉鲁肽、度拉糖肽和索玛鲁肽，具有更好的效力、药效或功效、具有更小的潜在副作用的风险、具有更好的体重减轻和抑制饮食效果、具有更长 或相当的体内持续作用时间或体内半衰期的化合物，以为糖尿病患者提供更优的用药选项。
另一方面，随着全世界2型糖尿病人群快速增加，对于给药更简单的更有效的药物存在着更大的需求。例如包含胰岛素和GLP-1肽两种活性成分的复方制剂可能是非常有效的治疗剂。因此目前也仍然需要能够协同实现具有更好的物理和化学稳定性、更长的作用时间、以及药效更好的复方制剂。
发明内容
为克服或改善现有技术的至少一个缺点、或提供有用的替代品，本发明第一方面提供了新的GLP-1化合物(也称GLP-1衍生物)。所述新的GLP-1化合物相比于利拉鲁肽、度拉糖肽、索玛鲁肽等已上市的GLP-1衍生物，具有更好的效力、药效或功效、更小的潜在副作用的风险、更好的减轻体重的效果、更长的体内持续作用时间或体内半衰期，更好或相当的GLP-1受体结合亲和力，以及具有更好或相当的DPP-IV稳定性。且本发明的长效GLP-1化合物和本发明提供的长效胰岛素的药物组合物或组合制剂不仅不会削弱所述GLP-1化合物和所述胰岛素化合物的物理稳定性，而且，组合制剂具有比单药制剂更好的物理稳定性。与其他长效GLP-1化合物的组合制剂(例如利拉鲁肽和德谷胰岛素的组合制剂)相比，本发明的组合制剂的物理稳定性是出乎意料的。此外，组合制剂相比于单药制剂，还使得所述GLP-1化合物和酰化胰岛素的化学稳定性增加。本发明的GLP-1化合物，及本发明提供的含所述GLP-1化合物和胰岛化合物的组合制剂均能够很好地实现长的药代动力学(下文也称PK)特征，使得对糖尿病患者一周两次、一周一次、两周一次、或更低频率的皮下治疗成为可能。
本发明第一方面提供的GLP-1化合物为式B的化合物，或其药学上可接受的盐、酰胺或酯：
[Acy-(L1) r-(L2) q]-G1  (B)，
其中G1为在对应于GLP-1(7-37)(SEQ ID NO：1)的位置34处具有Arg及在位置8处具有Ala或Gly的GLP-1类似物，[Acy-(L1) r-(L2) q]是连接至所述GLP-1类似物的位置26的Lys残基的ε氨基上的取代基，其中
r为1-10的整数，q为0或1-10的整数；
Acy是包含20-24个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除；
L1是选自下述的氨基酸残基：γGlu、αGlu、βAsp、αAsp、γ-D-Glu、α-D-Glu、β-D-Asp或α-D-Asp；
L2是中性的、包含亚烷基二醇的氨基酸残基；
Acy、L1、和L2之间以酰胺键连接；和
L1和L2在式(B)中出现的顺序可以独立地互换。
在一个实施方案中，G1为[Gly8,Arg34]GLP-1-(7-37)肽或[Arg34]GLP-1-(7-37)肽，优选为[Gly8，Arg34]GLP-1-(7-37)肽。
在一个实施方案中，r为1、2、3、4、5或6，优选地，r为1、2、3或4，优选地，r为1或2，优选地，r为1。
在另一个实施方案中，q为0、1、2、3、4、5、6、7或8，优选地，q为0、1、2、3或4，更优选，q为0、1、或2。
在一个实施方案中，Acy是包含20-23个碳原子的脂肪族二酸，优选Acy是包含20、21、或22个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除。
在一个实施方案中，L2是：-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 3-O-CH 2-CO-、或-HN-(CH 2) 4-O-(CH 2) 4-O-CH 2-CO-；优选L2是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-。
在一个实施方案中，L1选自γGlu或βAsp，优选L1为γGlu。
在一个实施方案中，Acy是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-或HOOC-(CH 2) 22-CO-，Acy是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
在一个实施方案中，式(B)中Acy、L1、和L2之间依次以酰胺键连接，L2的C末端连接至所述GLP-1类似物的位置26的Lys残基的ε氨基上。
在一个实施方案中，本发明第一方面所述化合物选自下述化合物：
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[23-羧基二十三烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[23-羧基二十三烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、或
N-ε 26-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽。
在一个实施方案中，本发明第一方面所述化合物选自下述化合物：
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[20-羧基二十烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[22-羧基二十二烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[20-羧基二十烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(2-[2-(2-[4-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-[2-(2-[2-(4-[22-羧基二十二烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
N-ε 26-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、或
N-ε 26-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽。
在一个实施方案中，本发明第一方面所述化合物选自下述化合物：
N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、或N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽。
本发明第二方面提供了一种药物制剂，其包含本发明第一方面所述的化合物和可药用赋形剂。
在一个实施方案中，所述可药用赋形剂选自缓冲剂、防腐剂、等渗剂、稳定剂和螯合剂中的一种或多种。在另一个实施方案中，所述可药用赋形剂为缓冲剂、防腐剂和等渗剂。
在一个实施方案中，所述药物制剂包含：本发明第一方面所述的化合物、等渗剂、防腐剂和缓冲剂。优选地，在所述药物制剂中，本发明第一方面所述的化合物为N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、或N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽。
在一个实施方案中，所述等渗剂选自氯化钠、丙二醇、甘露醇、山梨醇、甘油、葡萄糖和木糖醇的一种或多种，优选为丙二醇、甘露醇或氯化钠。
在另一个实施方案中，所述防腐剂选自苯酚、间甲酚、对-羟基苯甲酸甲酯、对-羟基苯甲酸丙酯、2-苯氧乙醇、对-羟基苯甲酸丁酯、2-苯乙醇、和苯甲醇中的一种或多种，优选为苯酚或间甲酚。
在另一个实施方案中，所述缓冲剂选自醋酸钠、碳酸钠、柠檬酸盐、甘氨酰甘氨酸、组氨酸、甘氨酸、赖氨酸、精氨酸、磷酸二氢钠、磷酸氢二钠、磷酸钠，和三(羟甲基)-氨基甲烷中的一种或多种，优选为醋酸钠、柠檬酸盐、磷酸二氢钠、或磷酸氢二钠。
在一个实施方案中，所述制剂的pH为约6.0至约10.0，优选为约6.5至约10.0，优选为约6.5至约9.5，优选为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
在一个实施方案中，所述药物制剂含有如下成分：
约0.1-1.2mM、优选约0.2-1mM、优选约0.3-0.7mM、更优选约0.48-0.6mM的本发明第一方面所述的化合物；
约10-1500mM、优选约13-800mM、优选约65-400mM、优选约90-240mM、优选约150-250mM、优选约180-200mM、更优选约183-195mM的等渗剂，优选，所述等渗剂选自丙二醇、甘油、甘露醇或氯化钠中的一种或多种；
约1-200mM、优选约5-150mM、优选约10-100mM、优选约20-85mM、优选约30-75mM、优选约45-60mM、更优选约50-60mM的防腐剂，优选，所述防腐剂选自苯酚或间甲酚中的一种或多种；
约3-35mM、优选约5-20mM、更优选约5-15mM、更优选约7-10mM的缓冲剂，所述缓冲剂选自醋酸钠、柠檬酸盐、磷酸二氢钠或磷酸氢二钠中的一种或多种；和
所述药物制剂的pH为约6.0至约10.0，优选为约6.5至约9.5，优选为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
在另一个实验方案中，所述药物制剂包含：约0.3-0.7mM、更优选为约0.48-0.6mM的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；约180-200mM、更优选约183-195mM的丙二醇；约45-60mM、更优选约50-60mM的苯酚；约5-15mM的缓冲剂，优选约7-10mM的磷酸氢二钠；和所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、进一步优选为约7.3至约8.3。
在另一个实验方案中，所述药物制剂包含：约0.5mM的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；约184mM的丙二醇；约58.5mM的苯酚；约10mM的磷酸氢二钠；和所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
在另一个实验方案中，所述药物制剂包含：约2.0mg/ml的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；约14mg/ml的丙二醇；约5.5mg/ml的苯酚；约1.42mg/ml的磷酸氢二钠；和
所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
本发明第三方面提供了一种药物组合物，其包含本发明第一方面所述的GLP-1化合物、和酰化胰岛素。
在一个实施方案中，所述酰化胰岛素是B29K(N(ε)-二十二烷二酰基-γGlu-OEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素；或B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素。
在一个实施方案中，所述酰化胰岛素为下述的胰岛素：所述酰化胰岛素的胰岛素母体为天然存在的胰岛素或胰岛素类似物，且包含至少一个赖氨酸残基，所述酰化胰岛素的酰基部分与所述胰岛素母体的赖氨酸残基或N-末端氨基酸残基的氨基相连接，所述酰基部分如式(A)所示：
III-(II) m-(I) n-(A)，其中,m为0或1-10的整数，n为5-20的整数；I是中性的、包含亚烷基二醇的氨基酸残基；II是酸性的氨基酸残基；III是包含20-24个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除；III、II、和I之间以酰胺键连接；和II和I在式(A)中出现的顺序可以独立地互换。
在一个实施方案中，n为5-15，优选地，n为5、6、7、8、9、10、11、12、13或14，优选地，n为5、6、7、8、9、10、11或12，优选地，n为5、6、7、8、9、或10，优选地，n为5、6、7、8或9，优选地，n为5、6、7、或8。
在另一个实施方案中，m为1-6，优选地，m为1、2、3、或4，优选地，m为1或2，优选地，m为1。
在又一个实施方案中，III是包含20-23个碳原子的脂肪族二酸，优选III是包含20、21、或22个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除。
在另一个实施方案中，所述胰岛素母体包含一个赖氨酸残基。
在一个实施方案中，I是：-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-CH 2-O-CH 2-CO-、 -HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 3-O-CH 2-CO-、或-HN-(CH 2) 4-O-(CH 2) 4-O-CH 2-CO-；优选I是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-。
在另一个实施方案中，II是选自下述的氨基酸残基：γGlu、αGlu、βAsp、αAsp、γ-D-Glu、α-D-Glu、β-D-Asp或α-D-Asp；优选地，II选自γGlu或βAsp。
在另一个实施方案中，III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-或HOOC-(CH 2) 22-CO-，优选地III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
在一个实施方案中，式(A)通过I的C末端与所述胰岛素母体的赖氨酸残基或N-末端氨基酸残基的氨基连接。
在一个实施方案中，所述酰基部分与所述胰岛素母体的赖氨酸残基的ε氨基相连接。
在一个实施方案中，所述胰岛素母体的赖氨酸残基位于B29位处。
在一个实施方案中，所述胰岛素母体选自下述胰岛素或胰岛素类似物：desB30人胰岛素(SEQ ID NO:4和SEQ ID NO:5，分别表示A链和B链)；A14E,B16H,B25H,desB30人胰岛素(SEQ ID NO:6和SEQ ID NO:7，分别表示A链和B链)；A14E,B16E,B25H,desB30人胰岛素(SEQ ID NO:8和SEQ ID NO:9，分别表示A链和B链)；人胰岛素(SEQ ID NO:10和SEQ ID NO:11，分别表示A链和B链)；A21G人胰岛素(SEQ ID NO:12和SEQ ID NO:13，分别表示A链和B链)；A21G,desB30人胰岛素(SEQ ID NO:14和SEQ ID NO:15，分别表示A链和B链)；或B28D人胰岛素(SEQ ID NO:16和SEQ ID NO:17，分别表示A链和B链)。
在一个实施方案中，所述酰化胰岛素选自下述胰岛素：B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基 -6xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素； A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-7xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu), desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷 二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG), desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二 烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二 烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基 -γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基 -γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-24xOEG),desB30人胰岛素。
发明人出乎意料地发现，本发明第一方面所述的化合物和酰化胰岛素的药物组合物不仅不会削弱所述化合物的物理稳定性，而且，组合制剂具有比单药制剂更好的物理稳定性。与其他长效胰岛素衍生物(例如德谷胰岛素和利拉鲁肽)的组合制剂相比，本发明的组合制剂的物理稳定性是出乎意料的。此外，组合制剂相比于单药制剂，还使得所述酰化胰岛素的化学稳定性增加。
本发明第四方面提供了本发明第一方面所述的化合物、本发明第二方面所述的药物制剂或本发明第三方面所述的药物组合物用作药物的用途。
在一个实施方案中，本发明第一方面所述的化合物、本发明第二方面所述的药物制剂或本发明第三方面所述的药物组合物用于治疗或预防高血糖症、糖尿病、和/或肥胖症。
本发明第五方面提供了本发明第一方面所述的化合物、本发明第二方面所述的药物制剂或本发明第三方面所述的药物组合物在制备用于治疗或预防高血糖症、糖尿病、和/或肥胖症的药物中的用途。
本发明第六方面提供了治疗或预防高血糖症、糖尿病、和/或肥胖症的方法，所述方法包括施用有效量的本发明第一方面所述的化合物、本发明第二方面所述的药物制剂或本发明第三方面所述的药物组合物，所述疾病包括但不限于例如高血糖症、糖尿病和肥胖症。
附图说明
图1a示出了本发明实施例1-3的标题化合物、利拉鲁肽、和溶媒(vehicle)对db/db小鼠的降糖效果和作用时间，其中纵坐标的百分率是指，以给药前基线血糖为基准，给药后每个监测点的血糖与之相比所得的相应时间点的血糖百分率(下同)。
图1b与图1a相对应地示出了本发明实施例1-3的标题化合物、利拉鲁肽、和溶媒对db/db小鼠的降糖效果的AUC。
图2a示出了本发明实施例2的标题化合物、索玛鲁肽、和溶媒对db/db小鼠的降糖效果和作用时间。
图2b与图2a相对应地示出了本发明实施例2的标题化合物、索玛鲁肽、和溶媒对db/db小鼠的降糖效果的AUC。
图3a示出了本发明实施例3-4的标题化合物、利拉鲁肽、和溶媒对db/db小鼠的降糖效果和作用时间。
图3b与图3a相对应地示出了本发明实施例3-4的标题化合物、利拉鲁肽、和溶媒对db/db小鼠的降糖效果的AUC。
图4a示出了本发明实施例1-3的标题化合物、对照例3-4的标题化合物、和溶媒对db/db小鼠的降糖效果和作用时间。
图4b与图4a相对应地示出了本发明实施例1-3的标题化合物、对照例3-4的标题化合物、和溶媒对db/db小鼠的降糖效果的AUC。
图5a示出了本发明实施例11的标题化合物在100μg/kg和300μg/kg剂量时、对照例2的标题化合物、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠或正常小鼠(正常对照)降糖效果和作用时间。
图5b与图5a相对应地示出了本发明实施例11的标题化合物、对照例2的标题化合物、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠或正常小鼠(正常对照)降糖效果的AUC。
图5c示出了本发明实施例11的标题化合物、对照例2的标题化合物、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠或正常小鼠(正常对照)的减重效果。
图6a示出了本发明实施例11的标题化合物、对照例2的标题化合物、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠或正常小鼠(正常对照)首次给药后48小时进行ipGTT时的降糖效果。
图6b与图6a相对应地示出了本发明实施例11的标题化合物在、对照例2的标题化合物、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL 小鼠或正常小鼠(正常对照)首次给药后48小时进行ipGTT时的降糖效果的△AUC。
图7a示出了本发明实施例2的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的降糖效果。
图7b与图7a相对应地示出了本发明实施例2的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的降糖效果的△AUC。
图7c示出了本发明实施例2的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的食物摄入量的控制效果。
图7d示出了本发明实施例2的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的饮水量的控制效果
图8a示出了本发明实施例11的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的长期降糖效果。
图8b与图8a相对应地示出了本发明实施例11的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的长期降糖效果的AUC。
图8c示出了本发明实施例11的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的长期减重效果。
图8d示出了本发明实施例11的标题化合物、对照例2的标题化合物和溶媒对的db/db小鼠的长期食物摄入量的控制效果。
图8e示出了本发明实施例11的标题化合物、对照例2的标题化合物和溶媒对db/db小鼠的长期饮水量的控制效果。
图9a示出了本发明实施例11的标题化合物、实施例2的标题化合物、度拉糖肽和溶媒对Kkay小鼠的降糖效果。
图9b与图9a相对应地示出了本发明实施例11的标题化合物、实施例2的标题化合物、度拉糖肽和溶媒对Kkay小鼠的降糖效果的AUC。
图9c示出了本发明实施例11的标题化合物、实施例2的标题化合物、度拉糖肽和溶媒对Kkay小鼠的降HbA1c效果。
图10a示出了本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)对db/db小鼠或正常小鼠(正常对照)中的长期降糖效果。
图10b与图10a相对应地示出了本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)对db/db小鼠或正常小鼠(正常对照)中的长期降糖效果的△AUC。
图10c示出了施用本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)在db/db小鼠或正常小鼠(正常对照)中注射前、第三次、第五次和第十一次注射后的随机血糖值。
图10d示出了本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)对db/db小鼠或正常小鼠(正常对照组)首次给药后48小时进行ipGTT时的降糖效果。
图10e与图10d相对应地示出了本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)对db/db小鼠或正常小鼠(正常对照组)首次给药后48小时进行ipGTT时的降糖效果的AUC。
图11a示出了本发明实施例11的标题化合物、度拉糖肽、溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠或正常小鼠(正常对照组)的长期减重效果。
图11b示出了本发明实施例11的标题化合物、度拉糖肽和溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL小鼠的长期食物摄入量的控制效果。
图11c示出了本发明实施例11的标题化合物、度拉糖肽和溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL雌性小鼠的卵巢周围脂肪减少效果。
图11d示出了本发明实施例11的标题化合物、度拉糖肽和溶媒(模型对照组)对高脂饮食诱导的肥胖C57BL雄性小鼠的附睾脂肪减少效果。
具体实施方式
定义
GLP-1类似物、和GLP-1衍生物
本文所用的术语“GLP-1类似物”或“GLP-1的类似物”是指作为人胰高血糖素样肽-1(GLP-1(7-37))变体的肽或化合物，其中GLP-1(7-37)的一个或多个氨基酸残基被替换、和/或其中一个或多个氨基酸残基被缺失、和/或其中增加了一个或多个氨基酸残基。具体地，GLP-1(7-37)的序列如序列表中的SEQ ID NO：1所示。具有SEQ ID NO：1所示序列的肽也可称为“天然”GLP-1或“天然”GLP-1(7-37)。
在序列表中，SEQ ID NO：1的第一个氨基酸残基(组氨酸)编号为1。然而，在下文中，依据本领域已建立的习惯，该组氨酸残基编号定为7，并且其后的氨基酸残基也随之编号，结尾是37号甘氨酸。因此，通常，本文所涉及到的GLP-1(7-37)序列的氨基酸残基编号或位置编号是开始于位置7的His和结束于位置37的Gly的序列。
[Gly8，Arg34]GLP-1-(7-37)肽，是在对应于GLP-1(7-37)(SEQ ID NO：1)位置8和位置34的位置处分别具有Gly和Arg的GLP-1类似物，[Arg34]GLP-1-(7-37)肽，是在对应于GLP-1(7-37)(SEQ ID NO：1)位置34的位置处具有Arg的GLP-1类似物。具体地，[Gly8，Arg34]GLP-1-(7-37)肽和[Arg34]GLP-1-(7-37)肽的氨基酸序列分别如序列表中的SEQ ID NO：2和SEQ ID NO：3所示。
在GLP-1肽或其类似物的情况下，本文所用的术语“衍生物”是指经化学修饰的GLP-1肽或类似物，其中一个或多个取代基已与所述肽共价连接。取代基也可称为侧链。
除非另有说明，否则当提及与赖氨酸残基酰化时，理解为与其ε-氨基进行。
本发明的式(B)的GLP-1衍生物可存在不同的立体异构体形式，其具有相同分子式和所连接的原子序列，但仅在其原子空间的三维方向上不同。除非另有说明，否则本发明涉及要求保护的衍生物的所有立体异构体形式。
术语“肽”当用于例如本发明的GLP-1类似物时，是指包含通过酰胺(或肽)键相互连接的一系列氨基酸的化合物。
在一个具体的实施方案中，肽很大程度上或主要由通过酰胺键相互连接的氨基酸组成(例如摩尔质量的至少50％、60％、70％、80％或至少90％)。在另一个具体的实施方案中，肽由通过肽键相互连接的氨基酸组成。
氨基酸是含有氨基和羧酸基的分子，任选含有一个或多个额外基团，通常称为侧链。
术语“氨基酸”包含蛋白质性的氨基酸(由遗传密码所编码，包括天然氨基酸和标准氨基酸)、以及非蛋白质性(在蛋白质中未发现，和/或在标准遗传密码中未编码)、和合成氨基酸。非蛋白质源性氨基酸是可以通过肽键整合进肽的部分，但不是蛋白质源性氨基酸。合成的非蛋白质源性氨基酸包括通过化学合成产生的氨基酸，即通过遗传密码编码的氨基酸的D-异构体例如D-丙氨酸和D-亮氨酸、Aib(α-氨基异丁酸)、Abu(α-氨基丁酸)、3-氨甲基苯甲酸、邻氨基苯甲酸、脱氨基-组氨酸、氨基酸的β类似物例如β-丙氨酸等、D-组氨酸、脱氨基-组氨酸、2-氨基-组氨酸、β-羟基-组氨酸、和高组氨酸(homohistidine)等。
不由遗传密码所编码的氨基酸的非限制性实例是γ-羧基谷氨酸、鸟氨酸、D-丙氨酸、D-谷氨酰胺和磷酸丝氨酸。合成氨基酸的非限制性实 例是氨基酸的D-异构体，例如D-丙氨酸和D-亮氨酸、Aib(α-氨基异丁酸)、β-丙氨酸和des-氨基-组氨酸(desH，替代名称咪唑丙酸，缩写Imp)。
在下文中，并未标明旋光异构体的所有氨基酸都理解为是指L-异构体(除非另有说明)。
药学上可接受的盐、酰胺或酯
本发明的GLP-1衍生物、类似物和中间产物可以呈药学上可接受的盐、酰胺或酯的形式。盐可以是碱式盐、酸式盐，或者中性盐。在水中碱式盐产生氢氧离子，酸式盐产生水合氢离子。本发明衍生物的盐可以用分别与阴离子基团或阳离子基团反应的添加的阳离子或阴离子来形成。这些基团可位于肽部分内和/或在本发明衍生物的侧链内。
本发明衍生物的阴离子基团的非限制性实例包括侧链(如果有的话)以及肽部分中的游离羧基。肽部分通常包括C-端的游离羧酸，并且它也可包括在内部酸性氨基酸残基例如Asp和Glu上的游离羧基。
肽部分的阳离子基团的非限制性实例包括N-端的游离氨基(如果有的话)以及在内部碱性氨基酸残基例如His、Arg和Lys上的任何游离氨基。
本发明衍生物的酯可以是例如通过游离羧酸基团与醇或酚反应而形成，其导致至少一个羟基被烷氧基或芳氧基取代。酯的形成可涉及肽的C-端的游离羧基，和/或侧链的任何游离羧基。
本发明衍生物的酰胺可以是例如通过游离羧酸基团与胺或取代胺反应，或通过游离或取代氨基与羧酸反应而生成。酰胺的形成可涉及肽的C-端的游离羧基、侧链的任何游离羧基、肽的N-端的游离氨基、和/或在肽和/或侧链中的任何游离或取代的肽氨基。
在一个具体的实施方案中，本发明的GLP-1化合物或GLP-1衍生物呈药学上可接受的盐的形式。在另一个具体的实施方案中，呈药学上可接受的酰胺的形式，优选在肽的C-端具有酰胺基。在再进一步的具体的实施方案中，肽或衍生物呈药学上可接受的酯的形式。
本发明的GLP-1(7-37)的肽和GLP-1类似物的制备方法是本领域公知的。例如，可以通过经典的肽合成，例如使用t-Boc或Fmoc化学法的固相肽合成或其他完善的技术，来生产本发明衍生物的GLP-1肽部分(或其片段)以及本发明的GLP-1类似物，参见，例如，Greene和Wuts,“Protective Groups in Organic Synthesis”,John Wiley&Sons,1999，Florencio Zaragoza“Organic Synthesis on solid Phase”,Wiley-VCH Verlag  GmbH,2000，以及由W.C.Chan和P.D.White编著的“Fmoc Solid PhasePeptide Synthesis”,Oxford University Press,2000。
在一个实施方案中，本发明的完整GLP-1类似物例如[Gly8，Arg34]GLP-1-(7-37)肽可以通过重组方法产生，即通过培养宿主细胞，该宿主细胞含有编码该类似物的DNA序列并能够在允许该肽表达的条件下在合适的营养培养基中表达该肽。适合表达这些肽的宿主细胞的非限制性实例是：大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyce scerevisiae)以及哺乳动物BHK或CHO细胞系。在一些实施方案中，生产工艺方法的这种完全重组发酵步骤是满足需要的，例如出于生产经济性方面的考虑。
将含有GPL-1化合物主链的融合蛋白包涵体经过变性、复性，得到含有正确构象的融合蛋白，经酶切、调沉、离心等一系列处理后，得到含量较高的GLP-1化合物主链。通过离子交换层析纯化，处理后得到纯度较高的GLP-1化合物主链。
术语“赋形剂”广义上是指除了活性治疗成分之外的任何成分。赋形剂可以是惰性物质、无活性物质和/或并非药物活性物质。
赋形剂可用于多种目的，例如作为载体、溶媒、稀释剂、片剂助剂、和/或用于改善给药、和/或活性物质的吸收。
药物活性成分与不同赋形剂的配制是本领域已知的，参见例如Remington：The Science and Practice of Pharmacy(例如第19版(1995)，和任何更新版本)。
赋形剂的非限制性实例是：溶剂、稀释剂、缓冲剂、防腐剂、等渗剂、螯合剂和稳定剂。
本发明的GLP-1衍生物和类似物具有GLP-1活性。具有GLP-1活性是指与GLP-1受体结合并引发信号转导途径而产生促胰岛素作用或其它生理效应的能力。
在一个具体的实施方案中，效力、功效和/或活性是指体外功效，即在功能性GLP-1受体测定中的表现，更尤其是在表达克隆的人GLP-1受体的细胞系中刺激cAMP形成的能力。
在另一个具体的实施方案中，本发明的衍生物在体内是强效的，其可在任何合适的动物模型以及在临床试验中按照本领域已知方式来测定。例如糖尿病性db/db小鼠是合适动物模型的一个实例，在这样的小鼠体内可测定血糖降低效应，例如按照本发明的实施例部分所述。
术语“胰岛素”包括天然存在的胰岛素，例如人胰岛素，以及其胰岛素类似物、胰岛素衍生物。
术语“胰岛素类似物”包含这样的多肽，其具有在形式上可以通过缺失和/或置换(替换)在天然胰岛素中存在的一个或多个氨基酸残基和/或添加至少一个氨基酸残基，而衍生自天然存在的胰岛素(例如人胰岛素)的结构的分子结构。优选地，置换的氨基酸残基是可编码氨基酸残基。
此处，术语“胰岛素衍生物”指的是已被化学方式修饰过的天然存在的胰岛素或胰岛素类似物，该修饰可以是例如在胰岛素骨架的一个或多个位置上引入侧链或者氧化或还原胰岛素上氨基酸残基的基团或者将游离羧基转化成酯基或酰化游离氨基或羟基。本发明的酰化胰岛素即属于胰岛素衍生物。
术语“胰岛素母体”是指胰岛素衍生物或酰化胰岛素的胰岛素部分(在本文中也称作母体胰岛素)，例如在本发明中是指酰化胰岛素的没有附加酰基的部分。胰岛素母体可以为天然存在的胰岛素，诸如人胰岛素或猪胰岛素。另一方面，母体胰岛素可以是胰岛素类似物。
此处，术语“氨基酸残基”包含从其中氢原子已从氨基中去除和/或羟基已从羧基中去除和/或氢原子已从巯基中去除的氨基酸。不精确地，氨基酸残基可以叫做氨基酸。
除非另有说明，本文提及的氨基酸都是L-氨基酸。
此处，术语亚烷基二醇包含寡和聚亚烷基二醇部分以及单亚烷基二醇部分。单亚烷基二醇和聚亚烷基二醇包括例如基于单和聚乙二醇、基于单和聚丙二醇、和基于单和聚丁二醇的链，即基于重复单位-CH 2CH 2O-、-CH 2CH 2CH 2O-或-CH 2CH 2CH 2CH 2O-的链。亚烷基二醇部分可以是单分散(具有定义明确的长度/分子量)以及多分散(具有定义较不明确的长度/平均分子量)。单亚烷基二醇部分包括在每个末端处包含不同基团的-OCH 2CH 2O-、-OCH 2CH 2CH 2O-或-OCH 2CH 2CH 2CH 2O-。
术语“脂肪酸”包括直链或支链脂族羧酸，其具有至少两个碳原子并为饱和或不饱和的。脂肪酸的非限定实例为例如肉豆蔻酸、棕榈酸、硬脂酸和二十烷酸。
此处，术语“脂肪族二酸”包括直链或支链脂族二羧酸，其具有至少两个碳原子并为饱和或不饱和的。脂肪族二酸的非限定实例为己二酸、辛二酸、癸二酸、十二烷二酸、十四烷二酸、十六烷二酸、十七烷二酸、十八烷二酸、二十烷二酸、二十二烷二酸和二十四烷二酸。
在本文中，胰岛素或GLP-1化合物的命名按照以下原则进行：按相对于人胰岛素的突变和修饰(例如酰化)、或天然GLP-1(7-37)的突变和修饰(例如酰化)给予名称。对于酰基部分的命名，按照IUPAC命名法和在其它情况下按肽命名法进行命名。例如，命名下述酰基部分：
Figure PCTCN2020141057-appb-000001
可例如命名为“二十烷二酰基-γGlu-OEG-OEG”、“二十烷二酰基-γGlu-2xOEG”或“二十烷二酰基-gGlu-2xOEG”、“19-羧基十九烷酰基-γGlu-2xOEG”或“19-羧基十九烷酰基-γGlu-OEG-OEG”，其中OEG表示基团-NH(CH 2) 2O(CH 2) 2OCH 2CO-(即，2-[2-(2-氨基乙氧基)乙氧基]乙酰基)的简写，γGlu(以及gGlu)是呈L构型的氨基酸γ谷氨酸的简写表示。或者，酰基部分可按照IUPAC命名法(OpenEye,IUPAC格式)命名。根据该命名法，本发明的上述酰基部分被称为以下名称：“[2-[2-[2-[2-[2-[2-[(4S)-4-羧基-4-(19-羧基十九烷酰基氨基)丁酰基]-氨基]-乙氧基]-乙氧基]乙酰基]氨基]乙氧基]乙氧基]乙酰基]”或“[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基]”。
例如，本发明实施例6的胰岛素(具有下文给出的序列/结构)被称为“B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素”、“B29K(Nε-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素”、或“B29K(Nε-二十烷二酰基-gGlu-5xOEG)，desB30人胰岛素”以表示人胰岛素中位置B29的氨基酸K已通过在B29的赖氨酸残基的ε氮(称为Nε或(N(ε))上被残基二十烷二酰基-gGlu-2xOEG酰化而修饰，且人胰岛素中位置B30的氨基酸T已被缺失。又例如，对照例5的胰岛素(具有下文给出的序列/结构)被称为“A14E,B16H,B25H,B29K(Nε二十烷二酰基-gGlu-2xOEG),desB30人胰岛素”或“A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-2xOEG), desB30人胰岛素”，表示人胰岛素中位置A14的氨基酸Y已被突变为E，人胰岛素中位置B16的氨基酸Y已被突变为H，人胰岛素中位置B25的氨基酸F已被突变为H，人胰岛素中位置B29的氨基酸K已通过在B29的赖氨酸残基的ε氮(称为Nε)上被残基二十烷二酰基-gGlu-2xOEG酰化而修饰，和人胰岛素中位置B30的氨基酸T已被缺失。
Figure PCTCN2020141057-appb-000002
本文中“nxPEG”表示-NH(CH 2CH 2O) nCH 2CO-,其中n为整数。例如，“12xPEG”表示基团-NH(CH 2CH 2O) 12CH 2CO-。
胰岛素是由胰脏中β-细胞分泌的多肽激素，由A和B两条多肽链构成，所述A链和B链由两个链间二硫键连接。此外，所述A链特征为有一个链内二硫键。
主要有三种方法用于在微生物中制备人胰岛素。两种涉及大肠杆菌，一种通过在细胞质中表达融合蛋白(Frank et al.(1981)in Peptides:Proceedings of the 7th American Peptide Chemistry Symposium(Rich&Gross,eds.),Pierce Chemical Co.,Rockford,III.第729-739页)，另一种是使用信号肽以使之能够分泌至周质空间(Chan et al..(1981)PNAS 78:5401-5404)。第三种方法是利用酿酒酵母使胰岛素前体分泌到培养基中(Thim et al.(1986)PNAS 83:6766-6770)。现有技术公开了许多在大肠杆菌或酿酒酵母中表达的胰岛素前体的方法，参照例如美国专利第5,962,267号、WO95/16708、EP0055945、EP0163529、EP0347845和EP0741188。
胰岛素类似物的载体的构建、表达、处理和纯化可使用本领域技术人员公知的技术进行。例如可以通过美国专利第6500645号中所公开的众所周知的技术，通过在合适的宿主细胞中表达编码目标胰岛素类似物 的DNA序列来制备所述胰岛素类似物。例如也可以通过下述文献中报道的方法来制备胰岛素类似物：Glendorf T,
Figure PCTCN2020141057-appb-000003
AR,Nishimura E,Pettersson I,&Kjeldsen T:Importance of the Solvent-Exposed Residues of the Insulin B Chainα-Helix for Receptor Binding；Biochemistry 2008 47 4743-4751。该文献中使用重叠延伸PCR将突变引入胰岛素编码载体。胰岛素类似物作为具有Ala-Ala-Lys小C-肽的前胰岛素样融合蛋白在酿酒酵母菌株MT663中表达。使用水解无色杆菌(A.lyticus)内切蛋白酶，单链前体经酶促转化为双链desB30类似物。
经过分离的胰岛素类似物可通过本领域公知的酰化方法在所需位置酰化，这样的胰岛素类似物的实例在例如公布号为CN1029977C、CN1043719A和CN1148984A的中国专利申请中已有描述。
编码的各胰岛素类似物多肽的核酸序列可由已建立的标准方法通过合成来制备，例如由Beaucage等(1981)Tetrahedron Letters 22：1859-1869中所描述的方法、或Matthes等(1984)EMBO Journal 3：801-805中描述的方法。
以下通过实施例对本发明作进一步的说明。需要注意的是，这些实施例不构成对本发明保护范围的限制。
实施例
缩略语
cAMP是环磷酸腺苷；
BHK是幼仓鼠肾细胞；
DNA是脱氧核糖核酸；
Na 2HPO 4是磷酸氢二钠；
NaOH是氢氧化钠；
OEG是氨基酸残基-NH(CH 2) 2O(CH 2) 2OCH 2CO-；
OSu是琥珀酰亚胺基-1-基氧基-2,5-二氧代-吡咯烷-1-基氧基；
OtBu是氧叔丁基；
HCl是氯化氢；
γGlu或gGlu是γL-谷氨酰基；
NHS是N-羟基琥珀酰亚胺；
DCC是二环己基碳二亚胺；
AEEA是2-(2-(2-氨基乙氧基)乙氧基)乙酸；
OH是氢氧根；
Gly是甘氨酸；
Arg是精氨酸；
TFA是三氟乙酸；
HbA1c是糖化血红蛋白。
实施例1
标题化合物：N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽(化合物1)
Figure PCTCN2020141057-appb-000004
1、N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基 丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8， Arg34]GLP-1-(7-37)肽的制备
通过一般的蛋白质重组表达方法制备[Gly8，Arg34]GLP-1-(7-37)肽(具体方法参见Molecular Cloning:A Laboratory Manual(Fourth Edition)，Michael R.Green，Cold Spring Harbor Press，2012)。使[Gly8，Arg34]GLP-1-(7-37)肽(5g，1.48mmol)溶解于100mM Na 2HPO 4水溶液(150mL)中，并且加入乙腈(100mL)，并用1N NaOH将pH调整至pH 10-12.5。使叔丁基二十烷二酰-γGlu(2xOEG-OSu)-OtBu(1.59g,1.63mmol)溶解于乙腈(50mL)中，并且缓慢加入[Gly8，Arg34]GLP-1-(7-37)肽溶液中。使pH维持在10-12.5。在120分钟后，将反应混合物加入水(150mL)中，并且用1N HCl水溶液将pH调整至5.0。通过离心分离沉淀，并且冻干。将粗产物加入三氟乙酸(60mL)与二氯甲烷(60ml)混合溶液中，并且在室温下搅拌30分钟。将混合物浓缩至约30ml，倾注到冰冷的正庚烷(300mL)中，通过过滤分离沉淀的产物，并用正庚烷洗涤2次。真空干燥后，真空干燥后，产物通过离子交换层析(Ressource Q，在42.5％乙醇中的0.25％-1.25％乙酸铵梯度，pH7.5)、反相层析(乙腈，水，TFA)纯化，将纯化的级分合并，用1N HCl将pH调整至5.2，分离沉淀物，并冻干得到标题化合物。
LC-MS(电喷雾)：m/z＝1028.79[M+4H] 4+
2、中间体叔丁基二十烷二酰-γ Glu-(2xOEG-OSu)-OtBu的制备
2.1叔丁基二十烷二酰-OSu
氮气保护条件下，将二十烷二酸单叔丁酯(20g，50.17mmo1)和NHS(5.77g，50.17mmo1)在二氯甲烷(400mL)中混合，加入三乙胺(13.95mL)，将得到的浑浊混合物在室温下搅拌，然后加入DCC(11.39g，55.19mmo1)，将其进一步搅拌过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜，得到24.12g(收率97％)的叔丁基二十烷二酰-OSu。
LC-MS(Scie×100API)：m/z＝496.36(M+1) +
2.2叔丁基二十烷二酰-γGlu-OtBu
将叔丁基二十烷二酰-OSu(24.12g，48.66mmol)溶于二氯甲烷(250mL)搅拌，依次加入H-Glu-OtBu(10.88g，53.53mmo1)，三乙胺(12.49mL)，水(25mL)，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)，分液，下层有机相加入饱和盐水进行水洗，分液后下层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到27.27g(收率96％)的叔丁基二十烷二酰-γGlu-OtBu。
LC-MS(Scie×100API)：m/z＝584.44(M+1) +
2.3叔丁基二十烷二酰-γGlu(OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-OtBu(27.27g，46.71mmol)溶于二氯甲烷(300mL)，加入三乙胺(11.99mL)搅拌10分钟，再加入NHS(5.38g，50.17mmo1)，接着加入DCC(10.60g，51.38mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，加入甲基叔丁基醚，搅拌30分钟，抽滤，滤饼真空干燥过夜,得到25.76g(收率81％)的叔丁基二十烷二酰-γGlu-(OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝681.46(M+1) +
2.4叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu
将叔丁基二十烷二酰-γGlu-(OSu)-OtBu(25.76g，37.83mmol)溶于二氯甲烷(250mL)搅拌，依次加入2xAEEA(11.66g，37.83mmo1)，三乙胺(9.71mL)，水(25mL)，将其加热得到澄清溶液，将该溶液在室 温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)分液，下层有机相加入饱和盐水进行水洗，分液后下层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到30.75g(收率93％)的叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu。
LC-MS(Scie×100API)：m/z＝874.59(M+1) +
2.5叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu(30.75g，35.18mmol)溶于二氯甲烷(300mL)，加入三乙胺(9.03mL)搅拌10分钟，再加入NHS(4.05g，35.18mmo1)，接着加入DCC(7.98g，38.70mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥、过滤，将滤液减压浓缩至几乎干燥，真空干燥过夜，得到31.09g(收率91％)的叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝971.61(M+1) +
实施例2
标题化合物：N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽(化合物2)
Figure PCTCN2020141057-appb-000005
以与实施例1第1部分类似的步骤制备N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝992.52[M+4H] 4+
中间体 叔丁基二十烷二酰-γGlu-(OEG-OSu)-OtBu以与实施例1第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝826.54(M+1) +
实施例3
标题化合物：N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Gly8,Arg34]GLP-1-(7-37)肽(化合物3)
Figure PCTCN2020141057-appb-000006
以与实施例1第1部分类似的步骤制备 N-ε 26-(19-羧基十九烷酰基氨 基)-4(S)-羧基丁酰基-[Gly8,Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝956.25[M+4H] 4+
中间体 叔丁基二十烷二酰-γGlu-(OSu)-OtBu以与实施例1第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝681.46(M+1) +
实施例4
标题化合物：N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽(化合物4)
Figure PCTCN2020141057-appb-000007
以与实施例1第1部分类似的步骤制备 N-ε 26-(19-羧基十九烷酰基氨 基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝959.75[M+4H] 4+
中间体 叔丁基二十烷二酰-γGlu-(OSu)-OtBu以与实施例1第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝681.46(M+1) +
对照例1
对照化合物利拉鲁肽，按照专利CN1232470A的实施例37进行制备。
对照例2
对照化合物索玛鲁肽，按照专利CN101133082A的实施例4进行制备。
对照例3
标题化合物：N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽
Figure PCTCN2020141057-appb-000008
以与实施例1第1部分类似的步骤制备 N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17- 羧基十七烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基) 乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝1021.78[M+4H] 4+
对照例4
标题化合物：N-ε 26-(17-羧基十七烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽
Figure PCTCN2020141057-appb-000009
以与实施例1第1部分类似的步骤制备 N-ε 26-(17-羧基十七烷酰基氨 基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝949.24[M+4H] 4+
中间体 叔丁基十八烷二酰-γGlu-(OSu)-OtBu以与实施例1第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝653.43(M+1) +
实施例5：在db/db小鼠中的药效学研究
本研究的目的在于在糖尿病情况下证实本发明GLP-1衍生物对高血糖(BG)的调控效应。
在肥胖的2型糖尿病(T2DM)小鼠模型(db/db小鼠)上，在单剂量研究中测试实施例1-4和对照例1-4的标题化合物(也称GLP-1衍生物)。以100μg/kg的不同剂量测试所述GLP-1衍生物的降低血糖的药效效果。
8-9周龄雄性db/db(BKS/Lepr)小鼠于屏障环境内饲养于合适规格的饲养盒中，自由获取标准食物和纯化水，环境条件控制在相对湿度为40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
于当日实验开始前，在时间约上午约9：30点评估基础血糖，并对小鼠称重。根据随机血糖和体重将小鼠匹配分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下注射GLP-1衍生物100μg/kg，其中溶媒包含：丙二醇14mg/ml、苯酚5.5mg/ml、磷酸氢二钠1.133mg/ml，所述溶媒的pH为8.12。
将所述GLP-1衍生物溶解在溶媒中至20μg/ml的给药浓度，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药方式，颈背部皮下注射给药一次。在大约上午10:30(时间0)给予相应的GLP-1衍生物，给药期间动物自由摄食和饮水，在给药后2、4、6、8、10、12、24、48和72小时评估小鼠血糖。用酒精棉球清洁鼠尾部，使用一次性采血针从尾巴采集血滴，用血糖仪及配套试纸(罗氏)进行测定。给药24、48和72h分别测量小鼠摄食量和每只小鼠体重。
以给药前基线血糖为基准，给药后每个监测点的血糖与之相比得相应时间点的血糖百分率，对于每个单剂量的GLP-1衍生物绘制血糖百分率对时间的剂量反应曲线，为了定量说明GLP-1衍生物对血糖的影响，对于每个单独的剂量响应曲线，计算从0至72小时的血糖百分率-时间的曲线下面积(AUC 0-72h)。其中，AUC是时间-血糖百分率曲线的曲线下面积，AUC值越小，表明降糖效果越好，药效越好。
图1a-4b显示了本发明的GLP-1衍生物具有预料不到的增加的药效,例如实施例1-4的标题化合物对db/db小鼠的降糖效果明显优于利拉鲁肽、和对照例3-4的化合物。特别是本发明实施例2的化合物的降糖效果优于索玛鲁肽。此外，本发明的GLP-1衍生物例如实施例1-4的化合物在db/db小鼠中的有效作用时间相比于利拉鲁肽和对照例3-4的化合物有明显延长，特别是实施例2的化合物在db/db小鼠中的降糖有效作用时间长于索玛鲁肽。
实施例6
B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素(化合物5)
Figure PCTCN2020141057-appb-000010
1、des(B30)人胰岛素的合成
des(B30)人胰岛素根据中国专利CN1056618C实施例101中所述 方法制备。
2、目标胰岛素的制备
将desB30人胰岛素(5g，0.876mmol)溶解于100mM Na 2HPO 4水溶液(150mL)中，并且加入乙腈(100mL)，并用1N NaOH将pH调整至pH 10-12.5。使叔丁基二十烷二酰-γGlu-(5xOEG-OSu)-OtBu(1.36g，0.964mmol)溶解于乙腈(50mL)中，并且缓慢加入胰岛素溶液中。使pH维持在10-12.5。在120分钟后，将反应混合物加入水(150mL)中，并且用1N HCl水溶液将pH调整至5.0。通过离心分离沉淀，并冻干。将粗产物加入三氟乙酸(60mL)与二氯甲烷(60ml)混合溶液中，并且在室温下搅拌30分钟。将混合物浓缩至约30ml，倾注到冰冷的正庚烷(300mL)中，通过过滤分离沉淀的产物，并用正庚烷洗涤2次。真空干燥后，通过离子交换层析(Ressource Q，在42.5％乙醇中的0.25％-1.25％乙酸铵梯度，pH7.5)、反相层析(乙腈，水，TFA)纯化，将纯化的级分合并，用1N HCl将pH调整至5.2，并分离沉淀物，冻干、得到标题化合物化合物5。
LC-MS(电喷雾)：m/z＝1377.53[M+5H] 5+
3、中间体叔丁基二十烷二酰-γGlu-(5xOEG-OSu)-OtBu的制备
3.1叔丁基二十烷二酰-OSu
氮气保护条件下，将二十烷二酸单叔丁酯(20g，50.17mmo1)和NHS(5.77g，50.17mmo1)在二氯甲烷中混合，加入三乙胺(13.95mL)，将得到的浑浊混合物在室温下搅拌，然后加入DCC(11.39g，55.19mmo1)，并进一步搅拌过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减 压浓缩至几乎干燥，真空干燥过夜,得到24.12g(收率97％)的叔丁基二十烷二酰-OSu。
LC-MS(Scie×100API)：m/z＝496.36(M+1) +
3.2叔丁基二十烷二酰-γGlu-OtBu
将叔丁基二十烷二酰-OSu(24.12g，48.66mmol)溶于二氯甲烷(250mL)搅拌，依次加入H-Glu-OtBu(10.88g，53.53mmo1)，三乙胺(12.49mL)，水，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)，分液，下层有机相加入饱和盐水进行水洗，分液后下层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到27.27g(收率96％)的叔丁基二十烷二酰-γGlu-OtBu。
LC-MS(Scie×100API)：m/z＝584.44(M+1) +
3.3叔丁基二十烷二酰-γGlu-(OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-OtBu(27.27g，46.71mmol)溶于二氯甲烷(300mL)，依次加入三乙胺(11.99mL)搅拌10分钟，再加入NHS(5.38g，50.17mmo1)，接着加入DCC(10.60g，51.38mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，加入甲基叔丁基醚，搅拌30分钟，抽滤，滤饼真空干燥过夜,得到25.76g(收率81％)的叔丁基二十烷二酰-γGlu-(OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝681.46(M+1) +
3.4叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu
将叔丁基二十烷二酰-γGlu-(OSu)-OtBu(25.76g，37.83mmol)溶于二氯甲烷(250mL)搅拌，依次加入2xAEEA(11.66g，37.83mmo1)，三乙胺(9.71mL)，水(25mL)，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)，分液，下层有机相加入饱和盐水进行水洗，分液后下层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到30.75g(收率93％)的叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu。
LC-MS(Scie×100API)：m/z＝874.59(M+1) +
3.5叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu(30.75g，35.18mmol)溶于二氯甲烷(300mL)，加入三乙胺(9.03mL)搅拌10分钟，再加入NHS(4.05g，35.18mmo1)，接着加入DCC(7.98g，38.70mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水进行水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜，得到31.09g(收率91％)的叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝971.61(M+1) +
3.6叔丁基二十烷二酰-γGlu-(5xOEG-OH)-OtBu
将叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu(31.09g，32.01mmol)溶于二氯甲烷(350mL)搅拌，依次加入3xAEEA(14.52g，32.01mmo1)，三乙胺(8.90mL)，水(25mL)，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)，分液，下层有机相加入饱和盐水进行水洗，分液后下层有机相用无水硫酸钠干燥，过滤、将滤液减压浓缩至几乎干燥，真空干燥过夜。得到38.99g(收率93％)的叔丁基二十烷二酰-γGlu-(5xOEG-OH)-OtBu。
LC-MS(Scie×100API)：m/z＝1309.81(M+1)+
3.7叔丁基二十烷二酰-γGlu-(5xOEG-OSu)-OtBu
氮气保护条件下，将叔丁基二十烷二酰-γGlu-(5xOEG-OH)-OtBu(38.99g，29.77mmol)溶于二氯甲烷(400mL)，加入三乙胺(8.28mL)搅拌10分钟，再加入NHS(3.43g，29.77mmo1)，接着加入DCC(6.76g，32.75mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水洗涤，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜，得到38.11g(收率91％)的叔丁基二十烷二酰-γGlu-(5xOEG-OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝1406.83(M+1) +
实施例7：
B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物6)
Figure PCTCN2020141057-appb-000011
以与实施例6第2部分类似的步骤制备化合物6。
LC-MS(电喷雾)：m/z＝1406.28[M+5H] 5+
中间体 叔丁基二十烷二酰-γGlu-(6xOEG-OSu)-OtBu以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1551.90(M+1) +
实施例8：
B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素(化合物7)
Figure PCTCN2020141057-appb-000012
以与实施例6第2部分类似的步骤制备化合物7。
LC-MS(电喷雾)：m/z＝1464.30[M+5H] 5+
中间体 叔丁基二十烷二酰-γ Glu-(8xOEG-OSu)-OtBu以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1814.02(M+1) +
实施例9：
B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物8)
Figure PCTCN2020141057-appb-000013
以与实施例6第2部分类似的步骤制备化合物8。
LC-MS(电喷雾)：m/z＝1411.88[M+5H] 5+
中间体 叔丁基二十二烷二酰-γGlu-(6xOEG-OSu)-OtBu的制备以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1579.94(M+1) +
实施例10：
B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素(化合物9)
Figure PCTCN2020141057-appb-000014
以与实施例6第2部分类似的步骤制备化合物9。
LC-MS(电喷雾)：m/z＝1469.91[M+5H] 5+
中间体 叔丁基二十二烷二酰-γ Glu-(8xOEG-OSu)-OtBu的制备以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1870.08(M+1) +
实施例11：
标题化合物：N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽(化合物10)
Figure PCTCN2020141057-appb-000015
以与实施例1第1部分类似的步骤制备N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽
LC-MS(电喷雾)：m/z＝1035.80[M+4H] 4+
中间体 叔丁基二十二烷二酰-γGlu-(2xOEG-OSu)-OtBu以与实施例1第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝999.64(M+1) +
实施例12：体外效力或活性
本实施例的目的是测试本发明的GLP-1衍生物的体外效力或活性
复苏表达GLP-1R的细胞，使用ham’s-F12培养基，将所述细胞接种至25mL细胞培养瓶中，37℃，5％CO 2培养过夜。实验当天分别配制本发明的实施例11的标题化合物(化合物10)和利拉鲁肽至150μg/mL，然后梯度稀释样品至750ng/ml、150ng/ml、30ng/ml、6ng/ml、1.2ng/ml、0.24ng/ml、0.048ng/ml、0.0096ng/ml和0.00192ng/ml。调整细胞浓度至1×10 5个细胞/ml，每孔加入200μl细胞和200μl稀释后样品，混匀后吸取100μl至新96孔板中，3复孔，在细胞培养箱中，培养4小时后加入荧光素酶试剂，振荡混匀后从96孔板中转至新96孔白色平底板，酶标仪读取信号值，数据用GraphPad Prism 6处理，计算EC 50。体外效力实验在不同日期重复4次。
表1：体外效力
实验对象 EC 50(nM)
利拉鲁肽 0.439
化合物10 0.677
由实验结果可知，本发明的GLP-1衍生物具有令人满意的体外效力，其体外活性与利拉鲁肽接近，这证实了其具有GLP-1受体激动活性。
实施例13：在高脂饮食诱导肥胖C57BL小鼠中的药效学实验
本研究的目的在于证实本发明的GLP-1衍生物在高脂饮食诱导肥胖C57BL小鼠中对血糖的调节效果及减重效果。
将5周龄、体重17-22g的C57BL小鼠(雌雄各半)，于屏障环境内饲养于合适规格的饲养盒中(3-5只/盒)，高脂饮食诱导组自由获取高脂饲料和纯化水，正常对照组自由获取标准食物和纯化水，环境条件控制在相对湿度40％-60％，温度为22℃-24℃，饲养10周，选择体重超过正常对照组小鼠体重的30％-50％的小鼠进行药效评价。
于当日实验开始前，在时间-1/1h(上午9:30)评估基础血糖，并对小鼠称重。根据随机血糖和体重将高脂饮食诱导组小鼠匹配分配到溶媒组(即模型对照组)或治疗组，接受如下处理：皮下注射溶媒，或皮下注射对照化合物索玛鲁肽100μg/kg，或皮下注射本发明实施例11的标题化合物100μg/kg和300μg/kg。其中溶媒包含：丙二醇14mg/ml、苯酚5.5mg/ml、磷酸氢二钠1.133mg/ml，所述溶媒的pH为7.4。
采用皮下给药方式，颈背部皮下给药一次(5μl/g体重)。在大约上午10:30(时间0)给予GLP-1衍生物，在给药后3、6、24、48和72小时评估小鼠血糖。同时，每天监测测小鼠体重。
对每个单剂量的GLP-1衍生物绘制Δ血糖-时间曲线。其中，Δ是指给定时间的实际血糖减去基线，其中基线为在时间0时的血糖。因此，在这些曲线中，y＝0表示基线。对于每个单独的剂量响应曲线，计算从0至监测终点的血糖-时间曲线下面积差(ΔAUC)，ΔAUC值越小，表明降糖效果越好，药效越好。
首次给药48小时后进行一次腹腔糖耐量试验(ipGTT)实验，步骤如下：于指定时间点尾尖采血测定空腹血糖(0min)，之后腹腔给予葡萄糖溶液(200mg/ml，10ml/kg)，然后于糖负荷后30min、60min和120min测定血糖。
用酒精棉球清洁鼠尾部，使用一次性采血针从尾巴采集血滴，用血糖仪(罗氏)及配套试纸进行测定。
对于每个单剂量的GLP-1衍生物绘制血糖对时间的剂量响应曲线、每日体重变化对时间的剂量响应曲线。为了更直观及定量说明本发明的GLP-1衍生物对血糖的影响，对于每个单独的剂量响应曲线，计算从0至监测终点的相对血糖-时间曲线下面积差(ΔAUC)。其中，ΔAUC值越小，表明降糖效果越好，药效越好。
图5a-6b显示了本发明的GLP-1衍生物具有预料不到的增加的药效,例如实施例11的化合物10对高脂饮食诱导的肥胖C57BL小鼠的降糖效果在同剂量下与已上市的对照化合物索马鲁肽没有明显差异，甚至由定量的图5b和6b可以看出，本发明的GLP-1衍生物的降糖效果还略优于索玛鲁肽。尤其在药后72h时，同剂量的化合物10组血糖平均值低于同剂量的索玛鲁肽组血糖平均值。此外，本发明的GLP-1衍生物的降糖效果具有剂量依赖性，随着本发明的GLP-1剂量的增加，其降糖效果也随之明显提高。
图6a-6b显示，在ipGTT实验中，实施例11的化合物10，相比于溶媒，在高脂饮食诱导肥胖C57BL小鼠的首次给药后48小时进行ipGTT实验后，对血糖具有有明显的抑制作用，且略优于同剂量的索马鲁肽的降糖效果。
图5c显示本发明的GLP-1衍生物例如实施例11的化合物10具有优异的体重减轻效果，其减重效果优于索马鲁肽。
实施例14：在II型糖尿病db/db小鼠中的药效学研究
本研究的目的在于在糖尿病情况下证实本发明的GLP-1衍生物对血糖的调节效果。
在db/db小鼠上，以0.3、1、3、10、30和100nmol/kg的不同剂量测试实施例11的标题化合物和对照化合物利拉鲁肽的降低血糖效果，求算ED 50。
8-9周龄雄性db/db(BKS/Lepr)小鼠于屏障环境内饲养于合适规格的饲养盒中，自由获取标准食物和纯化水，环境条件控制在在相对湿度40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
于当日实验开始前，在时间上午9:00评估基础血糖，并对小鼠称重。根据随机血糖和体重将糖尿病小鼠匹配分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下注射实施例11的化合物或对照组化合物利拉鲁肽0.3、1、3、10、30和100nmol/kg，其中溶媒包含：丙二醇14mg/ml，苯酚5.5mg/ml，磷酸氢二钠1.133mg/ml，所述溶媒的pH为7.4。
采用皮下给药方式(50μl/10g体重)，颈背部皮下注射给药一次。在大约上午10:00(时间0)给予实施例11化合物，在给药后1、2、3、6、12、24、48和72小时评估小鼠血糖。
用酒精棉球清洁鼠尾部，使用一次性采血针从尾巴采集血滴，用血糖仪(罗氏)及配套试纸进行测定。
对于每个单剂量的GLP-1衍生物绘制Δ血糖对时间的剂量响应曲线。Δ是指给定时间的实际血糖减去基线，其中基线为在时间0时的血糖。为了说明GLP-1衍生物对血糖的影响，对于每个单独的剂量响应曲线，计算从0至72小时的Δ血糖的曲线下面积ΔAUC，并针对ΔAUC计算有效剂量50％(ED 50，产生在基线与最大效应之间的一半响应的GLP-1衍生物剂量)。以下表2中示出了所获得的ED 50值。
表2：在db/db小鼠中对血糖影响的ED 50值
样品名称 ED 50(nmol/kg)
利拉鲁肽 9.68
化合物10 8.42
由试验结果可知，本发明的化合物10的体内降糖药效明显优于利拉鲁肽。
实施例15在II型糖尿病db/db小鼠中的药效学研究
本研究的目的在于证实本发明GLP-1衍生物对血糖、摄食量和饮水量的控制。
在II型糖尿病db/db小鼠上，在单剂量研究中测试实施例2的标题化合物和对照化合物索马鲁肽。
8-9周龄雄性db/db(BKS/Lepr)小鼠于屏障环境内饲养于合适规格的饲养盒中，自由获取标准食物和纯化水，环境条件控制在在相对湿度40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
于当日实验开始前，在时间约上午9:00评估基础血糖，并对小鼠称重。根据随机血糖和体重将糖尿病小鼠匹配分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下注射实施例2的化合物或对照组化合物索马鲁肽100μg/kg，其中溶媒包含丙二醇14mg/ml，苯酚5.5mg/ml，和磷酸氢二钠1.133mg/ml，pH7.4。
将所述GLP-1衍生物溶解在溶媒中至20μg/ml的给药浓度，采用皮下给药方式(50μl/10g体重)，颈背部皮下注射给药一次。在大约上午10:00(时间0)给予实施例2化合物，在给药后1、2、3、6、12、24、48和72小时评估小鼠血糖。用酒精棉球清洁鼠尾部，使用一次性采血针从尾巴采集血滴，用血糖仪(罗氏)及配套试纸进行测定。同时，每天测量小鼠摄食量及饮水量。
对于每个单剂量的GLP-1衍生物绘制血糖对时间的剂量响应曲线、摄食量对时间的剂量响应曲线、及饮水量对时间的剂量响应曲线。为了说明本发明的GLP-1衍生物对血糖的影响，对于每个单独的剂量响应曲线，计算从0至监测终点的血糖-时间的曲线下面积差(ΔAUC)。其中，ΔAUC值越小，表明降糖效果越好，药效越好。
图7a-图7d显示了本发明的GLP-1衍生物在给药后具有预料不到的增加的降糖药效、对食物摄入量和饮水量的提高的抑制效果。这进一步证明了实施例2的标题化合物在给药后对db/db小鼠的降糖效果优于同剂量下的索马鲁肽。此外，实施例2的标题化合物可以有效的控制食物摄入量和饮水量，其效果优于索马鲁肽，提示本发明的GLP-1衍生物具有更好的体重减轻效果。
实施例16在II型糖尿病db/db小鼠中的长期药效学研究
本研究的目的在于证实本发明GLP-1衍生物对II型糖尿病db/db小鼠的长期降糖效果、减重、及控制饮食效果。
在II型糖尿病db/db小鼠上测试实施例11的GLP-1衍生物和对照化合物索玛鲁肽。GLP-1衍生物以100和300μg/kg的不同剂量及索玛鲁肽以100μg/kg的剂量给予小鼠，测定所述GLP-1衍生物及对照化合物索玛鲁肽降低血糖、降低体重、减少食物摄入量和饮水量的效果。
8-9周龄雄性db/db(BKS/Lepr)小鼠于屏障环境内饲养于合适规格的饲养盒中，自由获取标准食物和纯化水，环境条件控制在在相对湿度40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
于当日实验开始前，在时间大约上午9:00评估基础血糖，并对小鼠称重。根据随机血糖和体重将糖尿病小鼠匹配分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下注射GLP-1衍生物100和300μg/kg，或皮下注射对照化合物索玛鲁肽100μg/kg。其中溶媒包含：丙二醇14mg/ml，苯酚5.5mg/ml，磷酸氢二钠1.133mg/ml，所述溶媒的pH值为7.4。
采用皮下给药方式(50μl/10g体重)，颈背部皮下注射给药，在大约上午10:00(时间0)给予GLP-1衍生物，分别于第0、3、7、10、13、16、19、22、25、28天给药，在每次给药前及最后一次给药后72小时评估小鼠血糖。第0-17天每天测量小鼠体重、摄食量和饮水量，第17天后每3天监测一次小鼠体重、摄食量和饮水量。
图8a-8f显示了本发明的GLP-1衍生物在长期给药后仍具有预料不到的增加的降糖药效、提高的体重减轻效果、及抑制摄食和饮水量的效果。如图8a和8b显示，相比于同剂量的索马鲁肽，实施例11的化合物10在长期给药后对db/db小鼠具有更优的降糖效果。图8c-8d显示，相比于同剂量的索马鲁肽，本发明的GLP-1衍生物例如实施例11的标题化合物具有更好的体重减轻效果、及抑制摄食和饮水量的效果。
实施例17在II型糖尿病Kkay小鼠中的长期药效学研究
本研究的目的在于证实本发明GLP-1衍生物对II型糖尿病Kkay小鼠的降糖效果
在II型糖尿病Kkay小鼠上测试实施例11的化合物10、实施例2的化合物2、和对照化合物度拉糖肽(又称杜拉鲁肽)。化合物10和化合物2以100和300μg/kg的不同剂量、度拉糖肽以600μg/kg的剂量给予小鼠，测定本发明的GLP-1衍生物及对照化合物度拉糖肽降低血糖效果和HbA1c的效果。
12-14周龄雄性Kkay小鼠于屏障环境内饲养于合适规格的饲养盒中，自由获取标准食物和纯化水，环境条件控制在在相对湿度40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
于当日实验开始前，在时间大约上午9:00评估基础血糖，并对小鼠称重。根据随机血糖和体重将糖尿病小鼠匹配分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下注射本发明的GLP-1衍生物100和300μg/kg，或皮下注射对照化合物度拉糖肽600μg/kg。其中溶媒包含：丙二醇14mg/ml，苯酚5.5mg/ml，磷酸氢二钠1.133mg/ml，pH7.4。
采用皮下给药方式(50μl/10g体重)，颈背部皮下注射给药，在大约上午10:00(时间0)给予本发明的GLP-1衍生物、度拉糖肽或溶媒，每2天给药1次，连续给药16次，在首次给药后3h、6h，1天和2天评估小鼠血糖，末次给药48h后取EDTA抗凝检测HbA1c。
图9a-9b显示了本发明的GLP-1衍生物给药后具有预料不到的增加的降糖药效,实施例11和实施例2的标题化合物对Kkay小鼠的降糖效果明显优于度拉糖肽。图9c显示本发明的GLP-1衍生物对II型糖尿病Kkay小鼠降低HbA1c的效果明显优于度拉糖肽。
实施例18：药代动力学
本实施例的目的在于说明本发明化合物的体内药代动力学性质。
SD大鼠的药代动力学
SD大鼠32只，每组8只(雌雄各半)，分为化合物10低剂量组、化合物10中剂量组、和化合物10高剂量组，分别皮下注射给药15、90、540μg/kg，以及化合物10静脉注射组，经静脉注射给药90μg/kg。化合物10低中高剂量组分别于给药前(0min)、给药后1、3、5、8、12、16、24、36、48、72、96、120h采血测定血药浓度，化合物10静脉注射组于给药前(0min)、给药后1min、10min、1、3、5、8、12、24、48、72、96、120h采血测定血药浓度。使用WinNonLin v6.4软件的非房室模型计算药代参数C max、T max、T 1/2、AUC 0-t、MRT，试验结果示出在表3中。
表3：向SD大鼠皮下注射化合物10后药代动力学参数
Figure PCTCN2020141057-appb-000016
Figure PCTCN2020141057-appb-000017
C max＝最大实测血浆浓度，T max＝最大实测血药浓度对应的时间，T 1/2＝末端消除半衰期，AUC 0-t＝0-t时间-血糖浓度时间曲线下面积，MRT＝平均驻留时间
食蟹猴中的药代动力学
食蟹猴24只，每组6只(雌雄各半)，分为化合物10低剂量组、化合物10中剂量组、化合物10高剂量组，分别皮下注射给予10、60、360μg/kg，以及化合物10静脉注射组，经静脉注射60μg/kg化合物10。化合物10低中高剂量组分别于给药前(0min)、给药后1、3、6、8、10、12、16、24、48、72、120、168、240h采血测定血药浓度，化合物10静脉注射组于给药前(0min)、给药后1min、10min、1、3、6、8、10、12、24、48、72、120、168、240h采血测定血药浓度。使用WinNonLin v6.4软件的非房室模型计算药代参数，C max、T max、T 1/2、AUC 0-t、MRT，试验结果示出在表4中。
表4：向食蟹猴皮下注射化合物10后药代动力学参数
Figure PCTCN2020141057-appb-000018
由上述实验结果可知，本发明的GLP-1衍生物化合物10在大鼠和食蟹猴体内均表现出较长的半衰期、较大的AUC 0-t、较长的MRT。且本发明的GLP-1衍生物均具有剂量依赖性，其药效随着剂量的增加而提高。
实施例19
本实验的目的在于测量本发明的GLP-1衍生物制剂的化学稳定性。
GLP-1衍生物制剂
将化合物10溶解于5.68mg/ml的磷酸氢二钠溶液中至8mg/ml的最终浓度，根据在下表中各组分的量，依次加入含有丙二醇和苯酚的辅液，调节pH至下表中的值，产生最终GLP-1化合物浓度为2mg/ml。
本实施例中制剂的化学稳定性能够用在37℃存储27天后，高分子量蛋白(HMWP)相对于0天时的变化来显示，同时也能够用在37℃存储28天后测得的有关物质的量的变化来表示。
高分子量蛋白(HMWP)的测定
通过高效液相色谱法(HPLC)测定 高分子量蛋白(HMWP)的含 量，在Waters TSKgel G2000SWXL(7.8*300mm)，5μm柱上，在柱温为30℃、样品池温度为5℃时，用流动相以0.5ml/min的流速进行测试，其中，所述流动相包含300ml异丙醇，400ml冰醋酸和300ml纯化水。检测波长为276nm，进样量为25μl。表5示出了，在37℃存储27天相对于第0天时的HMWP的增加量。
有关物质的量的测定
通过高效液相色谱法(HPLC)测定GLP-1衍生物有关杂质的含量，在Waters Kromasil 100-3.5-C8(4.6*250mm)柱上，在柱温为35℃、样品池温度为5℃时，用洗脱相以1.0ml/min的流速进行测试。洗脱用由以下组成的流动相进行：
A相包含90mM磷酸二氢钾和10％乙腈(v/v)，pH2.4
B相为75％(v/v)乙腈。
梯度：0-5min的75％/25％A/B至55％/45％A/B的线性变化，5-12min至50％/50％A/B的线性变化，12-42min至40％/60％A/B的线性变化，42-60min的10％/90％A/B的线性变化，60-61min至75％/25％A/B的线性变化，61-70min的85％/15％A/B的等度梯度。
检测波长为214nm，流速为1.0ml/min，进样量为15μl。表5示出了，在37℃存储28天相对于第0天时有关物质的增加量。
表5
Figure PCTCN2020141057-appb-000019
Figure PCTCN2020141057-appb-000020
有上表可知，制剂在pH值6.5-8.4之间时，均具有良好的化学稳定性，并且在pH值处于7.0-8.0之间时，制剂的化学稳定性最好。
实施例20
本实验的目的在于测量本发明的GLP-1衍生物制剂的化学稳定性。
根据下表6和表7中各个组分的量，按照与实施例19类似的步骤，配制表6和表7中的GLP-1衍生物制剂。并按照与实施例19类似的步骤测定HMWP和有关物质的变化。下表6和表7示出了不同配方的GLP-1衍生物制剂的HMWP和有关物质的变化。
表6
Figure PCTCN2020141057-appb-000021
表7
Figure PCTCN2020141057-appb-000022
Figure PCTCN2020141057-appb-000023
由上表可知，本发明的上述GLP-1衍生物制剂中HMWP的量和有关物质的量随着时间的变化增加非常缓慢，表明上述GLP-1衍生物制剂均具有优异的化学稳定性。
对照例5
A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素(对照化合物5)
Figure PCTCN2020141057-appb-000024
1、A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素的制备
通过常规的制备胰岛素类似物的方法制备A14E,,B16H,B25H,desB30人胰岛素(具体方法参见Glendorf T,
Figure PCTCN2020141057-appb-000025
AR,Nishimura E,Pettersson I,&Kjeldsen T:Importance of the Solvent-Exposed Residues of the Insulin B Chainα-Helix for Receptor Binding；Biochemistry 2008 47 4743-4751)。将A14E,B16H,B25H,desB30人胰岛素(5g，0.888mmol)溶解于100mM Na 2HPO 4水溶液(150mL)中，并且加入乙腈(100mL)，并用1N NaOH将pH调整至pH 10-12.5。使叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu(0.948g，0.976mmol)溶解于乙腈(50mL)中，并且缓慢加入胰岛素溶液中。使pH维持在10-12.5。在120分钟后，将反 应混合物加入水(150mL)中，并且用1N HCl水溶液将pH调整至5.0。通过离心分离沉淀，并冻干。将冻干的粗产物加入三氟乙酸(60mL)与二氯甲烷(60ml)混合溶液中，并在室温下搅拌30分钟。将混合物浓缩至约30ml，倾注到冰冷的正庚烷(300mL)中，并且通过过滤分离沉淀的产物，并且用正庚烷洗涤2次。真空干燥后，通过离子交换层析((Ressource Q，在42.5％乙醇中的0.25％-1.25％乙酸铵梯度，pH7.5)、反相层析(乙腈，水，TFA)纯化，将纯化的级分合并，用1N HCl将pH调整至5.2，并分离沉淀物，冻干、得到对照化合物5。
LC-MS(电喷雾)：m/z＝1063.6852[M+6H] 6+
2、中间体叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu的制备：以 与实施例1第3部分类似的步骤进行。
2.1叔丁基二十烷二酰-OSu
氮气保护条件下，将二十烷二酸单叔丁酯(20g，50.17mmo1)和NHS(5.77g，50.17mmo1)在二氯甲烷中混合，加入三乙胺(13.95mL)，使得到的浑浊混合物在室温下搅拌，然后加入DCC(11.39g，55.19mmo1)，并进一步搅拌过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水洗涤，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜,得到24.12g(收率97％)的叔丁基二十烷二酰-OSu。
LC-MS(Scie×100API)：m/z＝496.36(M+1) +
2.2叔丁基二十烷二酰-γGlu-OtBu
将叔丁基二十烷二酰-OSu(24.12g，48.66mmol)溶于二氯甲烷(250mL)搅拌，依次加入H-Glu-OtBu(10.88g，53.53mmo1)，三乙胺(12.49mL)，水，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL)，分液，下层有机相加入饱和盐水洗，分液后下层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到27.27g(收率96％)的叔丁基二十烷二酰-γGlu-OtBu。
LC-MS(Scie×100API)：m/z＝584.44(M+1) +
2.3叔丁基二十烷二酰-γGlu-(OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-OtBu(27.27g，46.71mmol)溶于二氯甲烷(300mL)，加入三乙胺(11.99mL)搅拌10min，再加入NHS(5.38g，50.17mmo1)，接着加入DCC(10.60g，51.38mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残 余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，加入甲基叔丁基醚，搅拌30min，抽滤，滤饼真空干燥过夜,得到25.76g(收率81％)的叔丁基二十烷二酰-γGlu-(OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝681.46(M+1) +
2.4叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu
将叔丁基二十烷二酰-γGlu-(OSu)-OtBu(25.76g，37.83mmol)溶于二氯甲烷(250mL)搅拌，依次加入2xAEEA(11.66g，37.83mmo1)，三乙胺(9.71mL)，水(25mL)，将其加热得到澄清溶液，将该溶液在室温下搅拌4小时。然后加入10％柠檬酸水溶液(200mL),分液，下层有机相加入饱和盐水洗，分液后下层层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜。得到30.75g(收率93％)的叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu。
LC-MS(Scie×100API)：m/z＝874.59(M+1) +
2.5叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
氮气保护条件下，将叔丁基二十烷二酰-γGlu-(2xOEG-OH)-OtBu(30.75g，35.18mmol)溶于二氯甲烷(300mL)，加入三乙胺(9.03mL)搅拌10分钟，再加入NHS(4.05g，35.18mmo1)，接着加入DCC(7.98g，38.70mmol)。在室温下搅拌混合物过夜。过滤，将得到的滤液浓缩至几乎干燥，将残余物与冷的水和乙酸乙酯混合，搅拌20分钟，分液，上层有机相加入饱和食盐水洗，分液后上层有机相用无水硫酸钠干燥，过滤后将滤液减压浓缩至几乎干燥，真空干燥过夜，得到31.09g(收率91％)的叔丁基二十烷二酰-γGlu-(2xOEG-OSu)-OtBu。
LC-MS(Scie×100API)：m/z＝971.61(M+1) +
实施例21
A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物11)
Figure PCTCN2020141057-appb-000026
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝1160.3997[M+6H] 6+
中间体叔丁基二十烷二酰-γGlu-(6xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1551.90(M+1) +
实施例22
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物12)
Figure PCTCN2020141057-appb-000027
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝1165.0674[M+6H] 6+
中间体叔丁基二十二烷二酰-γGlu-(6xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1579.94(M+1) +
实施例23
A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物13)
Figure PCTCN2020141057-appb-000028
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝1305.4716[M+6H] 6+
中间体叔丁基二十烷二酰-γGlu-(12xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝2423.35(M+1) +
实施例24
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物14)
Figure PCTCN2020141057-appb-000029
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝1310.1425[M+6H] 6+
中间体叔丁基二十二烷二酰-γGlu-(12xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝2451.38(M+1) +
实施例25 GLP-1受体结合
本实施例的目的是测试本发明的GLP-1衍生物的体外受体结合亲和力，以及白蛋白的存在如何潜在地影响结合。受体结合是GLP-1衍生物对人GLP-1受体的亲和力的量度。
本发明的GLP-1衍生物及对照化合物与人GLP-1受体的结合亲和力测定如下：即通过测定它们从受体中取代 125I-GLP-1的能力。为了测定GLP-1衍生物与白蛋白(HSA)的结合，用低浓度白蛋白(0.005％(w/v))、以及用高浓度白蛋白(2％(w/v))进行测定。结合亲和力IC 50的变化表明GLP-1衍生物与白蛋白结合，由此在动物模型中预测GLP-1衍生物的潜在延长的药物动力学特征。
对于存在低HSA(0.005％(w/v))的受体结合试验，向测定板的每个孔中添加50μl的测定缓冲液。对于存在高HSA(2％(w/v))的受体结合试验， 向测定板的每个孔中添加50μl的8％(w/v)白蛋白储备液。用pH7.3的10mM Na 2HPO 4配制测试化合物，参考对照品GLP-1(7-37)，用超纯水配制成1mM储备液。0.005％HSA条件下，将所有测试化合物和参考对照品用测定缓冲液稀释至2μM，然后进行4倍连续梯度稀释，共10个浓度梯度。2％HSA条件下，将参考对照品GLP-1(7-37)稀释至2μM，利拉鲁肽稀释至20μM，化合物10和索玛鲁肽稀释至800μM，然后所有样品进行4倍连续梯度稀释，共10个浓度梯度。将25μl不同浓度测试化合物或参考对照品分别添加至测定板的适当孔中。将细胞膜蛋白等份解冻并稀释至其工作浓度(40μg/mL)，向测定板的每个孔中添加50μl含细胞膜的溶液。通过向测定板的每个孔中添加25μl的[ 125I]-GLP-1的600pM溶液来开始孵育。将测定板在室温下孵育1h。孵育完毕后，用细胞收集器将反应液收集到GF/C过滤板上，用洗板缓冲液洗6次，50℃干燥箱干燥1h。加入50μl闪烁液，并封闭，使用Microbeta2读数。利用GraphPad Prism中的非线性回归分析，用该软件计算IC 50值并以nM为单位报告。对每个测试化合物最少重复三次。所报告的值为每种测试化合物的所有测量值的平均值。
表8：GLP-1受体结合亲和力
Figure PCTCN2020141057-appb-000030
“比率”是指[(IC 50/nM)高HSA]/[(IC 50/nM)低HSA]
通常，在低白蛋白浓度时与GLP-1受体的结合应当尽可能好，这对应于低IC 50值。在高白蛋白浓度时IC 50值是白蛋白对GLP-1衍生物与GLP-1受体结合的影响的度量。正如所知，GLP-1衍生物也与白蛋白结合，这通常是所需的效应，这种效应将延长它们的血浆寿命。因此，在高白蛋白时IC 50值通常高于在低白蛋白时的IC 50值，对应于与GLP-1受体的结合降低，这是由与GLP-1受体结合竞争的白蛋白结合所致。
因此，可采用高比率(IC 50值(高白蛋白)/IC 50值(低白蛋白))，作为目标衍生物与白蛋白结合良好(由此可确定具有长的半衰期)、并且自身与GLP-1受体也结合良好(IC 50值(高白蛋白)高，IC 50值(低白蛋白)低)的指 示。
由上表可以看出本发明的GLP-1衍生物的比率高于对照化合物索玛鲁肽、利拉鲁肽及GLP-1(7-37)，预示着本发明的化合物具有更长的半衰期、并且自身与GLP-1受体也结合良好。
实施例26在II型糖尿病db/db小鼠中的长期药效学研究
参照实施例16的类似的实验步骤，在II型糖尿病db/db小鼠中进行长期药效学研究，不同之处在于所用的对照化合物为度拉糖肽，度拉糖肽的给药剂量为300μg/kg。采用皮下给药方式(50μl/10g体重)，颈背部皮下注射给药，在大约上午10:00(时间0)给予GLP-1衍生物，分别于第0、3、6、9、12、15、18、21、24、27、30天给药。在首次给药后3、6、9、12、24、48和72小时评估小鼠血糖，并计算血糖-时间曲线下面积的变化(ΔAUC)。给药前、第3、5、11次给药后48h禁食6h监测空腹血糖。首次给药后48h进行腹腔糖耐量试验(ipGTT)实验，步骤如下：于指定时间点尾尖采血测定空腹血糖(0min)，之后腹腔给予葡萄糖溶液(200mg/ml，10ml/kg)，然后于糖负荷后30min、60min和120min测定血糖。用酒精棉球清洁鼠尾部，使用一次性采血针从尾巴采集血滴，用血糖仪(罗氏)及配套试纸进行测定，绘制时间血糖曲线，并计算时间-血糖曲线下面积(AUC)。
图10a-10e显示了本发明的GLP-1衍生物在长期给药后仍具有预料不到的增加的降糖药效。如图10a和10b显示，相比于度拉糖肽，实施例11的化合物10在给药后对db/db小鼠具有更优的降糖效果。如图10c显示，相比于度拉糖肽，化合物10在长期给药后对db/db小鼠具有更优的降糖效果。如图10d-10e显示，相比于度拉糖肽，本发明的GLP-1衍生物对血糖具有更明显的抑制作用，优于度拉糖肽的降糖效果。
实施例27在高脂饮食诱导肥胖C57BL小鼠中的药效学实验
参照实施例13的类似的实验步骤，在高脂饮食诱导肥胖C57BL小鼠中进行药效学实验，不同之处在于所用的对照化合物为度拉糖肽，度拉糖肽的给药剂量为300μg/kg。
采用皮下给药方式，颈背部皮下给药一次(5μl/g体重)，每3天给药一次，共11次。在大约上午10:30(时间0)给予GLP-1衍生物，在给药后3、6、9、12、24、48和72小时评估小鼠血糖。同时，每3天监测小鼠体重与摄食量。试验结束时称量皮下脂肪、肾周脂肪、生殖器周围脂肪。
图11a-11d显示了本发明的GLP-1衍生物具有预料不到的提高的体重减轻效果、控制饮食效果、及降脂效果。
实施例28
B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物15)
Figure PCTCN2020141057-appb-000031
以与实施例6第2部分类似的步骤制备化合物B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
LC-MS(电喷雾)：m/z＝1585.98[M+5H] 5+
中间体 叔丁基二十二烷二酰-γGlu-(12xOEG-OSu)-OtBu以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝2451.38(M+1) +
实施例29
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素(化合物16)
Figure PCTCN2020141057-appb-000032
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝1247.47[M+7H] 7+
中间体叔丁基二十二烷二酰-γGlu-(18xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝3320.83(M+1) +
实施例30
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-24xOEG),desB30人胰岛素(化合物17)
Figure PCTCN2020141057-appb-000033
与对照例5第1部分类似的步骤制备化合物A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-24xOEG),desB30人胰岛素
LC-MS(电喷雾)：m/z＝873.35[M+11H] 11+
中间体叔丁基二十二烷二酰-γGlu-(24xOEG-OSu)-OtBu的制备以与对照例5第2部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝4192.27(M+1) +
实施例31
B29K(N(ε)-二十二烷二酰基-γGlu-OEG),desB30人胰岛素(化合物18)
Figure PCTCN2020141057-appb-000034
以与实施例6第2部分类似的步骤制备化合物B29K(N(ε)-二十二烷二酰基-γGlu-OEG),desB30人胰岛素。
LC-MS(电喷雾)：m/z＝1266.8122[M+5H] 5+
中间体 叔丁基二十二烷二酰-γGlu-(OEG-OSu)-OtBu以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝854.57(M+1) +
实施例32
B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素(化合物19)
Figure PCTCN2020141057-appb-000035
以与实施例6第2部分类似的步骤制备化合物B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素。
LC-MS(电喷雾)：m/z＝1354.8667[M+5H] 5+
中间体 叔丁基二十二烷二酰-γGlu-(12xPEG-OSu)-OtBu以与实施例6第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1294.83(M+1) +
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。

Claims (25)

  1. 式B的化合物，或其药学上可接受的盐、酰胺或酯：
    [Acy-(L1) r-(L2) q]-G1  (B)，
    其中G1为在对应于GLP-1(7-37)(SEQ ID NO：1)的位置34处具有Arg及位置8处具有Ala或Gly的GLP-1类似物，[Acy-(L1) r-(L2) q]是连接至所述GLP-1类似物的位置26的Lys残基的ε氨基上的取代基，其中
    r为1-10的整数，q为0或1-10的整数；
    Acy是包含20-24个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除；
    L1是选自下述的氨基酸残基：γGlu、αGlu、βAsp、αAsp、γ-D-Glu、α-D-Glu、β-D-Asp或α-D-Asp；
    L2是中性的、包含亚烷基二醇的氨基酸残基；
    Acy、L1、和L2之间以酰胺键连接；和
    L1和L2在式(B)中出现的顺序可以独立地互换。
  2. 如权利要求1所述的化合物，其中，
    G1为[Gly8，Arg34]GLP-1-(7-37)(SEQ ID NO：2)肽或[Arg34]GLP-1-(7-37)(SEQ ID NO：3)肽，优选为[Gly8，Arg34]GLP-1-(7-37)肽；和/或
    r为1、2、3、4、5或6，优选地，r为1、2、3或4，优选地，r为1或2，优选地，r为1；和/或
    q为0、1、2、3、4、5、6、7或8，优选地，q为0、1、2、3或4，更优选，q为0、1、或2；和/或
    Acy是包含20-23个碳原子的脂肪族二酸，优选Acy是包含20、21、或22个碳原子的脂肪族二酸。
  3. 如权利要求1或2所述的化合物，其中，
    L2是：-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、 -HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 3-O-CH 2-CO-、或-HN-(CH 2) 4-O-(CH 2) 4-O-CH 2-CO-；优选L2是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；和/或
    L1选自γGlu或βAsp，优选L1为γGlu；和/或
    Acy是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-或HOOC-(CH 2) 22-CO-，优选地，Acy是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
  4. 如权利要求1-3任一项所述的化合物，其中，式(B)中Acy、L1、和L2之间依次以酰胺键连接，L2的C末端连接至所述GLP-1类似物的位置26的Lys残基的ε氨基上。
  5. 如权利要求1所述的化合物，所述化合物选自下述化合物：
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[23-羧基二十三烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[23-羧基二十三烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-(23-羧基二十三烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
    N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
    N-ε 26-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[20-羧基二十烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[22-羧基二十二烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[20-羧基二十烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[22-羧基二十二烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Arg34]GLP-1-(7-37)肽、
    N-ε 26-(20-羧基二十烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、或
    N-ε 26-(22-羧基二十二烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽；
    优选，所述化合物选自下述化合物：
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε 26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Gly8，Arg34]GLP-1-(7-37)肽、
    N-ε26-(19-羧基十九烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、
    N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽、或
    N-ε 26-[2-(2-[2-(4-[21-羧基二十一烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽。
  6. 药物制剂，所述药物制剂包含权利要求1-5任一项所述的化合物和可药用赋形剂。
  7. 如权利要求6所述的药物制剂，所述可药用赋形剂选自缓冲剂、防腐剂、等渗剂、稳定剂和螯合剂中的一种或多种；优选所述可药用赋形剂为缓冲剂、防腐剂和等渗剂。
  8. 如权利要求6-7任一项所述的药物制剂，其中所述等渗剂选自氯化钠、丙二醇、甘露醇、山梨醇、甘油、葡萄糖和木糖醇的一种或多种优选为丙二醇、甘露醇或氯化钠；和/或
    所述防腐剂选自苯酚、间甲酚、对-羟基苯甲酸甲酯、对-羟基苯甲酸丙酯、2-苯氧乙醇、对-羟基苯甲酸丁酯、2-苯乙醇、和苯甲醇中的一种或多种，优选为苯酚或间甲酚；和/或
    所述缓冲剂选自醋酸钠、碳酸钠、柠檬酸盐、甘氨酰甘氨酸、组氨酸、甘氨酸、赖氨酸、精氨酸、磷酸二氢钠、磷酸氢二钠、磷酸钠，和三(羟甲基)-氨基甲烷中的一种或多种，优选为醋酸钠、柠檬酸盐、磷酸二氢钠、或磷酸氢二钠。
  9. 如权利要求6-8任一项所述的药物制剂，其中，所述制剂的pH为约6.0至约10.0，优选为约6.5至约10.0，优选为约6.5至约9.5，优选为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
  10. 药物制剂，含有如下成分：
    约0.1-1.2mM、优选约0.2-1mM、优选约0.3-0.7mM、更优选约0.48-0.6mM的权利要求1-5任一项所述的化合物；
    约10-1500mM、优选约13-800mM、优选约65-400mM、优选约90-240mM、优选约150-250mM、优选约180-200mM、更优选约183-195mM的等渗剂，优选，所述等渗剂选自丙二醇、甘油、甘露醇或氯化钠中的一种或多种；
    约1-200mM、优选约5-150mM、优选约10-100mM、优选约20-85mM、优选约30-75mM、优选约45-60mM、更优选约50-60mM的防腐剂，优选，所述防腐剂选自苯酚或间甲酚中的一种或多种；
    约3-35mM、优选约5-20mM、更优选约5-15mM、更优选约7-10mM的缓冲剂，所述缓冲剂选自醋酸钠、柠檬酸盐、磷酸二氢钠或磷酸氢二钠中的一种或多种；和
    所述药物制剂的pH为约6.0至约10.0，优选为约6.5至约9.5，优选为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
  11. 药物制剂，其包含：
    约0.3-0.7mM、更优选为约0.48-0.6mM的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；
    约180-200mM、更优选约183-195mM的丙二醇；
    约45-60mM、更优选约50-60mM的苯酚；
    约5-15mM的缓冲剂，优选约7-10mM的磷酸氢二钠；和
    所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、进一步优选为约7.3至约8.3。
  12. 药物制剂，其包含：
    约0.5mM的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；
    约184mM的丙二醇；
    约58.5mM的苯酚；
    约10mM的磷酸氢二钠；和
    所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
  13. 药物制剂，其包含：
    约2.0mg/ml的N-ε 26-[2-(2-[2-(2-[2-(2-[4-(21-羧基二十一烷酰基氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽或N-ε 26-[2-(2-[2-(4-[19-羧基十九烷酰基氨基]-4(S)-羧基丁酰基氨基)乙氧基]乙氧基)乙酰基][Gly8，Arg34]GLP-1-(7-37)肽；
    约14mg/ml的丙二醇；
    约5.5mg/ml的苯酚；
    约1.42mg/ml的磷酸氢二钠；和
    所述药物制剂的pH为约6.5至约8.5，更优选为约7.0至约8.5、更优选为约7.0至约8.1、进一步优选为约7.3至约8.1。
  14. 药物组合物，所述药物组合物包含权利要求1-5任一项所述的化合物、和酰化胰岛素，优选，所述酰化胰岛素是B29K(N(ε)-二十二烷二酰基-γGlu-OEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素；或B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素；
    更优选，所述酰化胰岛素为下述酰化胰岛素，所述酰化胰岛素的胰岛素母体为天然存在的胰岛素或胰岛素类似物，且包含至少一个赖氨酸残基，所述酰化胰岛素的酰基部分与所述胰岛素母体的赖氨酸残基或N-末端氨基酸残基的氨基相连接，所述酰基部分如式(A)所示：
    III-(II) m-(I) n-  (A)，
    其中,
    m为0或1-10的整数，n为5-30(优选5-20)的整数；
    I是中性的、包含亚烷基二醇的氨基酸残基；
    II是酸性的氨基酸残基；
    III是包含20-24个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除；
    III、II、和I之间以酰胺键连接；和
    II和I在式(A)中出现的顺序可以独立地互换。
  15. 如权利要求14所述的药物组合物，其中，
    n为5-15的整数，优选地，n为5、6、7、8、9、10、11、12、13或14，优选地，n为5、6、7、8、9、10、11或12，优选地，n为5、6、7、8、9、或10，优选地，n为5、6、7、8或9，优选地，n为5、6、7、或8；和/或
    m为1-6的整数，优选地，m为1、2、3、或4，优选地，m为1或2，优选地，m为1；和/或
    III是包含20-23个碳原子的脂肪族二酸，优选III是包含20、21、或22个碳原子的脂肪族二酸，其中在形式上羟基已从所述脂肪族二酸的羧基之一中去除；和/或
    所述胰岛素母体包含一个赖氨酸残基。
  16. 如权利要求14或15所述的药物组合物，其中，
    I是：-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 4-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-(CH 2) 2-CO-、-HN-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 2-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 2-O-(CH 2) 2-O-(CH 2) 3-NH-CO-CH 2-O-CH 2-CO-、-HN-(CH 2) 3-O-(CH 2) 3-O-CH 2-CO-、或-HN-(CH 2) 4-O-(CH 2) 4-O-CH 2-CO-；优选I是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；和/或
    II是选自下述的氨基酸残基：γGlu、αGlu、βAsp、αAsp、γ-D-Glu、α-D-Glu、β-D-Asp或α-D-Asp；和/或
    III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-或HOOC-(CH 2) 22-CO-。
  17. 如权利要求14-16任一项所述的药物组合物，其中，式(A)通过I的C末端与所述胰岛素母体的赖氨酸残基或N-末端氨基酸残基的氨基连接。
  18. 如权利要求14-17任一项所述的药物组合物，其中，所述酰基部分与所述胰岛素母体的赖氨酸残基的ε氨基相连接。
  19. 如权利要求14-18任一项所述的药物组合物，其中，所述胰岛素母体的赖氨酸残基位于B29位处。
  20. 如权利要求14-19任一项所述的药物组合物，其中，所述胰岛素母体选自下述胰岛素或胰岛素类似物：desB30人胰岛素；A14E,B16H,B25H,desB30人胰岛素；A14E,B16E,B25H,desB30人胰岛素；人胰岛素；A21G人胰岛素；A21G,desB30人胰岛素；或B28D人胰岛素。
  21. 如权利要求14所述的药物组合物，其中，所述酰化胰岛素选自下述胰岛素：B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素； B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基 -γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-7xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)- 二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H, B29K(N(ε)-二十烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-αAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)- 二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-6xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-5xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基 -αGlu-αGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-8xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-7xOEG-γGlu-γGlu),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-7xOEG),desB30人胰岛素； A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-βAsp-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αGlu-αGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-αAsp-8xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-8xOEG), desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-7xOEG),desB30人胰岛素；或A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16E,B25H, B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十一烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十一烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十三烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十四烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-24xOEG),desB30人胰岛素；
    优选，所述酰化胰岛素选自下述胰岛素：B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基 -γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-7xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-11xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素；
    优选，所述酰化胰岛素选自下述胰岛素：B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-5xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG), desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；
    优选，所述酰化胰岛素选自下述胰岛素：A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  22. 权利要求1-5任一项所述的化合物、权利要求6-13任一项所述的药物制剂或权利要求14-21任一项所述的药物组合物，其用作药物。
  23. 权利要求1-5任一项所述的化合物、权利要求6-13任一项所述的药物制剂或权利要求14-21任一项所述的药物组合物，其用于治疗或预防高血糖症、糖尿病、和/或肥胖症。
  24. 权利要求1-5任一项所述的化合物、权利要求6-13任一项所述的药物制剂或权利要求14-21任一项所述的药物组合物在制备用于治疗或预防高血糖症、糖尿病、和/或肥胖症的药物中的用途。
  25. 治疗或预防高血糖症、糖尿病、和/或肥胖症的方法，包括施用治疗有效量的权利要求1-5任一项所述的化合物、权利要求6-13任一项所述的药物制剂或权利要求14-21任一项所述的药物组合物。
PCT/CN2020/141057 2019-12-30 2020-12-29 长效glp-1化合物 WO2021136303A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
CA3166496A CA3166496A1 (en) 2019-12-30 2020-12-29 Long-acting glp-1 compound
BR112022013042A BR112022013042A2 (pt) 2019-12-30 2020-12-29 Composto da fórmula b, formulação farmacêutica, composição farmacêutica, método para tratar ou prevenir hiperglicemia, diabetes e/ou obesidade
AU2020418207A AU2020418207A1 (en) 2019-12-30 2020-12-29 Long-acting GLP-1 compound
MX2022008139A MX2022008139A (es) 2019-12-30 2020-12-29 Compuestos de glp-1 de acción prolongada.
KR1020227026310A KR20220119731A (ko) 2019-12-30 2020-12-29 장기 지속형 glp-1 화합물
EP20909624.7A EP4086277A4 (en) 2019-12-30 2020-12-29 LONG-ACTING GLP-1 COMPOUND
JP2022540870A JP2023510218A (ja) 2019-12-30 2020-12-29 長時間作用型ｇｌｐ－１化合物
CN202080091240.0A CN114901680B (zh) 2019-12-30 2020-12-29 长效glp-1化合物
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WO2023083301A1 (zh) * 2021-11-12 2023-05-19 福建盛迪医药有限公司 Glp-1受体和gip受体双重激动剂的药物组合物及其用途
US11744873B2 (en) 2021-01-20 2023-09-05 Viking Therapeutics, Inc. Compositions and methods for the treatment of metabolic and liver disorders
WO2024179606A1 (zh) * 2023-03-02 2024-09-06 甘李药业股份有限公司 一种glp-1化合物的医药用途

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US11744873B2 (en) 2021-01-20 2023-09-05 Viking Therapeutics, Inc. Compositions and methods for the treatment of metabolic and liver disorders
WO2023083301A1 (zh) * 2021-11-12 2023-05-19 福建盛迪医药有限公司 Glp-1受体和gip受体双重激动剂的药物组合物及其用途
WO2024179606A1 (zh) * 2023-03-02 2024-09-06 甘李药业股份有限公司 一种glp-1化合物的医药用途

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