WO2021136302A1 - 胰岛素衍生物 - Google Patents

胰岛素衍生物 Download PDF

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WO2021136302A1
WO2021136302A1 PCT/CN2020/141056 CN2020141056W WO2021136302A1 WO 2021136302 A1 WO2021136302 A1 WO 2021136302A1 CN 2020141056 W CN2020141056 W CN 2020141056W WO 2021136302 A1 WO2021136302 A1 WO 2021136302A1
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Prior art keywords
γglu
human insulin
desb30 human
insulin
diacyl
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English (en)
French (fr)
Inventor
甘忠如
陈伟
张一宁
薛方凯
蔡玲玉
牛江红
穆彬
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Gan and Lee Pharmaceuticals Co Ltd
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Gan and Lee Pharmaceuticals Co Ltd
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Priority to CN202411113751.4A priority Critical patent/CN118772259A/zh
Priority to US17/758,108 priority patent/US20240025957A1/en
Priority to CN202411112792.1A priority patent/CN118894922A/zh
Priority to CN202080091328.2A priority patent/CN114901683B/zh
Priority to CN202411113581.XA priority patent/CN118754967A/zh
Priority to EP20910739.0A priority patent/EP4086280A4/en
Publication of WO2021136302A1 publication Critical patent/WO2021136302A1/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
    • A61K38/28—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of therapeutic peptides, in particular to novel insulin derivatives, their pharmaceutical preparations, their pharmaceutical compositions with long-acting GLP-1 compounds, their pharmaceutical compositions with fast-acting insulin, and the insulin derivatives , Medicinal use of pharmaceutical preparations and pharmaceutical compositions.
  • Insulin is a polypeptide hormone secreted by the beta cells of the pancreas.
  • Insulin is composed of two polypeptide chains named A chain and B chain, which are connected by two interchain disulfide bonds.
  • a chain and B chain In human, porcine and bovine insulin, the A chain and B chain contain 21 and 30 amino acid residues, respectively. However, between different species, there are differences in the amino acid residues present in different positions of the two chains.
  • the widespread use of genetic engineering has made it possible to prepare analogs of naturally occurring insulin by replacing, deleting and adding one or more amino acid residues.
  • Insulin can be used to treat diabetes and the diseases associated with or caused by it, and is necessary to maintain normal metabolic regulation.
  • natural insulins such as human insulin have a short action time, which necessitates frequent injections of patients, causing many injection-related discomforts. Therefore, people have been striving to obtain insulin derivatives or analogs with good efficacy, longer action time, and lower injection frequency to improve the inconvenience and discomfort caused by higher frequency insulin injections.
  • WO1995007931A1 discloses the marketed long-acting insulin detemir. Its molecular structure is characterized by the removal of the threonine at position 30 of the B chain of human insulin, and a 14-carbon fat monomer attached to the lysine residue at position 29 of the B chain. acid.
  • WO2005012347A2 discloses another long-acting insulin degludec, which has been on the market. Insulin degludec is a new type of ultralong-acting insulin that has a longer action time than insulin detemir. Its molecular structure is characterized by the removal of the 30th position of the human insulin B chain.
  • Amino acid the 16-carbon fatty diacid side chain is connected to the lysine residue at position B29 through a glutamic acid molecule.
  • CN101573133B and WO2009/010428 disclose PEG (PEGylated) extended insulin, which has a longer action time than conventional unmodified insulin.
  • WO2013086927A1 and WO2018/024186 disclose a long-acting acylated derivative of a human insulin analog.
  • the present invention provides novel insulin derivatives (such as acylated insulin).
  • novel insulin derivatives such as acylated insulin.
  • the new insulin derivatives for example, acylated insulin
  • the marketed insulin degludec trade name "Novota”
  • certain other insulin derivatives have Unexpectedly significantly increased potency, efficacy or efficacy, longer duration of action, longer in vivo half-life, good bioavailability, better safety, and more satisfactory physical stability, Chemical stability, and solubility.
  • the present invention provides an insulin derivative comprising an insulin parent, an albumin binding residue, and a linking group Lin, the insulin parent being a naturally-occurring insulin or an insulin analog, the insulin The protein binding residue is connected to the insulin parent via the linking group Lin, wherein,
  • the linking group Lin has at least 10, preferably at least 15, preferably at least 20, preferably at least 25, preferably at least 30, preferably at least 36, preferably at least 40, preferably 15-200, preferably 20 -200, preferably 25-180, preferably 30-180, preferably 42-180, preferably 54-180, preferably 59-180, preferably 61-180, preferably 66-180, preferably 72-120
  • a hydrophilic linking group with three carbon atoms; or the linking group Lin contains at least 5 neutral amino acid residues containing alkylene glycol; preferably, the linking group Lin contains at least 6 neutral amino acid residues.
  • the linking group Lin contains 5-20 neutral amino acid residues containing alkylene glycol; or, the linking group Lin contains At least 15, preferably at least 20, preferably at least 24, preferably at least 30, preferably at least 42, preferably 15-120, preferably 20-120, preferably 30-100, preferably 39-100, preferably 42 -Alkylene glycol of 80 carbon atoms; and
  • the albumin binding residue contains 20-40 carbon atoms, preferably the albumin binding residue contains a linear or branched lipophilic group with 20-40 carbon atoms, preferably the albumin binding residue is A fatty acid or aliphatic diacid containing 20-26 carbon atoms (more preferably a fatty acid or aliphatic diacid of 20-24 carbon atoms), wherein the hydroxyl group has been formally changed from the carboxyl group of the fatty acid and the aliphatic diacid Removed from one of the acid’s carboxyl groups; and
  • the insulin parent is not A14E, B16H , B25H, desB30 human insulin.
  • the inventors unexpectedly discovered that the combination of a certain length of albumin binding residues and a certain length of hydrophilic linking groups in the insulin derivatives of the present invention makes the insulin derivatives of the present invention relative to the existing ones.
  • Insulin derivatives while having an equivalent or longer action time, also have unexpectedly significantly increased pharmacodynamics, and in the presence of albumin, their ability to bind insulin receptors is significantly reduced by the influence of albumin. It has significantly improved binding capacity to insulin receptor.
  • the insulin parent comprises at least one lysine residue
  • the albumin binding residue is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the insulin parent via the linking group Lin. Phase connection.
  • the insulin derivative further comprises one or more linking group II, the linking group II is an acidic amino acid residue, and the linking group II is connected to the albumin binding residue And the linking group Lin, and/or between the linking group Lin and the insulin parent; preferably, the linking group II is connected between the albumin binding residue and the link Between the group Lin.
  • an insulin derivative which is an acylated insulin.
  • the insulin parent of the acylated insulin is a naturally-occurring insulin or an insulin analogue and contains at least one lysine residue.
  • the acyl moiety of the modified insulin 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 shown in formula (A):
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is an integer greater than or equal to 5, preferably n is an integer of 5-30;
  • I is a neutral amino acid residue containing alkylene glycol
  • III is a fatty acid or aliphatic diacid containing 20-26 (preferably 20-24) carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • the insulin parent is not A14E, B16H, B25H, desB30 human insulin;
  • the acyl moiety is represented by formula (A'):
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n'is an integer
  • I' is a neutral amino acid residue containing alkylene glycol
  • III is a fatty acid or aliphatic diacid containing 20-26 (preferably 20-24) carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • the total number of carbon atoms in (I ') n' is 15-200, preferably 20-200, preferably 25-180, preferably 30-180, preferably 42-180, preferably 54-180, preferably 59- 180, preferably 61-180, preferably 66-180, preferably 72-120; and
  • an insulin derivative which is an acylated insulin.
  • the insulin parent of the acylated insulin is a naturally-occurring insulin or an insulin analogue and contains at least one lysine residue.
  • the acyl moiety of the modified insulin 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 shown in formula (A):
  • n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
  • I is a neutral amino acid residue containing alkylene glycol
  • III is an aliphatic diacid containing 20-26 (preferably 20-24) carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid;
  • the insulin parent is not A14E, B16H, B25H, desB30 human insulin;
  • the acyl moiety is represented by formula (A'):
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n'is an integer
  • I' is a neutral amino acid residue containing alkylene glycol
  • III is an aliphatic diacid containing 20-26 (preferably 20-24) carbon atoms, wherein the hydroxyl group has been formally removed from one of the carboxyl groups of the aliphatic diacid;
  • the total number of carbon atoms in (I ') n' is 20-200, preferably 25-180, preferably 30-180, preferably 42-180, preferably 54-180, preferably 59-180, preferably 61- 180, preferably 66-180, preferably 72-120; and
  • n is an integer of 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; and/or
  • n is an integer of 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 20-26 (preferably 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 One of the carboxyl groups of the aliphatic diacid; and/or
  • the insulin matrix 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 -CO-, -HN-(CH 2 ) 2
  • 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; and/or
  • III is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 19 -CO-, HOOC-(CH 2 ) 20 -CO-, HOOC-(CH 2 ) 21 -CO-, HOOC-(CH 2 ) 22 -CO-, or HOOC-(CH 2 ) 24 -CO-, preferably, 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-.
  • the formula (A) is connected 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, or the formula (A') is connected to the C-terminus of I'through the The lysine residue of the insulin parent or the amino group of the N-terminal amino acid residue is connected.
  • 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: 1 and SEQ ID NO: 2, respectively representing the A chain and the B chain); A14E, B16H, B25H ,desB30 human insulin (SEQ ID NO: 3 and SEQ ID NO: 4, representing the A chain and B chain, respectively); A14E, B16E, B25H, desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 6, respectively, representing A chain and B chain); human insulin (SEQ ID NO: 7 and SEQ ID NO: 8, respectively representing the A chain and B chain); A21G human insulin (SEQ ID NO: 9 and SEQ ID NO: 10, respectively representing A Chain and B chain); A21G, desB30 human insulin (SEQ ID NO: 11 and SEQ ID NO: 12, representing the A chain and B chain, respectively); or B28D human insulin (SEQ ID NO: 13 and SEQ ID NO: 14, Denote A chain and B chain respectively);
  • the insulin parent is selected from the following insulins
  • 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 acylated insulin is selected from the following insulins: B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu -6xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanediacyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-7xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-8xOEG), desB30 human insulin; B29K( N( ⁇ )-docosanedioyl- ⁇ Glu-7xOEG), desB30 human insulin; B29K(N( ⁇ )-docosane
  • the acylated insulin is selected from the following insulins: B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG) ,desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-8xOEG), desB30 human insulin; or B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-8xOEG), desB30 human insulin .
  • an insulin derivative which is an acylated insulin
  • the insulin parent of the acylated insulin is A14E, B16H, B25H, desB30 human insulin or A14E, B16E, B25H, desB30 human insulin
  • the acyl moiety of the acylated insulin 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 represented by formula (C):
  • n1 is an integer of 5, 6, 7, 8, 9 or 10;
  • Y3 is a neutral amino acid residue containing alkylene glycol
  • Y2 is an acidic amino acid residue
  • Y1 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;
  • Y1, Y2, and Y3 are connected by an amide bond
  • n1 is not 10;
  • the acyl moiety is represented by formula (C'):
  • n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n1' is an integer of 5, 6, 7, 8, 9 or 10;
  • Y3' is a neutral amino acid residue containing alkylene glycol
  • Y2 is an acidic amino acid residue
  • Y1 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;
  • Y1, Y2, and Y3’ are connected by an amide bond
  • the total number of carbon atoms of (Y3') n1' is 15-100, preferably 20-100, preferably 25-90, preferably 30-80, preferably 30-59, preferably 30-54;
  • n1 is 5, 6, 7, 8 or 9, preferably, n1 is 5, 6, 7, or 8; and/or m1 is an integer of 1-6, preferably m1 is 1, 2, 3, or 4, preferably, m1 is 1 or 2, preferably, m1 is 1; and/or
  • Y1 is an aliphatic diacid containing 20-23 carbon atoms, preferably Y1 is an aliphatic diacid containing 20, 21, or 22 carbon atoms, in which the hydroxyl group has changed formally from among the carboxyl groups of the aliphatic diacid One is removed.
  • Y3 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
  • Y2 is an amino acid residue selected from: ⁇ Glu, ⁇ Glu, ⁇ Asp, ⁇ Asp, ⁇ -D-Glu, ⁇ -D-Glu, ⁇ -D-Asp or ⁇ -D-Asp, preferably Y2 is selected from ⁇ Glu Or ⁇ Asp; and/or
  • Y1 is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 19 -CO-, HOOC-(CH 2 ) 20 -CO-, HOOC-(CH 2 ) 21 -CO-, HOOC-( CH 2 ) 22 -CO-, or HOOC-(CH 2 ) 24 -CO-, preferably, Y1 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 Y1 is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 20 -CO- or HOOC-(CH 2 ) 22 -CO-.
  • the formula (C) is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the insulin parent through the C-terminus of Y3, or the formula (C') is connected to the C-terminus of Y3' The lysine residue of the insulin parent or the amino group of the N-terminal amino acid residue is connected.
  • the acyl moiety is linked to the epsilon amino group of the lysine residue of the insulin parent.
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; A14E, B16H, B25H , B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-5xOEG), desB30 Human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; A14E, B16H, B25H, B29K( N( ⁇ )-Eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin; A14E ,B16H,B25H,B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG),desB30 human insulin; A14E,B16H,B25H,B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-7xOEG ), desB30 human insulin; A14E, B16H, B25H,B29K(N
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin.
  • an insulin derivative which is an acylated insulin
  • the insulin parent of the acylated insulin is A14E, B16H, B25H, desB30 human insulin or A14E, B16E, B25H, desB30 human insulin
  • the acyl moiety of the acylated insulin 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 represented by formula (D):
  • n2 is 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
  • W3 is a neutral amino acid residue containing alkylene glycol
  • W2 is an acidic amino acid residue
  • W1 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;
  • W1, W2, and W3 are connected by an amide bond
  • the acyl moiety is represented by formula (D'):
  • n2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and n2' is 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
  • W3' is a neutral amino acid residue containing alkylene glycol
  • W2 is an acidic amino acid residue
  • W1 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;
  • W1, W2, and W3’ are connected by an amide bond
  • n2' The total number of carbon atoms of n2' is 30-180, 42-180, preferably 61-180, preferably 66-180, preferably 72-120.
  • n2 is 11, 12, 13, 14, 15, 16, 17, 18, or 19, preferably, n2 is 11, 12, 13, 14, 15, 16, 17, or 18, preferably , N2 is 11, 12, 13, 14, 15, or 16, preferably, n2 is 11, 12, 13, 14, or 15; and/or
  • n2 is an integer of 1-6, preferably m2 is 1, 2, 3, or 4, preferably m2 is 1 or 2, preferably m2 is 1; and/or
  • W1 is an aliphatic diacid containing 20-23 carbon atoms, preferably W1 is an aliphatic diacid containing 20, 21, or 22 carbon atoms, wherein the hydroxyl group has been formally changed from one of the carboxyl groups of the aliphatic diacid. One is removed.
  • W3 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 )
  • W2 is an amino acid residue selected from: ⁇ Glu, ⁇ Glu, ⁇ Asp, ⁇ Asp, ⁇ -D-Glu, ⁇ -D-Glu, ⁇ -D-Asp or ⁇ -D-Asp, preferably, W2 is selected from ⁇ Glu Or ⁇ Asp; and/or
  • W1 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 W1 is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 20 -CO- or HOOC-(CH 2 ) 22 -CO-.
  • the formula (D) is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the insulin parent through the C-terminus of W3 or the formula (D') is connected to the amino group of the lysine residue or the N-terminal amino acid residue through the C-terminus of W3'.
  • the lysine residue of the insulin parent or the amino group of the N-terminal amino acid residue is connected.
  • the acyl moiety is linked to the epsilon amino group of the lysine residue of the insulin parent.
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin; A14E, B16H, B25H , B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-11xOEG), desB30 human Insulin; A14E, B16H, B25H, B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N( ⁇ )-docosane diacyl - ⁇ Glu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N(
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-11xOEG), desB30 human insulin; A14E, B16H, B25H, B29K( N( ⁇ )-Eicosandioyl- ⁇ Glu-12xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-11xOEG), desB30 human insulin; A14E , B16H, B25H, B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K(N( ⁇ )-docosanedioyl- ⁇ Glu -18xOEG), desB30 human insulin;
  • the acylated insulin is selected from the following insulins: A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin; or A14E, B16H, B25H, B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin.
  • the acylated insulin is selected from the following insulins: A14E, B16E, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; A14E, B16E, B25H , B29K(N( ⁇ )-eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K(N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-5xOEG), desB30 Human insulin; A14E, B16E, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16E, B25H, B
  • a pharmaceutical composition comprising the insulin derivative according to any one of claims 1-25 or A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-10xOEG), desB30 human insulin, and one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition contains at least 1.5 moles of zinc ions/6 moles of acylated insulin; preferably contains at least 2.2 moles of zinc ions/6 moles of acylated insulin; preferably contains at least 3.5 moles of zinc ions/ 6 moles of acylated insulin; preferably containing at least 4.5 moles of zinc ions/6 moles of acylated insulin; preferably containing 2.2-12 moles of zinc ions/6 moles of acylated insulin; more preferably containing 4.5-10 moles of zinc ions /6 moles of acylated insulin; more preferably containing 4.5-8 moles of zinc ion/6 moles of acylated insulin; more preferably containing 4.5-7.5 moles of zinc ion/6 moles of acylated insulin; more preferably containing 4.5- 7.0 moles of zinc ions/6 moles of acylated insulin; more preferably containing 4.5-6.5 moles of zinc ions/6 moles of acylated insulin; and/or
  • the pharmaceutical composition has a pH of 6.5-8.5; preferably a pH of 6.8-8.2; preferably a pH of 7.0-8.2; preferably a pH of 7.2-7.6; more preferably a pH of 7.4 or 7.6.
  • the pharmaceutical composition further comprises glycerin, phenol, m-cresol, NaCl, and/or Na 2 HPO 4 ; preferably, the pharmaceutical composition further comprises glycerin, phenol, and NaCl; preferably, the The pharmaceutical composition further comprises glycerin, phenol, m-cresol, and NaCl; preferably, the pharmaceutical composition further comprises glycerin, phenol, NaCl and Na 2 HPO 4 ; more preferably, the pharmaceutical composition further comprises glycerin, phenol , M-cresol, NaCl and Na 2 HPO 4 .
  • the glycerin content does not exceed about 2.5% (weight/weight), preferably does not exceed about 2% (weight/weight), preferably about 0.3% to about 2% (weight/weight), preferably From about 0.5% to about 1.8% (weight/weight), preferably from about 0.7% to about 1.8% (weight/weight), more preferably from about 1% to about 1.8% (weight/weight); and/or
  • the content of the phenol is about 16 to 80 mM, preferably about 25-75 mM, preferably about 30-70 mM, 45-70 mM, preferably about 45-65 mM; preferably about 45 mM, about 46 mM, about 47 mM, about 48 mM, About 49mM, 50mM, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61mM, about 62mM, about 63mM, about 64mM, or about 65mM ;and / or
  • the content of the m-cresol is about 0-35mM, preferably about 0-19mM, preferably about 0-15mM, preferably about 0mM, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, About 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, or about 15mM; and/or
  • the content of the NaCl is about 0-150 mM, preferably about 5-120 mM, preferably about 10-120 mM, preferably about 10-100 mM, more preferably about 10-75 mM, more preferably about 10-50 mM, more preferably Is about 10-30mM; and/or
  • the content of the Na 2 HPO 4 is about 0-75 mM, preferably about 5-60 mM, preferably less than about 50 mM, more preferably less than about 25 mM, more preferably less than about 15 mM; and/or
  • the content of the insulin derivative is higher than about 0.3mM, preferably higher than about 0.6mM, preferably about 0.3-12mM, preferably about 0.6-9.0mM, preferably about 0.6-8.4mM, preferably about 0.6-7.2mM , Preferably about 0.6-6.0mM, preferably about 0.6-4.2mM, preferably about 0.6-3.6mM, preferably about 0.6-3.0mM, preferably about 0.6-2.4mM, preferably about 0.6-2.1mM, preferably It is about 0.6-1.2mM.
  • the insulin derivative is B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG ), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human Insulin; or B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-8xOEG), desB30 human insulin; B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-8xOEG), desB30 human insulin; B29K( N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin; A14E, B16H, B25H,
  • a pharmaceutical composition comprising about 0.6-4.2 mM of the insulin derivative of the present invention described above, about 1% to about 1.8% (weight/weight) of glycerol, about 45 -65mM phenol, about 4.5-6.5 moles of zinc ions/6 moles of insulin derivatives, about 10-120mM sodium chloride, about 0-15mM m-cresol, and have a pH of about 7.0-8.2, preferably, so
  • the insulin derivative is B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N(
  • a pharmaceutical composition comprising about 0.6 mM or 1.2 mM of the insulin derivative of the present invention described above, 1.7% (weight/weight) of glycerol, about 45 mM of phenol, about 10mM m-cresol, about 6.5 moles of zinc ion/6 moles of insulin derivative, about 20mM of sodium chloride, and having a pH of about 7.0-8.0, preferably, the insulin derivative is B29K(N( ⁇ ) -Eicosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N( ⁇ )-docosane two Acyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N( ⁇ )-( ⁇ )-Eicosane di
  • a pharmaceutical composition comprising about 0.6-4.2 mM of the insulin derivative of the present invention described above, about 1% to about 2% (preferably about 1.5%-1.7%) ( Weight/weight) glycerol, about 15mM-60mM (preferably about 30mM-60mM, more preferably about 45mM-60mM) phenol, about 1.5-7.0 (preferably about 2.2-4.5) moles of zinc ions/6 moles of insulin derivatives, About 10-120 mM (preferably about 20-50 mM) sodium chloride, about 0-25 mM (preferably about 0-15 mM, 0-10 mM) m-cresol, and have a pH of about 7.0-8.2, preferably, the insulin Derivatives are B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin;
  • a pharmaceutical composition comprising about 0.6mM-4.2mM of the insulin derivative of the present invention described above, about 1.5%-1.7% (weight/weight) of glycerol, about 45mM -60mM phenol, about 0-10mM m-cresol, about 2.2-2.5 moles of zinc ions/6 moles of insulin derivatives, about 20mM of sodium chloride, and have a pH of about 7.0-8.0, wherein the insulin Derivatives are A14E, B16H, B25H, B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K(N( ⁇ )-docosane diacyl - ⁇ Glu-6xOEG), desB30 human insulin; A14E, B16H, B25H, B29K (N( ⁇ )-docosanedioyl- ⁇ Glu-10xOEG), desB
  • the pharmaceutical composition further comprises an insulinotropic GLP-1 compound; preferably, the pharmaceutical composition further comprises an insulinotropic GLP-1 compound selected from: N- ⁇ 26 -(17- Carboxyhexadecanoylamino)-4(S)-carboxybutyryl-[Arg34]GLP-1-(7-37) peptide, N- ⁇ 26 -(17-carboxyheptadecanylamino)-4(S )-Carboxybutyryl-[Gly8,Arg34]GLP-1-(7-37) peptide, N- ⁇ 26 -[2-(2-[2-(2-[2-[2-[2-[2-[2-[4-(17 -Carboxyheptadecanylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy)acetamino)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1- (7-37) peptide, N- ⁇ 26 -[2-
  • the pharmaceutical composition further comprises the insulinotropic GLP-1 compound represented by 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: 15) 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 (SEQ ID NO: 16) or [Arg34] GLP-1-(7-37) peptide (SEQ ID NO: 17). ), preferably [Gly8,Arg34]GLP-1-(7-37) peptide; and/or
  • 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, in which the hydroxyl group has changed formally from among the carboxyl groups of the aliphatic diacid One 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; and/or
  • 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-, preferably, 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 insulinotropic GLP-1 compound is selected from the following compounds:
  • the insulinotropic GLP-1 compound is selected from the following compounds:
  • the insulinotropic GLP-1 compound is:
  • the pharmaceutical composition or combined preparation of the insulin derivative (e.g., acylated insulin) and insulinotropic GLP-1 compound of the present invention not only does not weaken the insulin derivative (e.g., acylated insulin). ), and the combination preparation has better physical stability than the single-drug preparation.
  • the physical stability of the combined preparation of the present invention is unexpected.
  • the combined preparation also increases the chemical stability of the insulin derivative (for example, acylated insulin) compared to the single-drug preparation.
  • the pharmaceutical composition further comprises fast-acting insulin.
  • the fast-acting insulin is selected from one or more of Asp B28 human insulin, Lys B28 Pro B29 human insulin, Lys B3 Glu B29 human insulin, human insulin and desB30 human insulin; preferably, The fast-acting insulin is Asp B28 human insulin, Lys B28 Pro B29 human insulin, Lys B3 Glu B29 human insulin, human insulin or desB30 human insulin.
  • the inventors unexpectedly found that the pharmaceutical composition comprising the insulin derivative (for example, acylated insulin) of the present invention and the double insulin component of insulin aspart is compared with the double insulin comprising deglubber and insulin aspart after administration.
  • the pharmaceutical composition of the insulin component has an unexpectedly increased hypoglycemic effect.
  • the dosage ratio of the insulin derivative (such as acylated insulin) and insulin aspart of the present invention is much smaller than that of insulin deglubber and insulin aspart. When the dosage is compared, it can still achieve a better or equivalent hypoglycemic effect.
  • the molar ratio of the insulin derivative to the fast-acting insulin is about 60:3 to about 0.5:3, preferably about 57:3 to about 1:3, preferably about 55: 3 to about 1.2:3, preferably about 50:3 to about 1.5:3, preferably about 40:3 to about 1.5:3, preferably about 30:3 to about 1.5:3, preferably about 27:3 to About 1.5:3, preferably about 25:3 to about 1.5:3, preferably about 22:3 to about 1.5:3, preferably about 20:3 to about 1.5:3, preferably about 17:3 to about 1.5 :3, preferably about 15:3 to about 1.5:3, preferably about 12:3 to about 1.5:3, preferably about 10:3 to about 1.5:3, preferably about 9:3 to about 1.5:3 , Preferably from about 8:3 to about 1.5:3, preferably from about 7:3 to about 1.5:3, preferably from about 6.9:3 to about 1.5:3, preferably from about 6.8:3 to about 1.5:3, preferably From about 6.5:3 to about 1.5:3,
  • the insulin derivative is B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG ), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human Insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-7xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-8xOEG), desB30 human insulin; B29K(N( ⁇ )-docosane diacyl- ⁇ Glu-7xOEG), desB30 human insulin; B29K (N( ⁇ )-docosane diacyl-
  • the pharmaceutical composition comprises about 0.09-0.36 mM insulin derivative, about 0.18 mM Asp B28 human insulin, about 0.85% to about 2.0% (weight/weight) glycerol, about 15-70 mM Phenol, about 8-14 moles of zinc ions/6 moles of insulin derivatives, about 10-120 mM sodium chloride, about 0-15 mM m-cresol, and have a pH of about 7.0-8.2, wherein the insulin derivative
  • the substance is B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-eicosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N( ⁇ )-docosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K (N( ⁇ )
  • the pharmaceutical composition comprises about 0.165-0.18mM insulin derivative, about 0.18mM Asp B28 human insulin, about 1.5%-1.7% (weight/weight) glycerol, about 20mM-30mM Phenol, about 9-12 moles of zinc ion/6 moles of insulin derivative, about 20mM-75mM sodium chloride, about 10mM-15mM m-cresol, and having a pH of about 7.0-8.2, wherein the insulin derivative B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K(N( ⁇ )-eicosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N( ⁇ ) )-Docosane diacyl- ⁇ Glu-5xOEG), desB30 human insulin; B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-6xOEG), desB30 human insulin; B29K(N( ⁇ )-
  • the insulin derivative or the pharmaceutical composition of the present invention for use as a medicine.
  • the insulin derivative or the pharmaceutical composition of the present invention is provided, which is used as a medicine for treating or preventing diabetes, hyperglycemia, and/or impaired glucose tolerance.
  • the insulin derivative or the pharmaceutical composition of the present invention which is used to treat or prevent diabetes, hyperglycemia, and/or impaired glucose tolerance.
  • the use of the insulin derivative or the pharmaceutical composition of the present invention in the preparation of a medicine is provided; preferably, the medicine is used for the treatment or prevention of diabetes, hyperglycemia, and / Or impaired glucose tolerance.
  • the medicament is used to treat diabetes, and the insulin derivative is administered to the same patient every other day or less frequently, and on average, for a period of at least 1 month, 6 months, or 1 year However, the insulin derivatives are not administered to the same patient more frequently.
  • the drug is used to treat diabetes, and the insulin derivative is administered twice a week or less frequently, and on average, during a period of at least 1 month, 6 months, or 1 year, The acylated insulin is not administered to the same patient at a higher frequency.
  • Another aspect of the present invention provides a method for treating or preventing diabetes, hyperglycemia, and/or impaired glucose tolerance, comprising administering a therapeutically effective amount of the insulin derivative or the pharmaceutical composition described above of the present invention.
  • the inventors unexpectedly discovered that the insulin derivatives of the present invention (for example, acylated insulin) have long pharmacokinetics (hereinafter also referred to as PK) characteristics, making subcutaneous treatment for diabetic patients twice a week, once a week or less frequently may.
  • PK pharmacokinetics
  • Another aspect of the present invention provides a method for increasing the binding ability of insulin derivatives to insulin receptor in the presence of albumin, the method comprising:
  • the insulin derivative is obtained by linking the albumin binding residue with the naturally-occurring insulin or insulin analog via the linking group Lin, wherein the linking group Lin has at least 10, preferably at least 15, preferably at least 20 One, preferably at least 25, preferably at least 30, preferably at least 36, preferably at least 40, preferably 15-200, preferably 20-200, preferably 25-180, preferably 30-180, preferably 42-180 Hydrophilic linking groups, preferably 54-180, preferably 59-180, preferably 61-180, preferably 66-180, preferably 72-120 carbon atoms; the albumin binding residue comprises 20 -40 carbon atoms, preferably the albumin binding residue contains a linear or branched lipophilic group with 20-40 carbon atoms, preferably the albumin binding residue is a fatty acid containing 20-26 carbon atoms Or an aliphatic diacid (more preferably a fatty acid with 20-24 carbon atoms or an aliphatic diacid), in which the hydroxyl group has been formally removed from
  • the derivative is obtained by modifying naturally occurring insulin or insulin analogues with formula (A) or formula (A'), III-(II) m -(I) n- (A), wherein,
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is an integer greater than or equal to 5, preferably n is an integer of 5-30;
  • I is a neutral amino acid residue containing alkylene glycol
  • III is an albumin binding residue containing a linear or branched lipophilic group with 20-40 carbon atoms, preferably III is a fatty acid or aliphatic diacid containing 20-26 carbon atoms (more preferably 20-24 Carbon-atom fatty acid or aliphatic diacid), wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • (A') is III-(II) m -(I') n' -(A'),
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n'is an integer
  • I' is a neutral amino acid residue containing alkylene glycol
  • III is an albumin binding residue containing a linear or branched lipophilic group with 20-40 carbon atoms, preferably III is a fatty acid or aliphatic diacid containing 20-26 carbon atoms (more preferably 20-24 Carbon-atom fatty acid or aliphatic diacid), wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • the total number of carbon atoms in (I ') n' is 15-200, preferably 20-200, preferably 25-180, preferably 30-180, preferably 42-180, preferably 54-180, preferably 59- 180, preferably 61-180, preferably 66-180, preferably 72-120.
  • the present invention provides a method for increasing the efficacy of insulin derivatives, the method comprising:
  • the insulin derivative is obtained by linking the albumin binding residue with the naturally-occurring insulin or insulin analog via the linking group Lin, wherein the linking group Lin has at least 10, preferably at least 15, preferably at least 20 One, preferably at least 25, preferably at least 30, preferably at least 36, preferably at least 40, preferably 15-200, preferably 20-200, preferably 25-180, preferably 30-180, preferably 42-180 Hydrophilic linking groups, preferably 54-180, preferably 59-180, preferably 61-180, preferably 66-180, preferably 72-120 carbon atoms; the albumin binding residue comprises 20 -40 carbon atoms, preferably the albumin binding residue contains a linear or branched lipophilic group with 20-40 carbon atoms, preferably the albumin binding residue is a fatty acid containing 20-26 carbon atoms Or an aliphatic diacid (more preferably a fatty acid with 20-24 carbon atoms or an aliphatic diacid), in which the hydroxyl group has been formally removed from
  • the derivative is obtained by modifying naturally occurring insulin or insulin analogues with formula (A) or formula (A'), III-(II) m -(I) n- (A), wherein,
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is an integer greater than or equal to 5, preferably n is an integer of 5-30;
  • I is a neutral amino acid residue containing alkylene glycol
  • III is an albumin binding residue containing a linear or branched lipophilic group with 20-40 carbon atoms, preferably III is a fatty acid or aliphatic diacid containing 20-26 carbon atoms (more preferably 20-24 Carbon-atom fatty acid or aliphatic diacid), wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and n'is an integer greater than or equal to 5;
  • I' is a neutral amino acid residue containing alkylene glycol
  • III is an albumin binding residue containing a linear or branched lipophilic group with 20-40 carbon atoms, preferably III is a fatty acid or aliphatic diacid containing 20-26 carbon atoms (more preferably 20-24 Carbon-atom fatty acid or aliphatic diacid), wherein the hydroxyl group has been formally removed from one of the carboxyl group of the fatty acid and the carboxyl group of the aliphatic diacid;
  • the total number of carbon atoms in (I ') n' is 15-200, preferably 20-200, preferably 25-180, preferably 30-180, preferably 42-180, preferably 54-180, preferably 59- 180, preferably 61-180, preferably 66-180, preferably 72-120.
  • the naturally-occurring insulin or insulin analogue comprises at least one lysine residue, the linking group Lin, formula (A) or formula (A') and the lysine residue of the insulin parent The amino group or the amino group of the N-terminal amino acid residue is connected.
  • n is an integer of 5-18, preferably n is an integer of 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; and/or
  • n is an integer of 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 20-26 (preferably 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 One of the carboxyl groups of the aliphatic diacid; and/or
  • the insulin matrix 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 -CO-, -HN-(CH 2 ) 2
  • 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; and/or
  • III is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 19 -CO-, HOOC-(CH 2 ) 20 -CO-, HOOC-(CH 2 ) 21 -CO-, HOOC-(CH 2 ) 22 -CO- or HOOC-(CH 2 ) 24 -CO-, preferably III is HOOC-(CH 2 ) 18 -CO-, HOOC-(CH 2 ) 20 -CO- or HOOC-(CH 2 ) 22 -CO-.
  • the formula (A) is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the natural insulin or insulin analog through the C-terminus of I or the formula (A') is passed through the I The C-terminus of 'is connected to the amino group of the lysine residue or the N-terminal amino acid residue of the natural insulin or insulin analogue.
  • formula (A) or formula (A') is linked to the epsilon amino group of the lysine residue of the insulin parent.
  • the lysine residue of the natural insulin or insulin analog is located at position B29.
  • the naturally occurring insulin or insulin analogue is selected from the following insulin or insulin analogues: desB30 human insulin; A14E, B16H, B25H, desB30 human insulin; A14E, B16E, B25H, desB30 human insulin; human insulin; A21G human insulin A21G, desB30 human insulin; or B28D human insulin; preferably, the insulin parent is desB30 human insulin or A14E, B16H, B25H, desB30 human insulin.
  • Figure 1a shows the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, insulin degludec, and vehicle on db/db mice.
  • Fig. 1b corresponding to Fig. 1a shows the AUC of the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, insulin degludec and vehicle on db/db mice.
  • Figure 2a shows the hypoglycemic effects of the compounds of Examples 1 and 2 of the present invention, the compound of Comparative Example 2 and the vehicle on db/db mice.
  • Figure 2b corresponds to Figure 2a showing the AUC of the hypoglycemic effect of the compounds of Examples 1 and 2 of the present invention, the compound of Comparative Example 2 and the vehicle on db/db mice.
  • Figure 3a shows the hypoglycemic effect and action time of the compounds of Examples 1-3 of the present invention and the vehicle on db/db mice.
  • Figure 3b corresponds to Figure 3a showing the AUC of the hypoglycemic effect of the compounds of Examples 1-3 and the vehicle of the present invention on db/db mice.
  • Figure 4a shows the hypoglycemic effect and action time of the compound of Example 2 of the present invention, the compound of Comparative Example 3 and the vehicle on db/db mice.
  • Figure 4b corresponds to Figure 4a showing the AUC of the hypoglycemic effect of the compound of Example 2 of the present invention, the compound of Comparative Example 3 and the vehicle on db/db mice.
  • Figure 5a shows the hypoglycemic effect and action time of the compound of Comparative Example 3-4 of the present invention and the vehicle on db/db mice.
  • Figure 5b corresponds to Figure 5a showing the AUC of the hypoglycemic effect of the compound of Comparative Example 3-4 of the present invention and the vehicle on db/db mice.
  • Fig. 6a shows the hypoglycemic effect and action time of the compounds of Example 2 and Examples 4-5 of the present invention and the vehicle on db/db mice.
  • Figure 6b corresponds to Figure 6a showing the AUC of the hypoglycemic effect of the compounds of Examples 2 and 4-5 of the present invention and the vehicle on db/db mice.
  • Figure 7a shows the hypoglycemic effect of the compound of Example 1 of the present invention and the vehicle on streptozotocin (STZ)-induced type I diabetes (T1DM) rats.
  • Figure 7b corresponds to Figure 7a showing the AUC of the hypoglycemic effect of the compound of Example 1 of the present invention and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 8a shows the hypoglycemic effects of the title compounds of Comparative Example 5, Examples 15 and 16 of the present invention, and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 8b corresponds to Figure 8a showing the AUC of the hypoglycemic effect of Comparative Example 5, the title compounds of Examples 15 and 16, and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 9a shows the hypoglycemic effect of the compounds of Examples 2 and 4 of the present invention and the vehicle on STZ-induced type I diabetes (T1DM) female rats.
  • Figure 9b corresponds to Figure 9a showing the AUC of the hypoglycemic effect of the compounds of Examples 2 and 4 of the present invention and the vehicle on STZ-induced type I diabetes (T1DM) female rats.
  • Figure 10a shows the hypoglycemic effect of the title compounds of Comparative Example 5, Examples 15 and 16 and the vehicle of the present invention on db/db mice.
  • Figure 10b and Figure 10a correspondingly show the AUC of the hypoglycemic effect of Comparative Example 5, Examples 15 and 16 of the present invention, and the vehicle on the hypoglycemic effect of db/db mice.
  • Figure 11a shows the hypoglycemic effect of the title compounds of Comparative Example 5, Examples 17 and 18, and the vehicle of the present invention on STZ-induced Type I diabetes (T1DM) rats.
  • Fig. 11b and Fig. 11a correspondingly show the AUC of the hypoglycemic effect of the control example 5, the title compounds of Examples 17 and 18, and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 12a shows the hypoglycemic effect of the title compound of Comparative Example 5 and Example 16 of the present invention and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Fig. 12b and Fig. 12a correspondingly show the AUC of the hypoglycemic effect of the title compound of Comparative Example 5 and Example 16 of the present invention and the vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 13a shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart di-insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart di-insulin components of the present invention, and a vehicle pair STZ
  • T1DM induced type I diabetes
  • Figure 13b and Figure 13a correspondingly show insulin aspart, a pharmaceutical composition containing deglubber and insulin aspart dual insulin components, and a pharmaceutical composition containing the acylated insulin and insulin aspart dual insulin components of the present invention
  • Figure 14a shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and the vehicle pair STZ
  • T1DM induced type I diabetes
  • Figure 14b and Figure 14a correspondingly show insulin aspart, a pharmaceutical composition comprising deglubber and insulin aspart dual insulin component, and a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin component of the present invention
  • Figure 15a shows insulin aspart, a pharmaceutical composition comprising deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and the vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice before the fourth dose of mouse blood glucose.
  • T1DM vehicle pair STZ Induced type I diabetes
  • Figure 15b shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and the vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice before the eighth dose of mouse blood glucose.
  • T1DM vehicle pair STZ Induced type I diabetes
  • Figure 15c shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice before the tenth dose of mouse blood glucose.
  • T1DM STZ Induced type I diabetes
  • Figure 16a shows insulin aspart, a pharmaceutical composition comprising deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and the vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice 1 hour after the fourth dose of mouse blood glucose.
  • T1DM vehicle pair STZ Induced type I diabetes
  • Figure 16b shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and the vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice with blood glucose 1 hour after the eighth dose.
  • T1DM vehicle pair STZ Induced type I diabetes
  • Figure 16c shows insulin aspart, a pharmaceutical composition comprising insulin deglubber and insulin aspart di-insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart di-insulin components of the present invention, and a vehicle pair STZ Induced type I diabetes (T1DM) C57/6J mice 1 hour after the tenth dose of mouse blood glucose.
  • T1DM vehicle pair STZ Induced type I diabetes
  • Figure 17 shows insulin aspart, a pharmaceutical composition comprising deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle pair STZ HbA1c-lowering effect in induced type I diabetes (T1DM) C57/6J mice.
  • T1DM induced type I diabetes
  • Figure 18a shows the hypoglycemic effect of the compound of Example 4 of the present invention, insulin degludec and vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 18b corresponds to Figure 18a showing the AUC of the hypoglycemic effect of the compound of Example 4 of the present invention, insulin degludec and vehicle on STZ-induced type I diabetes (T1DM) rats.
  • Figure 19a shows a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle for type I induction of STZ
  • T1DM diabetic
  • Figure 19b and Figure 19a correspondingly show a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle pair AUC of the hypoglycemic effect of STZ-induced type I diabetes (T1DM) C57/6J mice.
  • T1DM STZ-induced type I diabetes
  • Figure 20 shows a pharmaceutical composition comprising insulin deglubber and insulin aspart double insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart double insulin components of the present invention, and a vehicle for type I induction of STZ The effect of reducing HbA1c in diabetic (T1DM) C57/6J mice.
  • Figure 21a shows a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle for db/db mice The hypoglycemic effect.
  • Figure 21b and Figure 21a correspondingly show a pharmaceutical composition comprising insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle pair AUC of hypoglycemic effect in db/db mice.
  • Figure 22a shows a pharmaceutical composition containing insulin deglubber and insulin aspart dual insulin components, a pharmaceutical composition containing the acylated insulin and insulin aspart dual insulin components of the present invention, and the db/db small after the vehicle Random blood glucose of the rat.
  • Figures 22b and 22a respectively show after injection of a pharmaceutical composition containing deglubber and insulin aspart dual insulin components, a pharmaceutical composition containing the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle AUC of random blood glucose in db/db mice.
  • Figure 22c shows a pharmaceutical composition comprising insulin deglubber and insulin aspart di-insulin components, a pharmaceutical composition comprising the acylated insulin and insulin aspart di-insulin components of the present invention, and db/db small after the vehicle Fasting blood glucose of rats.
  • Figures 22d and 22c correspondingly show after injection of a pharmaceutical composition containing deglubber and insulin aspart dual insulin components, a pharmaceutical composition containing the acylated insulin and insulin aspart dual insulin components of the present invention, and a vehicle AUC of fasting blood glucose in db/db mice.
  • Figure 23 shows the receptor binding ability of the compound of Example 2 and the control compound 2 of the present invention in the presence of 2% HSA and 0% HSA, respectively.
  • Figure 24a shows the receptor binding ability of the compound of Example 17 and the control compound 5 of the present invention at a sample concentration of 12800 nM in the presence of 2% HSA and 0% HSA, respectively.
  • Figure 24b shows the receptor binding capacity of the compound of Example 17 and the control compound 5 of the present invention at a sample concentration of 25600 nM, in the presence of 2% HSA and 0% HSA, respectively.
  • Figure 25 shows the receptor binding ability of the compound of Example 41 and the control compound 2 of the present invention in the presence of 2% HSA and 0% HSA, respectively.
  • Figure 26a shows the receptor binding ability of the compound of Example 18, the compound of Example 42 and the control compound 5 of the present invention at a sample concentration of 12800 nM in the presence of 2% HSA and 0% HSA, respectively.
  • Figure 26b shows the receptor binding ability of the compound of Example 18, the compound of Example 42 and the control compound 5 of the present invention at a sample concentration of 25600 nM in the presence of 2% HSA and 0% HSA, respectively.
  • insulin includes naturally-occurring insulin, such as human insulin, as well as its insulin analogs and insulin derivatives.
  • insulin analogue encompasses polypeptides that have one or more amino acid residues and/or addition of one or more amino acid residues that can be formally deleted and/or replaced (substitution) in natural insulin, and A molecular structure derived from the structure of a naturally occurring insulin, such as human insulin.
  • the added and/or substituted amino acid residues may be codable amino acid residues, or other naturally occurring amino acid residues, or purely synthetic amino acid residues.
  • the added and/or substituted amino acid residues are codable amino acid residues.
  • 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 the insulin part of an acylated insulin (also referred to herein as the parent insulin), for example, in the present invention refers to an insulin derivative or an acylated insulin that has no side chain attached or attached The acyl part.
  • 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.
  • albumin binding residue refers to a residue capable of non-covalently binding to human serum albumin.
  • Albumin binding residues linked to insulin generally have a binding affinity for human serum albumin that is less than, for example, about 10 ⁇ M or even less than about 1 ⁇ M.
  • the albumin binding properties can be measured by the surface plasmon resonance described in the following literature: J. Biol. Chem. 277 (38), 35035-35042, (2002).
  • hydrophilic linking group refers to a linking group that contains at least 6 chemical moieties other than hydrogen atoms (moiety) that separates the insulin precursor from the albumin binding residues, and 30-50 of these non-hydrogen atoms % Is N or O.
  • Lipophilic refers to the ability of the group to dissolve in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene.
  • Lipophilic groups including but not limited to, for example, fats, fatty acids, fatty diacids, etc., usually have "lipid tails".
  • the lipid tails present in these lipophilic groups can be saturated or unsaturated, depending on Whether the lipid tail contains double bonds.
  • Lipid tails can also contain different lengths, such as tails having between 7-12 carbons (e.g., C 7-12 alkyl or C 7-12 alkenyl), tails having 13-22 carbons (e.g., C 13-12 alkyl or C 13-12 alkenyl), or a tail having 23-30 carbons (for example, C 23-30 alkyl or C 23-30 alkenyl).
  • tails having between 7-12 carbons e.g., C 7-12 alkyl or C 7-12 alkenyl
  • tails having 13-22 carbons e.g., C 13-12 alkyl or C 13-12 alkenyl
  • a tail having 23-30 carbons for example, C 23-30 alkyl or C 23-30 alkenyl.
  • 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, Behenedioic acid, and tetracosanedioic acid.
  • fast-acting insulin includes fast-acting naturally-occurring insulin, insulin analogs, and insulin derivatives.
  • Fast-acting insulin usually starts to work in, for example, 1 to 20 minutes, reaches a peak in about an hour, and continues to work for three to five hours.
  • basic insulin means insulin that has a longer duration of action than normal or normal human insulin.
  • chemical stability means that, chemically, the insulin derivative of the present invention is sufficiently stable in the desired formulation. That is, only chemical degradation products are formed in an amount that does not impair the shelf life of the final drug product.
  • Chemical degradation products include deamidation products, isoaspartate formation, dimer formation, racemization products, products resulting from dehydration processes and the like. Chemical stability can be determined by HPLC analysis of aged samples or formulations.
  • binding ability to insulin receptor refers to the interaction between insulin and insulin receptor, and the magnitude or intensity of this interaction can be measured by, for example, surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • when measuring with SPR when a solution containing insulin flows through a chip coated with insulin receptors, the interaction between insulin and insulin receptors will cause changes in the deflection angle of the SPR. This change is usually used as a relative response. Generally, the larger the relative response value, the higher the binding ability to insulin receptor.
  • High physical stability means that the tendency of fibrillation is less than 50% of that of human insulin. Fibrillation can be described by the lag time before fibril formation begins under given conditions.
  • Polypeptides with affinity for insulin receptor and IGF-1 receptor are polypeptides that can interact with insulin receptor and human IGF-1 receptor in a suitable binding assay. Such receptor assays are well known in the art.
  • pharmaceutical effect refers to the ability of a drug or active compound to produce a certain action or effect (for example, lowering blood sugar).
  • a drug or active compound for example, lowering blood sugar.
  • administering the same dose of the insulin derivative of the present invention will have a higher blood sugar lowering effect or effect than deglubber or other existing insulin derivatives.
  • diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other conditions that cause hyperglycemia.
  • the term is used for metabolic disorders, where the pancreas produces insufficient amounts of insulin, or where the cells of the body cannot respond appropriately to insulin, thereby preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.
  • Type 1 diabetes also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-type diabetes, is associated with major insulin resistance and therefore relative insulin deficiency and/or major insulin secretion defects with insulin resistance.
  • 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: 15 in the sequence list.
  • the peptide having the sequence shown in SEQ ID NO: 15 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.
  • GLP-1-(7-37) peptide which is a GLP with Gly and Arg at positions 8 and 34 corresponding to GLP-1(7-37) (SEQ ID NO: 15) -1 analogue.
  • [Arg34] GLP-1-(7-37) peptide is a GLP-1 analogue with Arg at the position corresponding to position 34 of GLP-1(7-37) (SEQ ID NO: 15).
  • 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: 16 and SEQ ID NO in the sequence list, respectively. : Shown at 17.
  • 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 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)
  • 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-[4-(19-carboxynonadenoylamino)-4 (S)-Carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl], or [2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[
  • the insulin of Comparative Example 2 of the present invention is called "B29K (N( ⁇ )-eicosandioyl- ⁇ Glu-2xOEG), desB30 human insulin", “B29K(N ⁇ -eicosane diacyl- ⁇ Glu-2xOEG), desB30 human insulin", or "B29K (N ⁇ -eicosane diacyl-gGlu-2xOEG), desB30 human insulin” to indicate the amino acid K at position B29 in human insulin It has been modified by the acylation of the eicosandioyl-gGlu-2xOEG residue on the ⁇ nitrogen of the lysine residue of B29 (called N ⁇ or (N( ⁇ )), and the position B30 in human insulin T amino acid has been deleted.
  • insulin for Comparative example 5 is referred to as "A14E, B16H, B25H, B29K (N ⁇ eicosane diacyl -gGlu-2xOEG), desB30 Human insulin” or "A14E, B16H, B25H, B29K (N( ⁇ )-eicosanedioyl- ⁇ Glu-2xOEG), desB30 human insulin” means that the amino acid Y at position A14 in human insulin has been mutated to E, human The amino acid Y at position B16 in insulin has been mutated to H, the amino acid F at position B25 in human insulin has been mutated to H, and the amino acid K at position B29 in human insulin has passed the epsilon nitrogen (called It is modified by the acylation of eicosane diacyl-gGlu-2xOEG on N ⁇ ), and the amino acid T at position B30 in human insulin has been deleted.
  • A14E, B16H, B25H, B29K N ⁇
  • nxPEG represents the group -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 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.
  • excipient in a broad sense refers to any component other than the active therapeutic ingredient.
  • the excipient may be an inert substance, an inactive substance and/or a non-pharmaceutically active substance.
  • Excipients can be used for various purposes, depending on the pharmaceutical composition, for example as a carrier, vehicle, diluent, tablet adjuvant, and/or for improving the administration and/or absorption of the active substance.
  • excipients include, but are not limited to, diluents, buffers, preservatives, tonicity regulators (also known as tonicity agents or isotonic agents), chelating agents, surfactants, protease inhibitors, wetting agents, emulsifiers , Antioxidants, fillers, metal ions, oily solvents, proteins and/or zwitterions and stabilizers.
  • compositions of pharmaceutically active ingredients and various excipients are known in the art, see, for example, Remington: The Science and Practice of Pharmacy (for example, the 19th edition (1995) and any later editions).
  • the patient prefers the time interval (time delay) from the administration of the acylated insulin of the present invention to the next administration of the acylated insulin of the present invention to have the same length or approximately the same length in days. It can even be expected that patients will prefer to administer acylated insulin once a week, that is, on the same day of the week, for example every Sunday. On average over a period of 1 month, 6 months, or 1 year, this would be acylated insulin administered every 6 days and not at a higher frequency. For some patients, on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every 5 days or approximately every 5 days and not more frequently.
  • acylated insulin For other patients, calculated on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every 4 days or approximately every 4 days and not more frequently. For other patients, calculated on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every 3 days or approximately every 3 days and not more frequently. Even other patients may find it advantageous to administer acyl groups on average over a period of 1 month, 6 months, or 1 year, twice a week, for example, at intervals of about 3-4 days between each administration. Insulin. For some patients, calculated on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every 2 days or approximately every 2 days and not more frequently.
  • acylated insulin For other patients, calculated on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every other day or approximately every other day and not more frequently. For some patients, calculated on average over a period of 1 month, 6 months, or 1 year, it may be necessary to administer acylated insulin every 7 days or approximately every 7 days and not more frequently. Even other patients may not administer acylated insulin at exactly the same length of time interval (in days) every week, month, or year. On average over a period of 1 month, 6 months, or 1 year, some patients may sometimes be given acylated insulin at intervals of 5 to 7 days and not at a higher frequency.
  • the diseases and conditions that are the main targets of the present invention are diabetes (type 1 or type 2) or other conditions characterized by hyperglycemia, but are generally metabolic diseases and conditions in which insulin metabolism is clinically relevant or beneficial , Such as pre-diabetes, impaired glucose tolerance, metabolic syndrome, obesity, cachexia, damage/death of ⁇ -cells in the body, excessive appetite, and inflammation. All these types of conditions are known or believed to benefit from the stable metabolic state of subjects suffering from the disease or condition.
  • any treatment regimen that includes administration of insulin can be modified by practicing the teachings of the present invention, meaning that such therapy will include administration of insulin according to the prolonged action provided herein.
  • 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
  • CH 3 CN is acetonitrile
  • Gly is glycine
  • Arg is arginine
  • TFA is trifluoroacetic acid
  • HbA1c is glycosylated hemoglobin
  • AUC is the area under the curve of time-glucose curve
  • RU Response Unit.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-5xOEG
  • desB30 human insulin compound 1
  • des(B30) human insulin was prepared according to the method described in Example 11 of Chinese Patent CN1056618C.
  • 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), and the precipitated product was separated by filtration and washed twice with n-heptane.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-6xOEG
  • desB30 human insulin compound 2
  • Compound 2 was prepared in a procedure similar to that of Example 1, Part 2.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-8xOEG
  • desB30 human insulin compound 3
  • B29K N( ⁇ )-docosanediacyl- ⁇ Glu-6xOEG
  • desB30 human insulin compound 4
  • Compound 4 was prepared in a procedure similar to that of Example 1, Part 2.
  • B29K N( ⁇ )-docosanedioyl- ⁇ Glu-8xOEG
  • desB30 human insulin compound 5
  • Compound 5 was prepared in a procedure similar to that of Example 1, Part 2.
  • the reference compound insulin degludec was prepared according to Example 4 of patent CN105820233A.
  • B29K N( ⁇ )-eicosandioyl- ⁇ Glu-2xOEG
  • desB30 human insulin control compound 2
  • control compound 2 was prepared in a procedure similar to that of Example 1 Part 2.
  • B29K N( ⁇ )-octadecane diacyl- ⁇ Glu-2xOEG
  • desB30 human insulin control compound 3
  • control compound 3 was prepared in a procedure similar to that of Example 1, Part 2.
  • B29K N( ⁇ )-octadecandioyl- ⁇ Glu-6xOEG
  • desB30 human insulin control compound 4
  • control compound 4 was prepared by a procedure similar to that of Example 1, Part 2.
  • the purpose of this study is to verify the acylated insulin of the present invention on the regulation of blood glucose (BG) in the case of diabetes.
  • acylated insulins of Examples 1-5 and the control compounds of Control Examples 1-4 were tested in a single-dose study.
  • the blood glucose lowering effect of the acylated insulin was tested at different doses of 9U/kg or 10U/kg.
  • mice 8-9 weeks old male db/db(BKS/Lepr) mice are raised 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 acylated insulin 9U/kg or 10U/kg, where the vehicle contained: glycerol 19.6mg/ml, phenol 1.5mg/ml, m-cresol 1.72mg/ml, zinc ion concentration 55 ⁇ g/ml, pH value 7.6.
  • vehicle contained: glycerol 19.6mg/ml, phenol 1.5mg/ml, m-cresol 1.72mg/ml, zinc ion concentration 55 ⁇ g/ml, pH value 7.6.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 1.8 or 2 U/ml, and the administration volume is 5 ml/kg (that is, 50 ⁇ l/10 g body weight).
  • Subcutaneous administration (S.C.) is adopted, and the drug is administered once subcutaneously on the back of the neck.
  • the acylated insulin was administered at approximately 10:30 in the morning (time 0).
  • time 0 time 0
  • the animals were fasted without water, and the blood glucose of the mice was evaluated 3, 6, 9, 12, and 15 hours after the administration.
  • the oral glucose tolerance test (OGTT) was started after the 15-hour blood glucose was detected in the test, and the blood glucose was measured 30min, 60min, 120min and 180min after intragastric administration of glucose solution (100mg/mL, 10mL/kg).
  • the OGTT experiment was carried out three times in a row. According to the results of the preliminary experiment, at the last OGTT experiment, the efficacy of the test compound was nearly disappeared, and it was terminated after 30 hours of blood glucose evaluation.
  • the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated.
  • AUC blood glucose-time curve
  • Figures 1a and 1b show that the acylated insulins of the present invention, such as compound 1, and compound 2, have a significantly better hypoglycemic effect on db/db mice than insulin deglu, and the effective action time is prolonged compared with insulin deglu.
  • Figures 2a and 2b show that the acylated insulins of the present invention such as compound 1 and compound 2 have significantly better hypoglycemic effect on db/db mice than the control compound 2.
  • the compound 1 and compound 2 of the present invention are compared with the control compound 2.
  • the efficacy of the drug was increased by 39.5% and 45.1% in the interval of 0 to 16.5 hours of administration, as shown in Table 1:
  • Table 1 Increased efficacy of the acylated insulin of the present invention relative to the control compound 2
  • Percentage increase in efficacy relative to control compound 2 [(AUC(test compound)-AUC(vehicle))/((AUC(reference compound 2)-AUC(vehicle))-1]*100%, where test Compound refers to the acylated insulin of the present invention
  • Figures 3a-3b show that compound 1, compound 2 and compound 3 of the present invention have very good pharmacological effects, and in db/db mice, they are still effective after 30 hours of monitoring, and have a significantly prolonged hypoglycemic effect. .
  • Figures 4a-5b show that the acylated insulin of the present invention such as compound 2 has significantly better hypoglycemic effect on db/db mice than the control compound 3 and the control compound 4.
  • Figures 6a-6b show that compound 4, compound 5 and compound 2 of the present invention all have very good pharmacological effects, and in db/db mice, they are still effective after 41 hours of monitoring, and have a significantly prolonged hypoglycemic effect time .
  • mice Male wistar rats aged 8w weeks, weighing 180-220g. Raised in a barrier environment in a feeding box of suitable specifications (5 per box), free access to standard food and purified water, the environmental conditions are controlled at a relative humidity of 40% to 60%, and a temperature of 22°C to 24°C. After the 4-day adaptation period, fasting for 12 hours, rats were intraperitoneally injected with streptozotocin (sigma) solution (10 mg/mL in 0.1 M citrate buffer) at 60 mg/kg. After administration, to prevent sudden hypoglycemia in rats, appropriate supplementation of glucose (20%) in drinking water, and withdrawal of supplementation after 12 hours. After 4 days of administration of streptozotocin, random blood glucose detection was performed, and the rats with blood glucose values above 20 mmol/L were selected as T1DM model rats for subsequent experiments.
  • streptozotocin Sigma
  • the basal blood glucose was evaluated at time -1/1h (9:30 am), and the rats were weighed. According to random blood glucose and body weight, the rats were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of acylated insulin, 3U/kg, where the vehicle contained: glycerol 19.6mg/ml, phenol 1.5mg/ ml, m-cresol 1.72 mg/ml, zinc ion concentration 55 ⁇ g/ml, and the pH of the solvent is 7.6.
  • vehicle contained: glycerol 19.6mg/ml, phenol 1.5mg/ ml, m-cresol 1.72 mg/ml, zinc ion concentration 55 ⁇ g/ml, and the pH of the solvent is 7.6.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 1.5 U/ml, and the administration volume is 2 ml/kg (ie, 0.2 ml/100 g body weight). It is administered subcutaneously, once subcutaneously on the back of the neck.
  • the acylated insulin was administered at approximately 9:30 in the morning (time 0), and the blood glucose of the rats was evaluated at 2, 4 hours after the administration.
  • An oral glucose tolerance test (OGTT) test was performed at 4 hours and 7 hours respectively (see below for details).
  • Detection time At the specified time point, blood was collected from the tail tip to determine fasting blood glucose (0min), then glucose solution (100mg/mL or 200mg/mL, 10mL/kg) was given by gavage, and then the glucose was measured at 30min, 60min, 120min and 180min after the glucose load blood sugar.
  • a dose-response curve of blood glucose versus time is drawn.
  • AUC blood glucose-time curve
  • Figures 7a-7b show that the acylated insulin of the present invention also has a very good hypoglycemic effect in type I diabetes (T1DM) rats, that is, it has a very good pharmacological effect.
  • [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 10 with 1N NaOH -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.
  • A14E, B16H, B25H, desB30 human insulin by conventional methods of preparing insulin analogs (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 13)
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-6xOEG), desB30 human insulin (compound 14)
  • A14E, B16H, B25H, B29K N( ⁇ )-docosanedioyl- ⁇ Glu-12xOEG), desB30 human insulin (compound 16)
  • the acylated insulin is dissolved in a solvent to a dosage of 33.5 U/ml, and the dosage is 1 ml/kg (that is, 0.1 ml/100 g body weight).
  • the acylated insulin was administered at approximately 9:30-10:00 (time 0) in the morning, and the blood glucose of rats was monitored at 3h, 6h, 9h, 24h, 48h, 72h, 96h, and 120h after administration.
  • acylated insulin control compound 5, compound 14, compound 13
  • a dose response curve of blood glucose versus time was drawn.
  • AUC blood glucose-time under-curve product
  • Figures 8a-8b show that the acylated insulin of the present invention has unexpectedly increased pharmacodynamics.
  • the title compounds of Examples 15 and 16 of Compound 13 and Compound 14 induce STZ I Type 2 diabetes (T1DM) rats all have better hypoglycemic effect, that is, have better efficacy.
  • Example 19 With reference to the similar experimental procedure of Example 19, the pharmacodynamic study was carried out in streptozotocin (STZ)-induced type I diabetes (T1DM) female rats. The difference is that the acylated insulin used is implemented
  • STZ streptozotocin
  • T1DM type I diabetes
  • Example 6 With reference to the similar experimental procedure of Example 6, on the obese diabetic mouse model (db/db mice), the title compound of Comparative Example 5, and the title compounds of Examples 15 and 16 (ie, the control compound) was tested in a single-dose study. 5. Compound 13 and Compound 14). The blood glucose lowering effect of the acylated insulin was tested at a dose of 9 U/kg.
  • mice 8-9 weeks old male db/db(BKS/Lepr) mice are raised 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 acylated insulin 9U/kg, where the vehicle used contained: phenol 5.65mg/ml, glycerol 15mg/ml , Disodium hydrogen phosphate 0.708 mg/ml, sodium chloride 0.585 mg/ml, and the pH of the solvent is 7.6.
  • vehicle contained: phenol 5.65mg/ml, glycerol 15mg/ml , Disodium hydrogen phosphate 0.708 mg/ml, sodium chloride 0.585 mg/ml, and the pH of the solvent is 7.6.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 1.8 U/ml, and the administration volume is 5 ml/kg (that is, 50 ⁇ l/10 g body weight).
  • Subcutaneous administration (S.C.) is adopted, and the drug is administered once subcutaneously on the back of the neck.
  • the acylated insulin was administered at approximately 10:30 in the morning (time 0).
  • time 0 time 0
  • the animals were fasted without water, and the blood glucose of the mice was evaluated 3h, 6h, 9h, and 21.5h after the administration.
  • the Oral Glucose Tolerance Test was started, and the blood glucose was measured 30min, 60min, 120min and 360min after intragastric administration of glucose solution (100mg/mL, 7.5mL/kg) ;
  • the second OGTT experiment was started after 330min of blood glucose was measured by the first OGTT, and the blood glucose was measured 30min, 90min, 210min and 360min after intragastric administration of glucose solution (50mg/mL, 10mL/kg).
  • the third OGTT experiment was started after the second OGTT measurement of blood glucose for 360 minutes.
  • the blood glucose was measured 30 minutes, 60 minutes, and 120 minutes after intragastric administration of glucose solution (50 mg/mL, 10 mL/kg).
  • glucose solution 50 mg/mL, 10 mL/kg.
  • the efficacy of the test compound Has not disappeared, the experiment was terminated after 36 hours of blood glucose assessment.
  • acylated insulin For each single dose of acylated insulin, a dose-response curve of blood glucose versus time is drawn. To illustrate the effect of acylated insulin on blood glucose, for each individual dose response curve, the area under the blood glucose-time curve (AUC) from 0 to the monitoring endpoint was calculated. Among them, the smaller the AUC value, the better the hypoglycemic effect and the better the drug effect.
  • Figures 10a-10b show that, compared to the control compound 5, the acylated insulin compound 14 and compound 13 of the present invention have significantly improved hypoglycemic effect in type 2 diabetic db/db mice.
  • the test was started 14 days after the model was built. Before the start of the experiment on the same day, the basal blood glucose was evaluated at time -1/1h (9:30 am), and the rats were weighed. According to random blood glucose and body weight, the rats 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 title compounds of control example 5, example 17 and 18 (ie control compound 5, compound 15, Compound 16), the dosage is 25 U/kg, wherein the solvent contains: phenol 5.65 mg/ml, glycerol 15 mg/ml, disodium hydrogen phosphate 0.708 mg/ml, sodium chloride 0.585 mg/ml, and the pH of the solvent is 7.6 .
  • the acylated insulin is dissolved in a solvent to an administration concentration of 25 U/ml, and the administration volume is 1 ml/kg (that is, 0.1 ml/100 g body weight).
  • Adopt subcutaneous administration method subcutaneous administration on the back of the neck, repeated administration 4 times, each time interval of 4 days, SD rats eat and drink freely during the test.
  • the acylated insulin was administered at approximately 9:30-10:00 (time 0) in the morning, and the blood glucose of rats was monitored after the first administration 3h, 6h, 9h, 24h, 48h, 72h, 96h, and 6h after each administration. The blood glucose of rats was monitored every 24h.
  • a dose-response curve of blood glucose versus time is drawn.
  • AUC blood glucose-time curve
  • the percentage increase of the drug effect relative to the control compound 5 [(AUC(test compound)-AUC(vehicle))/((AUC(reference compound 5)-AUC(vehicle))-1]*100%, where the test Compound refers to the acylated insulin of the present invention
  • the acylated insulin of the present invention has an unexpectedly increased hypoglycemic effect in type I diabetic (T1DM) rats after administration.
  • T1DM type I diabetic
  • the test was started 14 days after the model was built. Before the start of the experiment on the same day, the basal blood glucose was evaluated at time -1/1h (9:30 am), and the rats were weighed. According to random blood glucose and body weight, the rats were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of the title compound of control example 5 and example 16 (ie control compound 5, compound 14), dose It is 25 U/kg, wherein the solvent contains: phenol 5.65 mg/ml, glycerol 15 mg/ml, disodium hydrogen phosphate 0.708 mg/ml, sodium chloride 0.585 mg/ml, and the pH of the solvent is 7.6.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 25 U/ml, and the administration volume is 1 ml/kg (that is, 0.1 ml/100 g body weight).
  • Adopt subcutaneous administration method subcutaneous administration on the back of the neck, repeated administration 4 times, each time interval of 4 days, SD rats eat and drink freely during the experiment.
  • the acylated insulin was administered at approximately 9:30-10:00 (time 0) in the morning, and the blood glucose of rats was monitored after the first administration 3h, 6h, 9h, 24h, 48h, 72h, 96h, and 6h after each administration. The blood glucose of rats was monitored every 24h.
  • a dose-response curve of blood glucose versus time is drawn.
  • AUC blood glucose-time curve
  • the acylated insulin of the present invention has an unexpectedly increased hypoglycemic effect in type I diabetic (T1DM) rats after administration.
  • T1DM type I diabetic
  • composition comprising the acylated insulin and insulin aspart of the present invention can regulate blood glucose (BG) in streptozotocin (STZ) induced type I diabetes (T1DM) C57/6J mice effect.
  • BG blood glucose
  • STZ streptozotocin
  • T1DM type I diabetes
  • mice 4-6 weeks old male C57/6J mice (purchased from Vitality) are raised in a barrier environment in a breeding box of appropriate 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 fasted for 12 hours, and the mice were intraperitoneally injected with streptozotocin (sigma) solution (10 mg/mL in 0.1 M citrate buffer) at 150 mg/kg. Three days after the administration of streptozotocin, random blood glucose testing was performed, and the T1DM model mice with blood glucose values above 20mmol/L were selected for follow-up experiments.
  • streptozotocin Sigma
  • mice were tested for random blood glucose and weighed. According to random blood glucose and body weight, mice were assigned to vehicle group or treatment group. There were 5 groups, 8 mice in each group. Each group received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of insulin aspart (0.36U/kg), Subcutaneous injection of a pharmaceutical composition containing insulin deglubber and insulin aspart, wherein the injection doses of insulin deglubber and insulin aspart are respectively 0.84 U/kg and 0.36 U/kg, or subcutaneous injection containing the title compound of Example 4 of the present invention Two pharmaceutical compositions of compound 4 and insulin aspart, wherein when the two pharmaceutical compositions are injected, the injection doses of compound 4 are 0.82 U/kg and 0.64 U/kg, respectively, and the injection doses of insulin aspart are both 0.36 U/kg, wherein the solvent contains: glycerol 19.6 mg/ml, phenol 1.5 mg/ml, m-cresol 1.72 mg/ml, zinc ion concentration 55
  • the premix of compound 4 and insulin aspart was dissolved in the vehicle to an administration concentration of 0.072 U/mL (based on the concentration of insulin aspart in the premix), and the administration volume was 5 ml/kg (that is, 50 ⁇ l/10 g body weight).
  • Subcutaneous administration (S.C.) is adopted, and the drug is administered once subcutaneously on the back of the neck. The drug was administered at approximately 16:00 (time 0) in the afternoon.
  • the animals were fasted without water, and the blood glucose of the mice was evaluated at 0.5, 1, 2, 3, 6 and 15:00 after the administration.
  • Figures 13a-13b show that after administration of a pharmaceutical composition containing the acylated insulin and insulin aspart of the present invention, compared to the pharmaceutical composition containing deglubber and insulin aspart, in type I diabetic (T1DM) mice There is an unexpectedly increased hypoglycemic effect.
  • T1DM type I diabetic mice
  • composition containing the acylated insulin of the present invention and insulin aspart was tested against blood glucose in streptozotocin (STZ) induced type I diabetes (T1DM) C57/6J mice. BG)'s moderating effect.
  • mice were tested for random blood glucose and weighed. According to random blood glucose and body weight, mice were assigned to vehicle group or treatment group. There were 7 groups, 8 mice in each group. Each group received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of insulin aspart (3U/kg), or subcutaneous Inject a pharmaceutical composition containing insulin deglucol and insulin aspart, wherein the injection doses of insulin deglucol and insulin aspart are respectively 7U/kg and 3U/kg, or subcutaneously inject four drug combinations containing compound 4 and insulin aspart
  • the injection doses of compound 4 are 6.79 U/kg, 5.34 U/kg, 3.84 U/kg, and 2.39 U/kg, respectively, and the injection doses of insulin aspart are all 3 U /kg, wherein the solvent contains: glycerol 19.6 mg/ml, phenol 1.5 mg/ml, m-cresol 1.72 mg/ml, zinc ion concentration 55 ⁇ g/ml,
  • the premix of compound 4 and insulin aspart was dissolved in a vehicle to a concentration of 0.6 U/mL (calculated as the concentration of insulin aspart in the premix), and the administration volume was 5 ml/kg (that is, 50 ⁇ l/10 g body weight). It is administered by subcutaneous administration (S.C.) and subcutaneous injection on the back of the neck. The drug was administered at about 17:00 (time 0) every afternoon for 10 consecutive days. During the administration period, the mice were free to eat and drink. The blood glucose of the mice before the fourth, eighth, and tenth doses (0h) was evaluated, and the fourth, 8.
  • mice Random blood glucose of mice 1 hour after the tenth administration, random blood glucose before the eighth administration (0h) and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 16, 24 hours after the administration Blood glucose is tested. After the last administration, the mice were fasted for 1 hour, and blood was taken from the orbit to detect the percentage of glycosylated hemoglobin (Hb1Ac) in the whole blood.
  • Hb1Ac glycosylated hemoglobin
  • Figures 14a-17 show that after administration of a composition containing the acylated insulin and insulin aspart of the present invention, compared to a pharmaceutical composition containing deglubber and insulin aspart, in type I diabetic (T1DM) mice It has an unexpectedly increased hypoglycemic effect, and has a more excellent cumulative effect of hypoglycemic.
  • T1DM type I diabetic
  • Figures 14a and 14b show that a composition containing the acylated insulin and insulin aspart of the present invention, after administration, is in type I diabetes ( T1DM) mice have an unexpectedly increased hypoglycemic effect.
  • T1DM type I diabetes
  • Figures 15a-15c respectively show the blood glucose situation of mice in each administration group before the fourth, eighth, and tenth doses (0h), indicating that compared to the pharmaceutical composition containing deglubber and insulin aspart, the present invention is included
  • the pharmaceutical composition of acylated insulin and insulin aspart has better efficacy and a more excellent cumulative effect of lowering blood sugar.
  • Figures 16a-16c show the blood glucose situation of mice 1 hour after the fourth, eighth, and tenth administrations, respectively, indicating that the acylated insulin of the present invention is contained relative to the pharmaceutical composition containing deglubber and insulin aspart
  • the pharmaceutical composition with insulin aspart has better medicinal effects and a more excellent cumulative effect of lowering blood sugar.
  • Figure 17 shows that the composition comprising the acylated insulin and insulin aspart of the present invention has a better effect of reducing Hb1Ac after administration, compared with the pharmaceutical composition comprising insulin deglubber and insulin aspart, in compound 4
  • the dosage ratio of insulin aspart is much smaller than the dosage ratio of insulin degludec and insulin aspart, it can still achieve a better effect of reducing Hb1Ac.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • pH value is about 10-11.
  • add phenol, m-cresol, zinc acetate, glycerin and Sodium chloride produces an acylated insulin preparation with a final insulin concentration of 1.2mM (200U/ml or 8.46mg/ml), where the content of Zn is Zn/6 moles of acylated insulin (abbreviated as "Zn/6ins") To represent.
  • the chemical stability of the formulation in this example can be shown by the change in high molecular weight protein (HMWP) relative to day 0 after storage at 25°C and 37°C for 14 days and 20 days, and it can also be used at 25°C and 37°C.
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • High molecular weight protein content was determined by high performance liquid chromatography (HPLC), and the model specifications: Waters Xbride BEH 200A (7.8 * 300mm), 5 ⁇ m column, at a column temperature of 30 °C, temperature of the sample cell At 5°C, the test was performed with a mobile phase at a flow rate of 0.5 ml/min, where the mobile phase contained 600 ml of 0.1% arginine solution, 150 ml of glacial acetic acid and 250 ml of acetonitrile. The detection wavelength is 276nm, and the injection volume is 10ul. Table 3 shows the increase in HMWP on Day 14 and Day 20 relative to Day 0 at 25°C and 37°C.
  • the amount of HMWP in the above-mentioned acylated insulin preparations of the present invention increases very slowly with time, indicating that the above-mentioned acylated insulin preparations all have excellent chemical stability.
  • the Zn content is 6.5Zn/6ins
  • the amount of HMWP increases more slowly than when the Zn content is 5.5Zn/6ins.
  • the content of insulin-related substances was determined by high performance liquid chromatography (HPLC). On Waters Kromasil 300A-5 ⁇ m-C8 (4.6*250mm) column, when the column temperature is 40°C and the sample pool temperature is room temperature, the elution phase is used. The test was performed at a flow rate of 1.0 ml/min.
  • the elution phase consists of a mobile phase consisting of:
  • Phase A contains 0.1M anhydrous sodium sulfate, 0.1M sodium dihydrogen phosphate dihydrate, 10% acetonitrile (v/v), adjust the pH value to 5.0 with NaOH;
  • Phase B is 50% acetonitrile (v/v).
  • Table 4 shows the increase in related substances on the 14th and 20th day relative to the 0th day at 37°C.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the acylated insulin preparations in Table 5-7 were prepared according to the steps similar to those in Example 26.
  • the changes of HMWP and related substances were measured according to similar procedures as in Example 26.
  • Tables 5-7 below show the changes of HMWP and related substances of acylated insulin preparations of different formulations.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the acylated insulin preparations in Table 8 were prepared according to the steps similar to those in Example 26.
  • the changes of HMWP and related substances were measured according to similar procedures as in Example 26.
  • the following table shows the changes of HMWP and related substances of acylated insulin preparations of different formulations.
  • T1DM streptozotocin induced type I diabetes
  • the test was started 8 days after the model was built, and the basal blood glucose was monitored and the rats were weighed one day before the administration. According to random blood glucose and body weight, the rats were matched and assigned to the vehicle group or treatment group, and received the following treatment: subcutaneous injection of vehicle, or subcutaneous injection of insulin degludec (50U/kg), compound 4 (25U/kg or 40U/kg),
  • the solvent includes: phenol 60mM, glycerol 15mg/ml, m-cresol 10mM, sodium chloride 0.585mg/ml, and the pH value of the solvent is 7.4.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 25 U/ml or 40 U/ml, and the administration volume is 1 ml/kg (ie, 0.1 ml/100 g body weight).
  • Adopt subcutaneous administration method subcutaneous administration on the back of the neck, once every other day, repeated administration 11 times, SD rats freely eat and drink during the experiment.
  • the acylated insulin was administered at approximately 9:30-10:30 in the morning.
  • the blood glucose of rats was monitored after the first administration of 3h, 4h, 5h, 6h, 24h, and 48h. After each administration, the rats’ blood glucose was monitored at 4h, 24h, and 48h. Rat blood sugar.
  • a dose-response curve of blood glucose versus time is drawn.
  • AUC blood glucose-time curve
  • the acylated insulin of the present invention has an unexpectedly increased hypoglycemic effect in type I diabetic (T1DM) rats after administration compared to insulin deglu, and the hypoglycemic effect of compound 4 Significantly better than insulin deglu.
  • composition containing the acylated insulin of the present invention and insulin aspart was tested against blood glucose in streptozotocin (STZ) induced type I diabetes (T1DM) C57/6J mice. BG)'s moderating effect.
  • mice were weighed. According to random blood glucose and body weight, mice were assigned to vehicle group or treatment group. There were 8 groups, 9 mice in each group (5 males and 4 females).
  • each group received the following treatments: subcutaneous injection of vehicle or subcutaneous injection containing Germany
  • a pharmaceutical composition of insulin glucomannan and insulin aspart wherein the injection doses of insulin degludec and insulin aspart are 7 U/kg and 3 U/kg, respectively, or subcutaneously injected containing the title compound compound 4 and insulin aspart of Example 4 of the present invention
  • the six pharmaceutical compositions of, wherein when the six pharmaceutical compositions are injected, the injection doses of compound 4 are 1.49 U/kg, 1.99 U/kg, 2.45 U/kg, 2.85 U/kg, 3.43 U/kg, respectively , And 3.92U/kg, the injection dose of insulin aspart is 3U/kg, wherein the solvent contains: 60mM phenol, 10mM m-cresol, 15mg/ml glycerol, 15mM Na 2 HPO 4 , the pH of the above solvent is 7.6.
  • the premix of compound 4 and insulin aspart was dissolved in a vehicle to a concentration of 0.6 U/mL (calculated as the concentration of insulin aspart in the premix), and the administration volume was 5 ml/kg (that is, 50 ⁇ l/10 g body weight). It is administered by subcutaneous administration (S.C.) and subcutaneous injection on the back of the neck. The drug was administered at about 16:00 (time 0) every afternoon for 15 consecutive days. During the administration period, the mice were free to eat and drink.
  • the first, second, fifth, eighth, and fifteenth doses were evaluated before (0h) and administration Random blood glucose of the mice after 1 hour, and the second, fifth, eighth, and fifteenth doses before (0h) blood glucose and 0.5, 1, 2, 4, 6, 16, 20, 24 hours after the administration of random blood glucose Detection. After the last administration, the mice were fasted for 2 hours, and blood was taken from the orbit to detect the percentage of glycosylated hemoglobin (Hb1Ac) in the whole blood.
  • Hb1Ac glycosylated hemoglobin
  • Figure 19a and Figure 19b show that the composition containing the acylated insulin and insulin aspart of the present invention, after administration, is compared with the pharmaceutical composition containing deglubber and insulin aspart in type I diabetic (T1DM) mice There is an unexpectedly increased hypoglycemic effect.
  • T1DM type I diabetic mice
  • Figure 20 shows that a composition containing the acylated insulin and insulin aspart of the present invention has a better effect of reducing Hb1Ac after administration than the pharmaceutical composition containing insulin deglubber and insulin aspart.
  • the dosage ratio of insulin aspart is much smaller than the dosage ratio of insulin degludec and insulin aspart, it can still achieve a better effect of reducing Hb1Ac.
  • the purpose of this study is to verify the regulatory effect of the combination of the acylated insulin of the present invention and insulin aspart on blood glucose (BG) in an obese diabetic mouse model (db/db mice) in the case of diabetes.
  • mice 8-9 weeks old male db/db(BKS/Lepr) mice are raised 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 tested for random blood glucose, and the mice were weighed. According to random blood glucose and body weight, mice were assigned to vehicle group or treatment group. There were 5 groups, 8 mice in each group. Each group received the following treatments: subcutaneous injection of vehicle or subcutaneous injection of a drug combination containing insulin degludec and insulin aspart Insulin, wherein the injection doses of insulin degludec and insulin aspart are respectively 7U/kg and 3U/kg, or subcutaneous injection of three pharmaceutical compositions containing the title compound compound 4 of Example 4 of the present invention and insulin aspart, wherein When the three pharmaceutical compositions were injected, the injection doses of compound 4 were 2.0 U/kg, 2.4 U/kg, 3.84 U/kg, and the injection doses of insulin aspart were all 3 U/kg, and the solvent contained: 60 mM phenol , 10mM m-cresol, 15mg/ml glycerol, 15mM Na 2 HPO 4 , pH value is 7.6.
  • the acylated insulin is dissolved in a solvent to an administration concentration of 0.6 U/ml, and the administration volume is 5 ml/kg (that is, 50 ⁇ l/10 g body weight).
  • Adopt subcutaneous administration (S.C.) method subcutaneous injection administration on the back of the neck four times.
  • the acylated insulin was administered at approximately 09:30 in the morning (time 0).
  • time 0 time 0
  • the animals were fasted without water, and the blood glucose of the mice was evaluated 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours after the administration.
  • Figure 21a and Figure 21b show that the composition containing the acylated insulin and insulin aspart of the present invention, after administration, compared to the pharmaceutical composition containing insulin deglubber and insulin aspart, in an obese diabetic mouse model ( db/db mice) has an unexpectedly increased hypoglycemic effect.
  • the dosage ratio of compound 4 to insulin aspart is much smaller than the dosage ratio of insulin degludec and insulin aspart, it can still achieve better lowering effects.
  • Sugar effect When the dosage ratio of compound 4 to insulin aspart is much smaller than the dosage ratio of insulin degludec and insulin aspart, it can still achieve better lowering effects.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the title compound compound 4 of Example 4 was dissolved in 0.03% NaOH solution to a concentration of 2.4 mM, and then the pH value was adjusted to 7.4 with 4% NaOH solution. According to the amount of each component in the following table, the phenol, m-methyl After mixing the phenol, glycerin and sodium chloride, add to the compound 4 solution, adjust the pH to 7.4, and then add it to the compound 4 solution in three averages according to the amount of zinc acetate in the table below to adjust the pH to the final value.
  • acylated insulin preparation with a final insulin concentration of 1.2 mM (200 U/ml or 8.46 mg/ml) was produced, where the content of Zn was expressed as Zn/6 moles of acylated insulin (abbreviated as "Zn/6ins").
  • the chemical stability of the formulation in this example can be shown by the change in high molecular weight protein (HMWP) relative to day 0 after storage at 25°C and 37°C for 14 days and 21 days, and it can also be used after storage at 37°C for 21 days.
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • HPLC high performance liquid chromatography
  • the content of insulin-related substances was determined by high-performance liquid chromatography (HPLC). On a Waters Kromasil 100A-3.5 ⁇ m-C8 (4.6*250mm) column, the column temperature was 40°C and the sample pool temperature was 10°C. The dephasing is tested at a flow rate of 1.0 ml/min.
  • the elution phase consists of a mobile phase consisting of:
  • Phase A contains 0.1M anhydrous sodium sulfate, 0.1M sodium dihydrogen phosphate dihydrate, 10% acetonitrile (v/v), adjust the pH value to 3.0 with concentrated phosphoric acid;
  • Phase B is 60% acetonitrile (v/v).
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the acylated insulin preparations in Tables 11 and 12 were prepared according to the procedure similar to that in Example 32.
  • the changes of HMWP and related substances were measured according to the procedure similar to that in Example 32.
  • Tables 11 and 12 below show the changes of HMWP and related substances of acylated insulin preparations of different formulations.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the title compound compound 16 of Example 18 was dissolved in 0.08% NaOH solution to twice the final insulin concentration, and then the pH value was adjusted to 7.45 with 4% NaOH solution. According to the amount of each component in the table below, the phenol, After m-cresol, glycerol and sodium chloride were mixed, they were added to the compound 16 solution to adjust the pH to 7.4, and then added to the compound 16 solution in three averages according to the amount of zinc acetate in the table below to adjust the pH to 7.4. An acylated insulin preparation with a final insulin concentration of 1.2 mM (9.43 mg/ml) or 1.5 mM (11.74 mg/ml) was produced.
  • the chemical stability of the formulation in this example can be shown by the change in high molecular weight protein (HMWP) relative to day 0 after storage at 25°C and 37°C for 14 days and 21 days, and it can also be used at 25°C and 37°C.
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • Tables 13-15 show the increase in HMWP on Day 14 and Day 21 relative to Day 0 at 25°C and 37°C.
  • the content of insulin-related substances was determined by high-performance liquid chromatography (HPLC), on a Waters Kromasil 300A-5 ⁇ m-C4 (4.6*150mm) column, when the column temperature was 40°C and the sample pool temperature was 10°C, the elution was used
  • HPLC high-performance liquid chromatography
  • the phase is tested at a flow rate of 1.0 ml/min.
  • the elution phase consists of a mobile phase consisting of:
  • Phase A contains 0.18M anhydrous sodium sulfate, 10% acetonitrile (v/v), adjust the pH to 2.3 with 85% phosphoric acid;
  • Phase B is 75% acetonitrile (v/v).
  • Tables 16-17 show the increase in related substances on the 14th and/or 21st day relative to the 0th day at 25°C and 37°C.
  • the purpose of this experiment is to measure the chemical stability of the acylated insulin preparation of the present invention.
  • the title compound compound 16 of Example 18 was dissolved in 10 mM 50% final volume disodium hydrogen phosphate solution to twice the final insulin concentration, and then the pH value was adjusted to the final value with 4% NaOH, according to each group in the table below After mixing phenol, m-cresol, glycerin and sodium chloride, add to the compound 16 solution, adjust the pH value to the final value, and then add it to the compound 16 solution in three averages according to the amount of zinc acetate in the table below. , Adjust the pH value to the final value. An acylated insulin preparation with a final insulin concentration of 1.5 mM (11.74 mg/ml) was produced.
  • the HMWP was measured according to the procedure similar to that in Example 32, and the change of related substances was measured according to the procedure similar to that in Example 34.
  • Tables 19 and 20 below show the changes in HMWP and related substances of acylated insulin preparations of different formulations.
  • the purpose of this experiment is to measure the chemical stability of the combined preparation of acylated insulin and insulin aspart 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 14 days and 28 days.
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • HMWP high molecular weight protein
  • HPLC high performance liquid chromatography
  • the purpose of this experiment is to measure the chemical stability of the combined preparation of acylated insulin and insulin aspart of the present invention.
  • Combinations 6-10 were formulated according to the amounts of each component in Table 23 below. And the change of HMWP was measured according to the procedure similar to that in Example 36. Table 24 below shows the changes in HMWP of acylated insulin preparations of different formulations.
  • the purpose of this experiment is to measure the chemical stability of the combined preparation of acylated insulin and insulin aspart of the present invention.
  • Combinations 11 and 12 were formulated according to the amounts of each component in Table 25 below. And the change of HMWP was measured according to the procedure similar to that in Example 36. Table 26 below shows the changes in HMWP of acylated insulin preparations of different formulations.
  • the purpose of this study is to verify the effect of the composition comprising the acylated insulin and insulin aspart of the present invention on blood glucose (BG) in an obese diabetic mouse model (db/db mice) in the case of diabetes.
  • mice were tested for random blood glucose and the mice were weighed. According to random blood glucose and body weight, the mice were assigned to vehicle group or treatment group. There were 4 groups, 5 mice in each group.
  • the injections of the acylated insulin and insulin aspart are dissolved in a solvent to the corresponding administration concentration, and the administration volume is 5 ml/kg (that is, 50 ⁇ l/10 g body weight). It is administered subcutaneously (S.C.) once a day. During the administration period, the animals were free to eat and drink. The random blood glucose of mice was evaluated at 0.5, 1, 2, 3, 4, 6, and 8 hours after the 21st day of continuous administration and 0.5, 1, 2, after the 18th day of continuous administration. After 3, 4, 6, 8, and 10 hours, the mice were fasted for blood glucose.
  • Figures 22a to 22d show that the composition comprising the acylated insulin and insulin aspart of the present invention, after administration, is compared with the pharmaceutical composition comprising insulin deglubber and insulin aspart in an obese diabetic mouse model ( db/db mice) has an unexpectedly increased hypoglycemic effect.
  • the dosage ratio of compound 4 to insulin aspart is much smaller than the dosage ratio of insulin degludec and insulin aspart, it can still achieve better lowering effects. Glucose effect, and the hypoglycemic lasts longer.
  • C max peak concentration
  • T max peak time
  • T 1/2 terminal elimination half-life
  • AUC 0-t 0-t time-area under the blood glucose concentration time curve
  • AUC INF time from administration under the plasma concentration time curve Area to infinity
  • Vd apparent volume of distribution
  • Cl clearance rate
  • MRT average residence time
  • Compound 4 intravenous group and insulin degludec intravenous group were taken before administration and after administration 2min, 10min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 30h, 36h, 48h to determine the blood concentration of the compound.
  • Table 28 Pharmacokinetic parameters after subcutaneous injection of compound 4 and Degu into beagle dogs
  • the acylated insulin derivative compound 4 of the present invention exhibits a longer half-life and a more stable hypoglycemic effect in rats and beagle dogs.
  • the compound B29K (N( ⁇ )-docosanediacyl- ⁇ Glu-12xOEG), desB30 human insulin was prepared in a procedure similar to the second part of Example 1.
  • B29K N( ⁇ )-docosanedioyl- ⁇ Glu-OEG), desB30 human insulin (compound 20)
  • the compound B29K (N( ⁇ )-docosanediacyl- ⁇ Glu-OEG), desB30 human insulin was prepared in a similar procedure to that of Example 1, Part 2.
  • the compound B29K (N( ⁇ )-docosane diacyl- ⁇ Glu-12xPEG), desB30 human insulin was prepared by the similar steps as in the second part of Example 1.
  • the purpose of this test is to prove the binding ability of the insulin derivative of the present invention to the insulin receptor.
  • the surface plasmon resonance (SPR) method was used to compare the compound 2 of the present invention and the control compound 2 in the absence of human serum albumin (HSA) and the presence of 2% HSA, respectively, with the extracellular domain of his-tagged insulin receptor A ( IRA, Sino Biological) binding ability test.
  • HSA human serum albumin
  • IRA his-tagged insulin receptor A
  • the samples were diluted with Running buffer (Cytiva) or 2.0% HSA-containing operating buffer, respectively, so that the injection concentration of the sample and the control compound 2 sample were both 400 nM.
  • the NTA sensor chip (Cytiva) was selected, and the SPR analysis was performed on a Biacore T200 (Cytiva) at 25°C.
  • 0.5M NiCl 2 (Cytiva) was injected at a flow rate of 10 ⁇ L/min for 60 seconds, and then washed with HBS-EP buffer (Cytiva). Inject 3 ⁇ g/mL IRA receptor for 180s at a flow rate of 5 ⁇ L/min to bind the IRA receptor to the surface of the chip.
  • the tested insulin derivative sample was injected at a flow rate of 30 ⁇ L/min for 60 seconds, and dissociated for 60 seconds.
  • 350mM EDTA (Cytiva) was injected at a flow rate of 10 ⁇ L/min for 60s to regenerate the chip, and finally the HBS-P buffer (Cytiva) was washed and the next sample could be tested.
  • the response value at the 4s before the dissociation of the sample is selected as the result of the receptor binding ability test, and the test is repeated 3 times for each sample.
  • Figure 23 shows the receptor binding ability of Compound 2 and Control Compound 2 in the presence of 2% HSA (simulating physiological conditions), relative to 0% HSA. It can be seen from Figure 23 that in the presence of 2% HSA, compound 2 has a significantly improved receptor binding ability compared to the control compound 2. The effect of albumin on the receptor binding ability of the compound 2 of the present invention is significantly lower than that of the control. Compound 2.
  • the insulin derivatives of the present invention such as compound 2 have an unexpectedly significantly improved receptor binding capacity compared to the control compound 2, that is, the receptor binding ability of albumin to the insulin derivatives of the present invention
  • the effect of body binding capacity is significantly lower than that of the control compound 2.
  • the purpose of this test is to prove the binding ability of the insulin derivative of the present invention to the insulin receptor.
  • the insulin derivative compound 15 and the control compound 5 of the present invention were tested in the absence of human serum albumin (HSA) and in the presence of 2% HSA using a method similar to that of Example 46. The difference is that the injection concentrations of compound 15 and control compound 5 samples are both 12800 nM and 25600 nM, and the measurement results are shown in Figures 24a and 24b.
  • HSA human serum albumin
  • Figures 24a and 24b show the receptor binding ability of compound 15 and control compound 5 in the presence of 2% HSA (simulated physiological conditions), relative to 0% HSA. It can be seen from Figures 24a and 24b that in the presence of 2% HSA, compound 15 has an unexpectedly significantly improved receptor binding capacity compared to the control compound 5. Albumin has a significant effect on the receptor of the insulin derivative compound 15 of the present invention. The effect of binding capacity is significantly lower than that of the control compound 5.
  • the purpose of this test is to prove the binding ability of the insulin derivative of the present invention to the insulin receptor.
  • the insulin derivative compound 17 and the control compound 2 of the present invention were tested in the absence of human serum albumin (HSA) and in the presence of 2% HSA using a method similar to that of Example 46.
  • the measurement results are shown in Figure 25.
  • Figure 25 shows the receptor binding ability of Compound 17 and Control Compound 2 in the presence of 2% HSA (simulating physiological conditions), relative to 0% HSA. It can be seen from Figure 25 that in the presence of 2% HSA, compound 17 has a significantly improved receptor binding capacity compared to the control compound 2. The effect of albumin on the receptor binding capacity of the insulin derivative compound 17 of the present invention is significant Lower than the control compound 2.
  • the insulin derivatives of the present invention such as compound 17 have an unexpectedly significantly improved receptor binding capacity compared to the control compound 2, that is, the receptor binding ability of albumin to the insulin derivatives of the present invention
  • the effect of body binding capacity is significantly lower than that of the control compound 2.
  • the purpose of this test is to prove the binding ability of the insulin derivative of the present invention to the insulin receptor.
  • the insulin derivative compound 16, compound 18, and control compound 5 of the present invention were combined with IRA in the absence of human serum albumin (HSA) and in the presence of 2% HSA using a method similar to that in Example 46.
  • HSA human serum albumin
  • the difference is that the injection concentrations of compound 16, compound 18 and control compound 5 samples are all 12800 nM and 25600 nM, and the measurement results are shown in Figures 26a and 26b.
  • Figures 26a and 26b show the receptor binding ability of compound 16, compound 18 and control compound 5 in the presence of 2% HSA (simulated physiological conditions), relative to 0% HSA. It can be seen from Figures 26a and 26b that in the presence of 2% HSA, compound 16 and compound 18 have an unexpectedly significantly improved receptor binding capacity compared to the control compound 5.
  • the receptor binding capacity of albumin to the insulin derivative of the present invention is The effect of body binding capacity is significantly lower than that of the control compound 5.
  • A14E, B16H, B25H, desB30 human insulin B chain
  • A14E, B16E, B25H, desB30 human insulin B chain A14E, B16E, B25H, desB30 human insulin B chain:
  • A21G human insulin A chain A21G human insulin A chain:
  • A21G human insulin B chain A21G human insulin B chain:
  • A21G, desB30 human insulin A chain A21G, desB30 human insulin A chain:
  • A21G, desB30 human insulin B chain A21G, desB30 human insulin B chain:

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Abstract

一种胰岛素衍生物，其药物制剂、其与长效GLP-1化合物的药物组合物、及所述胰岛素衍生物、药物制剂和药物组合物的医药用途。所述胰岛素衍生物相比于德谷胰岛素或其它胰岛素衍生物，具有预料不到的显著增加的药效、具有更长的持续作用时间、更长的体内半衰期、良好的生物利用率、及具有更好的物理稳定性、化学稳定性。

Description

胰岛素衍生物 技术领域
本发明涉及治疗性肽领域，具体涉及新型胰岛素衍生物，其药物制剂、其与长效GLP-1化合物的药物组合物、其与快速起效的胰岛素的药物组合物、及所述胰岛素衍生物、药物制剂和药物组合物的医药用途。
背景技术
胰岛素是由胰的β细胞分泌的多肽激素。胰岛素由命名为A链和B链的2条多肽链组成，所述A链和B链通过2个链间二硫键连接在一起。在人、猪和牛胰岛素中，A链和B链分别包含21和30个氨基酸残基。然而，不同物种之间，在2条链的不同位置中存在的氨基酸残基存在差异。基因工程改造的广泛使用已使得可以通过替换、缺失和添加一个或多个氨基酸残基来制备天然存在的胰岛素的类似物。
胰岛素能够用于治疗糖尿病和与之关联或起因于其的疾病，在维持正常代谢调节中是必需的。但是人胰岛素等天然胰岛素作用时间较短，使得病人必须频繁注射，使患者产生许多注射相关的不适。因此，人们一直致力于获得药效好、作用时间更长、注射频率更低的胰岛素衍生物或类似物来改善因较高频率的胰岛素注射所带来的不便和不适。
WO1995007931A1公开了已上市的长效胰岛素地特胰岛素，其分子结构特点是去掉人胰岛素B链30位的苏氨酸，并在B链的第29位赖氨酸残基上连接有14碳脂肪单酸。WO2005012347A2公开了已上市的另一种长效胰岛素德谷胰岛素，德谷胰岛素是一种比地特胰岛素作用时间更长的新型超长效胰岛素，其分子结构特点是去掉人胰岛素B链30位苏氨酸，通过1个谷氨酸分子将16碳脂肪二酸侧链连接在B29位赖氨酸残基。CN101573133B和WO2009/010428公开了PEG(PEGylated)化延长的胰岛素，与常规的未修饰的胰岛素相比，PEG化延长的胰岛素具有更长的作用时间。WO2013086927A1和WO2018/024186公开一种长效的人胰岛素类似物的酰化衍生物。
然而，迄今为止，尚未有比每日皮下注射一次的频率更低的基础胰岛素产品获批上市。
因此，目前仍然需要相比于已上市的胰岛素(例如德谷胰岛素)或已知的胰岛素衍生物，具有更好的药效或功效、更长的作用时间、更低的施用频率、更优异的理化性质的胰岛素衍生物或类似物。
发明内容
本发明提供了新型胰岛素衍生物(例如酰化胰岛素)。发明人通过大量试验，出乎意料地发现所述新型胰岛素衍生物(例如酰化胰岛素)相比于已上市的德谷胰岛素(商品名“诺和达”)或其它某些胰岛素衍生物，具有预料不到的显著增加的效力、功效或药效、更长的持续作用时间、更长的体内半衰期、良好的生物利用率、更好的安全性、及具有更令人满意的物理稳定性、化学稳定性、和溶解度。
在一个方面，本发明提供了胰岛素衍生物，所述胰岛素衍生物包含胰岛素母体、白蛋白结合残基、和连接基团Lin，所述胰岛素母体为天然存在的胰岛素或胰岛素类似物，所述白蛋白结合残基经由连接基团Lin与所述胰岛素母体连接，其中，
所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；或者所述连接基团Lin包含至少5个中性的、包含亚烷基二醇的氨基酸残基；优选所述连接基团Lin包含至少6个中性的、包含亚烷基二醇的氨基酸残基；优选所述连接基团Lin包含5-20个中性的、包含亚烷基二醇的氨基酸残基；或者，所述连接基团Lin包含具有至少15个、优选至少20个、优选至少24个、优选至少30个、优选至少42个、优选15-120个、优选20-120个、优选30-100个、优选39-100个、优选42-80个碳原子的亚烷基二醇；和
所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和所述脂肪族二酸的羧基之一中去除；和
当所述连接基团Lin是具有60个碳原子的亲水性连接基团，且所述白蛋白结合残基是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
发明人通过大量实验，意外发现，本发明的胰岛素衍生物中的一定长度的白蛋白结合残基和一定长度的亲水性连接基团的组合，使得本发明的胰岛素衍生物相对于已有的胰岛素衍生物，在具有相当或更长的作用时间的同时，还具有预料不到的显著增加的药效、并且在白蛋白存在时，对胰岛素受体的结合能力受白蛋白的影响显著降低，对胰岛素受体具有显著提高的结合能力。
在一些实施方案中，所述胰岛素母体包含至少一个赖氨酸残基，所述白蛋白结合残基经由连接基团Lin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。
在一些实施方案中，所述胰岛素衍生物还包含一个或多个连接基团II，所述连接基团II是酸性的氨基酸残基，所述连接基团II连接在所述白蛋白结合残基与所述连接基团Lin之间、和/或连接在所述连接基团Lin和所述胰岛素母体之间；优选，所述连接基团II连接在所述白蛋白结合残基与所述连接基团Lin之间。
本发明的另一个方面，提供了胰岛素衍生物，其是酰化胰岛素，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0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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20-26个(优选20-24个)碳原子的脂肪酸或脂肪族二酸，其中在形式上羟基已从所述脂肪酸的羧基和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；
II和I在式(A)中出现的顺序可以独立地互换；
和
当m为1，n为10，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素；
或者
所述酰基部分如式(A’)所示：
III-(II) m-(I’) n’-  (A’)，
其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
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20-26个(优选20-24个)碳原子的脂肪酸或脂肪族二酸，其中在形式上羟基已从所述脂肪酸的羧基和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；
II和I’在式(A’)中出现的顺序可以独立地互换；和
(I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个；和
当m为1，(I’) n’的总碳原子数是60个，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
本发明的另一个方面，提供了胰岛素衍生物，其是酰化胰岛素，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0，1，2，3，4，5，6，7，8，9或10，n为5，6，7，8，9，10，11，12，13，14，15，16，17，18，19或20；
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20-26个(优选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；
II和I在式(A)中出现的顺序可以独立地互换；和
当m为1，n为10，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素；
或者
所述酰基部分如式(A’)所示：
III-(II) m-(I’) n’-  (A’)，
其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
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20-26个(优选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；
II和I’在式(A’)中出现的顺序可以独立地互换；和
(I’) n’的总碳原子数为20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个；和
当m为1，(I’) n’的总碳原子数是60个，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
在一些实施方案中，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20-26个(优选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。
在一些实施方案中，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-；或者，I’是HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-、-HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-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，优选地，II选自γGlu或βAsp；和/或
III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-、或HOOC-(CH 2) 24-CO-，优选，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-。
在一些实施方案中，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，或者式(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。
在一些实施方案中，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。
在一些实施方案中，所述胰岛素母体的赖氨酸残基位于B29位处。
在一些实施方案中，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:1和SEQ ID NO:2，分别表示A链和B链)；A14E,B16H,B25H,desB30人胰岛素(SEQ ID NO:3和SEQ ID NO:4，分别表示A链和B链)；A14E,B16E,B25H,desB30人胰岛素(SEQ ID NO:5和SEQ ID NO:6，分别表示A链和B链)；人胰岛素(SEQ ID NO:7和SEQ ID NO:8，分别表示A链和B链)；A21G人胰岛素(SEQ ID NO:9和SEQ ID NO:10，分别表示A链和B链)；A21G,desB30人胰岛素(SEQ ID NO:11和SEQ ID NO:12，分别表示A链和B链)；或B28D人胰岛素(SEQ ID NO:13和SEQ ID NO:14，分别表示A链和B链)；优选地，所述胰岛素母体为desB30人胰岛素；A14E,B16H,B25H,desB30人胰岛素；或A14E,B16E,B25H,desB30人胰岛素。
在一些实施方案中，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；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；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；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；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；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；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；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；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；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；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；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；或B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-13xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-13xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-14xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-14xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-15xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-15xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-16xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-16xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-17xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基 -γGlu-17xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-18xPEG),desB30人胰岛素；或B29K(N(ε)-二十烷二酰基-γGlu-18xPEG),desB30人胰岛素；
优选地，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；或B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素；
更优选地，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；或B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了胰岛素衍生物，其是酰化胰岛素，所述酰化胰岛素的胰岛素母体为A14E,B16H,B25H,desB30人胰岛素或A14E,B16E,B25H,desB30人胰岛素，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(C)所示：
Y1-(Y2) m1-(Y3) n1-  (C)，
其中,
m1为0，1，2，3，4，5，6，7，8，9或10，n1为5、6、7、8、9或 10的整数；
Y3是中性的、包含亚烷基二醇的氨基酸残基；
Y2是酸性的氨基酸残基；
Y1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；
Y1、Y2、和Y3之间以酰胺键连接；
Y2和Y3在式(C)中出现的顺序可以独立地互换；和
当Y1是包含20个碳原子的脂肪族二酸，且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时，且m1为1时，则n1不是10；
或者
所述酰基部分如式(C’)所示：
Y1-(Y2) m1-(Y3’) n1’-  (C’)，
其中,
m1为0，1，2，3，4，5，6，7，8，9或10，n1’为5、6、7、8、9或10的整数；
Y3’是中性的、包含亚烷基二醇的氨基酸残基；
Y2是酸性的氨基酸残基；
Y1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；
Y1、Y2、和Y3’之间以酰胺键连接；
Y2和Y3’在式(C’)中出现的顺序可以独立地互换；
(Y3’) n1’的总碳原子数为15-100个、优选20-100个、优选25-90个、优选30-80个、优选30-59个、优选30-54个；和
当Y1是包含20个碳原子的脂肪族二酸，且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时，且m1为1时，则(Y3’) n1’的总碳原子数不是60个。
在一些实施方案中，n1为5、6、7、8或9，优选地，n1为5、6、7、或8；和/或m1为1-6的整数，优选地，m1为1、2、3、或4，优选地，m1为1或2，优选地，m1为1；和/或
Y1是包含20-23个碳原子的脂肪族二酸，优选Y1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。
在一些实施方案中，Y3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Y3是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；或者，Y3’是-HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-；和/或
Y2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，Y2选自γGlu或βAsp；和/或
Y1是是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-、或HOOC-(CH 2) 24-CO-，优选，Y1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Y1是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
在一些实施方案中，式(C)通过Y3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，或者式(C’)通过Y3’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。
在一些实施方案中，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。
在一些实施方案中，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-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,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,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,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,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,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,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,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,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,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,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)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；
优选地，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-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)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；
优选地，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；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了胰岛素衍生物，其是酰化胰岛素，所述酰化胰岛素的胰岛素母体为A14E,B16H,B25H,desB30人胰岛素或A14E,B16E,B25H,desB30人胰岛素，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(D)所示：
W1-(W2) m2-(W3) n2-  (D)，
其中,
m2为0，1，2，3，4，5，6，7，8，9或10，n2为11，12，13，14，15，16，17，18，19或20；
W3是中性的、包含亚烷基二醇的氨基酸残基；
W2是酸性的氨基酸残基；
W1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；
W1、W2、和W3之间以酰胺键连接；和
W2和W3在式(D)中出现的顺序可以独立地互换；
或者
所述酰基部分如式(D’)所示：
W1-(W2) m2-(W3’) n2’-  (D’)，
其中,
m2为0，1，2，3，4，5，6，7，8，9或10，n2’为11，12，13，14，15，16，17，18，19或20；
W3’是中性的、包含亚烷基二醇的氨基酸残基；
W2是酸性的氨基酸残基；
W1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；
W1、W2、和W3’之间以酰胺键连接；和
W2和W3’在式(D’)中出现的顺序可以独立地互换；和
(W3’) n2’的总碳原子数为30-180个、42-180个、优选61-180个、优选66-180个、优选72-120个。
在一些实施方案中，n2为11、12、13、14、15、16、17、18或19，优选地，n2为11、12、13、14、15、16、17、或18，优选地，n2为11、12、13、14、15、或16，优选地，n2为11、12、13、14、或15；和/或
m2为1-6的整数，优选地，m2为1、2、3、或4，优选地，m2为1或2，优选地，m2为1；和/或
W1是包含20-23个碳原子的脂肪族二酸，优选W1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。
在一些实施方案中，W3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W3是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；或者，W3’是 -HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-CH 2-CO-；和/或
W2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，W2选自γGlu或βAsp；和/或
W1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W1是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
在一些实施方案中，式(D)通过W3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或者式(D’)通过W3’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。
在一些实施方案中，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。
在一些实施方案中，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-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,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-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,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-13xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-14xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-15xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-13xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-14xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-15xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基 -γGlu-17xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-19xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-20xOEG),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；
优选地，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-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；
优选地，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-12xOEG),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。
在一些实施方案中，所述酰化胰岛素选自下述胰岛素：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；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；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；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；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；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；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；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；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；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；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,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。
在本发明的另一个方面，提供了药物组合物，其包含权利要求1-25任一项所述的胰岛素衍生物或A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素、和一种或多种药学上可接受的赋形剂。
在一些实施方案中，所述药物组合物包含至少1.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少2.2摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少3.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少4.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含2.2-12摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-10摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-8摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-7.5摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-7.0摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-6.5摩尔锌离子/6摩尔的酰化的胰岛素；和/或
所述药物组合物具有的pH为6.5-8.5；优选pH为6.8-8.2；优选pH为7.0-8.2；优选pH为7.2-7.6；更优选pH为7.4或7.6。
在一些实施方案中，所述药物组合物进一步包含甘油、苯酚、间甲酚、NaCl、和/或Na 2HPO 4；优选，所述药物组合物进一步包含甘油、苯酚、和NaCl；优选，所述药物组合物进一步包含甘油、苯酚、间甲酚、和NaCl；优选，所述药物组合物进一步包含甘油、苯酚、NaCl和Na 2HPO 4；更优选，所述药物组合物进一步包含甘油、苯酚、间甲酚、NaCl和Na 2HPO 4。
在一些实施方案中，所述甘油的含量不超过约2.5％(重量/重量)，优选不超过约2％(重量/重量)，优选为约0.3％至约2％(重量/重量)，优选为约0.5％至约1.8％(重量/重量)，优选为约0.7％至约1.8％(重量/重量)，更优选为约1％至约1.8％(重量/重量)；和/或
所述苯酚的含量为约16至80mM，优选为约25-75mM，优选为约30-70mM，45-70mM，优选为约45-65mM；优选为约45mM、约46mM、约47mM、约48mM、约49mM、50mM、约51mM、约52mM、约53mM、约54mM、约55mM、约56mM、约57mM、约58mM、约59mM、约60mM、约61mM、约62mM、约63mM、约64mM、或约65mM；和/或
所述间甲酚的含量为约0-35mM,优选为约0-19mM,优选为约0-15mM，优选为约0mM、约1mM、约2mM、约3mM、约4mM、约5mM、约 6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、或约15mM；和/或
所述NaCl的含量为约0-150mM，优选为约5-120mM，优选为约10-120mM，优选为约10-100mM，更优选为约10-75mM，更优选为约10-50mM，更优选为约10-30mM；和/或
所述Na 2HPO 4的含量为约0-75mM，优选为约5-60mM，优选为低于约50mM，更优选为低于约25mM，更优选为低于约15mM；和/或
所述胰岛素衍生物的含量高于约0.3mM、优选高于约0.6mM、优选为约0.3-12mM、优选为约0.6-9.0mM、优选为约0.6-8.4mM、优选为约0.6-7.2mM、优选为约0.6-6.0mM、优选为约0.6-4.2mM、优选为约0.6-3.6mM、优选为约0.6-3.0mM、优选为约0.6-2.4mM、优选为约0.6-2.1mM、优选为约0.6-1.2mM。
在一些实施方案中，所述胰岛素衍生物为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；或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；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-6xOEG),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-10xOEG),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-12xOEG),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。
在本发明的另一个方面，提供了药物组合物，其包含约0.6-4.2mM的本发明上文所述的胰岛素衍生物、约1％至约1.8％(重量/重量)的甘油、约45-65mM的苯酚、约4.5-6.5摩尔锌离子/6摩尔的胰岛素衍生物、约10-120mM的氯化钠、约0-15mM间甲酚、和具有约7.0-8.2的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了药物组合物，其包含约0.6mM或1.2mM的本发明上文所述的胰岛素衍生物、1.7％(重量/重量)的甘油、约45mM的苯酚、约10mM的间甲酚、约6.5摩尔锌离子/6摩尔的胰岛素衍生物、约20mM的氯化钠、和具有约7.0-8.0的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了药物组合物，其包含约0.6-4.2mM的本发明上文所述的胰岛素衍生物、约1％至约2％(优选约1.5％-1.7％)(重量/重量)的甘油、约15mM-60mM(优选约30mM-60mM、更优选约45mM-60mM)的苯酚、约1.5-7.0(优选约2.2-4.5)摩尔锌离子/6摩尔的胰岛素衍生物、约 10-120mM(优选约20-50mM)的氯化钠、约0-25mM(优选约0-15mM、0-10mM)间甲酚、和具有约7.0-8.2的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了药物组合物，其包含约0.6mM-4.2mM的本发明上文所述的胰岛素衍生物、约1.5％-1.7％(重量/重量)的甘油、约45mM-60mM的苯酚、约0-10mM的间甲酚、约2.2-2.5摩尔锌离子/6摩尔的胰岛素衍生物、约20mM的氯化钠、和具有约7.0-8.0的pH值，其中所述胰岛素衍生物为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-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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在一些实施方案中，所述药物组合物还包含促胰岛素GLP-1化合物；优选地，所述药物组合物还包含选自下述的促胰岛素GLP-1化合物：N-ε 26-(17-羧基十七烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、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、N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(S)-羧基丁酰基氨基]乙氧 基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Aib8,Arg34]GLP-1-(7-37)肽、N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Gly8,Arg34]GLP-1-(7-37)肽。
在一些实施方案中，所述药物组合物还包含式(B)所示的促胰岛素GLP-1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：15)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、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。
在一些实施方案中，G1为[Gly8，Arg34]GLP-1-(7-37)肽(SEQ ID NO：16)或[Arg34]GLP-1-(7-37)肽(SEQ ID NO：17)，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，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。
在一些实施方案中，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-；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-。
在一些实施方案中，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。
在一些实施方案中，所述促胰岛素GLP-1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。
在一些实施方案中，所述促胰岛素GLP-1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、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；
优选地，所述促胰岛素GLP-1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。
发明人出乎意料地发现，本发明所述的胰岛素衍生物(例如酰化胰岛素)和促胰岛素GLP-1化合物的药物组合物或组合制剂不仅不会削弱所述胰岛素衍生物(例如酰化胰岛素)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，还使得所述胰岛素衍生物(例如酰化胰岛素)的化学稳定性增加。
在一些实施方案中，所述药物组合物还包含快速起效的胰岛素。
在一些实施方案中，所述快速起效的胰岛素选自Asp B28人胰岛素、Lys B28Pro B29人胰岛素、Lys B3Glu B29人胰岛素、人胰岛素和desB30人胰岛素中的一种或多种；优选，所述快速起效的胰岛素为Asp B28人胰岛素、Lys B28Pro B29 人胰岛素、Lys B3Glu B29人胰岛素、人胰岛素或desB30人胰岛素。
发明人出乎意料地发现，包含本发明的胰岛素衍生物(例如酰化胰岛素)和门冬胰岛素双胰岛素组分的药物组合物给药后，相比于包含德谷胰岛素和门冬胰岛素的双胰岛素组分的药物组合物，具有预料不到的增加的降糖药效，在本发明的胰岛素衍生物(例如酰化胰岛素)与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好或相当的降糖效果。
在一些实施方案中，所述胰岛素衍生物与所述快速起效的胰岛素的摩尔比为约60:3至约0.5:3、优选为约57:3至约1:3、优选为约55:3至约1.2:3、优选为约50:3至约1.5:3、优选为约40:3至约1.5:3、优选为约30:3至约1.5:3、优选为约27:3至约1.5:3、优选为约25:3至约1.5:3、优选为约22:3至约1.5:3、优选为约20:3至约1.5:3、优选为约17:3至约1.5:3、优选为约15:3至约1.5:3、优选为约12:3至约1.5:3、优选为约10:3至约1.5:3、优选为约9:3至约1.5:3、优选为约8:3至约1.5:3、优选为约7:3至约1.5:3、优选为约6.9:3至约1.5:3、优选为约6.8:3至约1.5:3、优选为约6.5:3至约1.5:3、优选为约6.3:3至约1.5:3、优选为约6:3至约1.5:3、优选为约5.8:3至约1.5:3、优选为约5.5:3至约1.5:3、优选为约5.3:3至约1.5:3、优选为约5:3至约1.5:3、优选为约4.8:3至约1.5:3、优选为约4.5:3至约1.5:3、优选为约4.2:3至约1.5:3、优选为约4:3至约1.5:3、优选为约3.9:3至约1.5:3、优选为约3.8:3至约1.5:3、优选为约3.5:3至约1.5:3、优选为约3.2:3至约1.5:3、优选为约3:3至约1.5:3、优选为约2.8:3至约1.5:3、优选为约2.5:3至约1.5:3、优选为约15:3至约2:3、优选为约12:3至约2:3、优选为约10:3至约2:3、优选为约9:3至约2:3、优选为约8:3至约2:3、优选为约7:3至约2:3、优选为约6.9:3至约2:3、优选为约6.8:3至约2:3、优选为约6.5:3至约2:3、优选为约6.3:3至约2:3、优选为约6:3至约2:3、优选为约5.8:3至约2:3、优选为约5.5:3至约2:3、优选为约5.3:3至约2:3、优选为约5:3至约2:3、优选为约4.8:3至约2:3、优选为约4.5:3至约2:3、优选为约4.2:3至约2:3、优选为约4:3至约2:3、优选为约3.9:3至约2:3、优选为约3.8:3至约2:3、优选为约3.5:3至约2:3、优选为约3.2:3至约2:3、优选为约3:3至约2:3、优选为约15:3至约2.4:3、优选为约12:3至约2.4:3、优选为约10:3至约2.4:3、优选为约9:3至约2.4:3、优选为约8:3至约2.4:3、优选为约7:3至约2.4:3、优选为约6.9:3至约2.4:3、优选为约6.8:3至约2.4:3、优选为约6.5:3 至约2.4:3、优选为约6.3:3至约2.4:3、优选为约6:3至约2.4:3、优选为约5.8:3至约2.4:3、优选为约5.5:3至约2.4:3、优选为约5.3:3至约2.4:3、优选为约5:3至约2.4:3、优选为约4.8:3至约2.4:3、优选为约4.5:3至约2.4:3、优选为约4.2:3至约2.4:3、优选为约4:3至约2.4:3、优选为约3.9:3至约2.4:3、优选为约3.8:3至约2.4:3、优选为约3.5:3至约2.4:3、优选为约3.2:3至约2.4:3、优选为约3:3至约2.4:3、更优选为约1.5:3、更优选为约2:3、更优选为约2.5:3、更优选为约2.75:3、更优选为约3:3。
在一些实施方案中，所述胰岛素衍生物为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；或B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在一些实施方案中，所述药物组合物包含约0.09-0.36mM的胰岛素衍生物、约0.18mM的Asp B28人胰岛素、约0.85％至约2.0％(重量/重量)的甘油、约15-70mM的苯酚、约8-14摩尔锌离子/6摩尔的胰岛素衍生物、约10-120mM的氯化钠、约0-15mM间甲酚、和具有约7.0-8.2的pH值，其中所述胰岛素衍生物为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)-二十二烷二酰基-γGlu-6xOEG), desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
在一些实施方案中，所述药物组合物包含约0.165-0.18mM的胰岛素衍生物、约0.18mM的Asp B28人胰岛素、约1.5％-1.7％(重量/重量)的甘油、约20mM-30mM的苯酚、约9-12摩尔锌离子/6摩尔的胰岛素衍生物、约20mM-75mM的氯化钠、约10mM-15mM间甲酚、和具有约7.0-8.2的pH值，其中所述胰岛素衍生物为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；或B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素。
在本发明的另一个方面，提供了本发明所述的胰岛素衍生物或所述的药物组合物，其用作药物。
在本发明的另一个方面，提供了本发明所述的胰岛素衍生物或所述的药物组合物，其用作用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱的药物。
在本发明的另一个方面，提供了本发明所述的胰岛素衍生物或所述的药物组合物，其用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱。
在本发明的另一个方面，提供了本发明所述的胰岛素衍生物或所述的药物组合物在制备药物中的用途；优选地，所述药物用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱。
在一些实施方案中，所述药物用于治疗糖尿病，所述胰岛素衍生物以每隔一天或更低频率给予同一患者，并且平均起来，在至少1个月、6个月或1年的时间期间，所述胰岛素衍生物不以更高频率给予同一患者。
在一些实施方案中，所述药物用于治疗糖尿病，所述胰岛素衍生物以一周两次或更低频率给予，并且平均起来，在至少1个月、6个月或1年的时间期间，所述酰化胰岛素不以更高频率给予同一患者。
本发明的另一个方面，提供了治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱的方法，包括施用治疗有效量的本发明如上所述的胰岛素衍生物或所述的药物组合物。
发明人意外发现，本发明的胰岛素衍生物(例如酰化胰岛素)具有长的药代动力学(下文也称PK)特征，使得对糖尿病患者一周两次、一周一次或更低频率的皮下治疗成为可能。
本发明的另一个方面，提供了使胰岛素衍生物在白蛋白存在时，对胰岛素受体结合能力提高的方法，所述方法包括：
经由连接基团Lin将白蛋白结合残基与天然存在的胰岛素或胰岛素类似物连接而得到所述胰岛素衍生物，其中所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基或所述脂肪族二酸的羧基之一中去除，
或者
用式(A)或式(A’)修饰天然存在的胰岛素或胰岛素类似物而得到所述衍生物，III-(II) m-(I) n-  (A)，其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
(A’)为III-(II) m-(I’) n’-  (A’)，
其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
II和I’在式(A’)中出现的顺序可以独立地互换；和
(I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个。
本发明的另一个方面，本发明提供了一种使胰岛素衍生物效力提高的方法，，所述方法包括：
经由连接基团Lin将白蛋白结合残基与天然存在的胰岛素或胰岛素类似物连接而得到所述胰岛素衍生物，其中所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基或所述脂肪族二酸的羧基之一中去除，
或者
用式(A)或式(A’)修饰天然存在的胰岛素或胰岛素类似物而得到所述衍生物，III-(II) m-(I) n-  (A)，其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
或者
III-(II) m-(I’) n’-  (A’)，其中,
m为0，1，2，3，4，5，6，7，8，9，或10，n’为大于等于5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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；和
(I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个。
在一些实施方案中，所述天然存在的胰岛素或胰岛素类似物包含至少一个赖氨酸残基，连接基团Lin、式(A)或式(A’)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。
在一些实施方案中，n为5-18的整数，优选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20-26个(优选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。
在一些实施方案中，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-；或者，I’是I-HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-、--HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-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，优选地，II选自γGlu或βAsp；和/或
III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-或HOOC-(CH 2) 24-CO-，优选地III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
在一些实施方案中，将式(A)通过I的C末端与所述天然胰岛素或胰岛素类似物的赖氨酸残基或N-末端氨基酸残基的氨基连接或将式(A’)通过I’的C末端与所述天然胰岛素或胰岛素类似物的赖氨酸残基或N-末端氨基酸残基的氨基连接。
在一些实施方案中，式(A)或式(A’)与所述胰岛素母体的赖氨酸残基的ε氨基相连接。
在一些实施方案中，所述天然胰岛素或胰岛素类似物的赖氨酸残基位于B29位处。
所述天然存在的胰岛素或胰岛素类似物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；优选地，所述胰岛素母体为desB30人胰岛素或A14E,B16H,B25H,desB30人胰岛素。
附图说明
图1a示出了本发明实施例1和2的化合物、德谷胰岛素、和溶媒(vehicle)对db/db小鼠的降糖效果。
图1b与图1a相对应地示出了本发明实施例1和2的化合物、德谷胰岛素和溶媒对db/db小鼠的降糖效果的AUC。
图2a示出了本发明实施例1和2的化合物、对照例2的化合物和溶媒对db/db小鼠的降糖效果。
图2b与图2a相对应地示出了本发明实施例1和2的化合物、对照例2的化合物和溶媒对db/db小鼠的降糖效果的AUC。
图3a示出了本发明实施例1-3的化合物、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1-3的化合物、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2的化合物、对照例3的化合物和溶媒对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2的化合物、对照例3的化合物和溶媒对db/db小鼠的降糖效果的AUC。
图5a示出了本发明对照例3-4的化合物、和溶媒对db/db小鼠的降糖效果和作用时间。
图5b与图5a相对应地示出了本发明对照例3-4的化合物、和溶媒对db/db小鼠的降糖效果的AUC。
图6a示出了本发明实施例2、实施例4-5的化合物和溶媒对db/db小鼠的降糖效果和作用时间。
图6b与图6a相对应地示出了本发明实施例2、实施例4-5的化合物和溶媒对db/db小鼠的降糖效果的AUC。
图7a示出了本发明实施例1的化合物、和溶媒对链脲佐菌素(STZ)诱导的I型糖尿病(T1DM)大鼠的降糖效果。
图7b与图7a相对应地示出了本发明实施例1的化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果的AUC。
图8a示出了本发明对照例5、实施例15和16的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果。
图8b与图8a相对应地示出了本发明对照例5、实施例15和16的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果的AUC。
图9a示出了本发明实施例2和4的化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)雌性大鼠的降糖效果。
图9b与图9a相对应地示出了本发明实施例2和4的化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)雌性大鼠的降糖效果的AUC。
图10a示出了本发明对照例5、实施例15和16的标题化合物、和溶媒对db/db小鼠的降糖效果。
图10b与图10a相应地示出了本发明对照例5、实施例15和16的标题化合物、和溶媒对db/db小鼠的降糖效果的AUC。
图11a示出了本发明对照例5、实施例17和18的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果。
图11b与图11a相应地示出了本发明对照例5、实施例17和18的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果的AUC。
图12a示出了本发明对照例5和实施例16的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果。
图12b与图12a相应地示出了本发明对照例5和实施例16的标题化合物、和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果的AUC。
图13a示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组 分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果。
图13b与图13a相应地示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果的AUC。
图14a示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果。
图14b与图14a相应地示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果的AUC。
图15a示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第四次给药前小鼠血糖。
图15b示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第八次给药前小鼠血糖。
图15c示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第十次给药前小鼠血糖。
图16a示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第四次给药后1小时小鼠血糖。
图16b示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第八次给药后1小时小鼠血糖。
图16c示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠第十次给药后1小时小鼠血糖。
图17示出了门冬胰岛素、包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降HbA1c效果。
图18a示出了本发明实施例4的化合物、德谷胰岛素和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果。
图18b与图18a相对应地示出了本发明实施例4的化合物、德谷胰岛素和溶媒对STZ诱导的I型糖尿病(T1DM)大鼠的降糖效果的AUC。
图19a示出了包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果。
图19b与图19a相应地示出了包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降糖效果的AUC。
图20示出了包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对STZ诱导的I型糖尿病(T1DM)C57/6J小鼠的降HbA1c效果。
图21a示出了包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒对db/db小鼠的降糖效果。
图21b与图21a相应地示出了包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分 的药物组合物、和溶媒对db/db小鼠的降糖效果的AUC。
图22a示出了注射包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒后db/db小鼠的随机血糖。
图22b与22a相应地示出了注射包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒后db/db小鼠的随机血糖的AUC。
图22c示出了注射包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒后db/db小鼠的禁食血糖。
图22d与22c相应地示出了注射包含德谷胰岛素和门冬胰岛素双胰岛素组分的药物组合物、包含本发明的酰化胰岛素和门冬胰岛素双胰岛素组分的药物组合物、和溶媒后db/db小鼠的禁食血糖的AUC。
图23示出了本发明实施例2的化合物和对照化合物2，分别在2％HSA和0％HSA存在时，受体结合能力的情况。
图24a示出了本发明实施例17的化合物和对照化合物5在进样浓度为12800nM，分别在2％HSA和0％HSA存在时，受体结合能力的情况。
图24b示出了本发明实施例17的化合物和对照化合物5在进样浓度为25600nM，分别在2％HSA和0％HSA存在时，受体结合能力的情况。
图25示出了本发明实施例41的化合物和对照化合物2分别在2％HSA和0％HSA存在时，受体结合能力的情况。
图26a示出了本发明实施例18的化合物、实施例42的化合物和对照化合物5在进样浓度为12800nM，分别在2％HSA和0％HSA存在时，受体结合能力的情况。
图26b示出了本发明实施例18的化合物、实施例42的化合物和对照化合物5在进样浓度为25600nM，分别在2％HSA和0％HSA存在时，受体结合能力的情况。
具体实施方式
定义
此处，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/或添加一个或多个氨基酸残基，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。添加和/或置换的氨基酸残基可以是可编码的氨基酸残基、或其它天然存在的氨基酸残基、或纯粹合成的氨基酸残基。优选地，添加和/或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胰岛素衍生物或酰化胰岛素的没有连接侧链或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。
术语“白蛋白结合残基”是指能与人血清白蛋白非共价结合的残基。与胰岛素连接的白蛋白结合残基通常对人血清白蛋白具有低于例如约10μM或甚至低于约1μM的结合亲和力。可通过以下文献所述的表面等离振子共振测量白蛋白结合性质：J.Biol.Chem.277(38)，35035-35042，(2002)。
本文中，“亲水性连接基团”是指包含至少6个非氢原子的化学部分(moiety)将胰岛素母体与白蛋白结合残基分开的连接基团，这些非氢原子中的30-50％是N或O。
“亲脂性”是指基团溶解于脂肪、油、脂质、以及亲脂性非极性溶剂(如己烷或甲苯)中的能力。亲脂性基团，包括但不限于例如脂肪、脂肪酸、脂肪二酸等，通常具有″脂质尾部″，存在于这些亲脂性基团中的脂质尾部可以为饱和的和不饱和的，取决于脂质尾部是否包含双键。脂质尾部也可包含不同长 度，例如具有7-12个碳之间的尾部(例如，C 7-12烷基或C 7-12烯基)、具有13-22个碳的尾部(例如，C 13-12烷基或C 13-12烯基)、或具有23-30个碳的尾部(例如，C 23-30烷基或C 23-30烯基)。
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。
本文中，快速起效的胰岛素包括快速起效的天然存在的胰岛素、胰岛素类似物和胰岛素衍生物。快速起效胰岛素通常在例如1至20分钟内开始发挥作用，在约一个小时后达到峰值，并且持续作用三至五小时。
术语“基础胰岛素”意指具有比常态或正常人胰岛素更长的作用持续时间的胰岛素。
本文中，术语“化学稳定性”是指，从化学上，本发明的胰岛素衍生物在所需制剂中足够稳定。也就是，仅形成不损害最终药物产物的保质期的量的化学降解产物。化学降解产物包括脱酰胺产物、异天冬氨酸酯形成、二聚体形成、外消旋化产物、从脱水过程等产生的产物。化学稳定性可通过老化样品或制剂的HPLC分析测定。
本文中，“对胰岛素受体的结合能力”是指，胰岛素与胰岛素受体之间的相互作用，这种相互作用的大小或强度可以用例如表面等离子共振(SPR)进行测定。例如，在用SPR测定时，当含有胰岛素的溶液流过包被有胰岛素受体的芯片时，胰岛素与胰岛素受体所产生的相互作用会导致SPR偏转角度发生 变化，这种变化通常用相对响应值来体现，通常相对响应值越大，表明对胰岛素受体的结合能力越高。
高物理稳定性意指原纤维化倾向小于人胰岛素原纤维化倾向的50％。原纤维化可以通过在给定条件下原纤维开始形成之前的滞后时间来描述。
具有胰岛素受体和IGF-1受体亲和力的多肽是在合适的结合测定中能够与胰岛素受体和人IGF-1受体相互作用的多肽。此种受体测定是本领域众所周知的。
本文中，“药效”或“效力”是指药物或活性化合物产生某种作用或效果(例如降低血糖)的能力。例如，施用同剂量的本发明的胰岛素衍生物，相对于德谷胰岛素或其它已有的胰岛素衍生物，会产生更高的降低血糖的效果或作用。
术语“糖尿病”包括1型糖尿病、2型糖尿病、妊娠糖尿病(在妊娠期间)和引起高血糖症的其它状态。该术语用于代谢紊乱，其中胰腺产生不足量的胰岛素，或其中身体的细胞不能适当地响应胰岛素，从而阻止细胞吸收葡萄糖。结果，葡萄糖在血液中聚集。
1型糖尿病，亦称为胰岛素依赖性糖尿病(IDDM)和青少年型糖尿病，由β-细胞破坏引起，通常导致绝对胰岛素缺乏。2型糖尿病，亦称为非胰岛素依赖性糖尿病(NIDDM)和成年型糖尿病，与主要的胰岛素抵抗和由此相对胰岛素缺乏和/或具有胰岛素抵抗的主要胰岛素分泌缺陷有关。
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：15所示。具有SEQ ID NO：15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：15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： 15)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：15)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：16和SEQ ID NO：17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。
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 PCTCN2020141056-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”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-[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]、或[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]。
例如，本发明对照例2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-2xOEG),desB30人胰岛素”、“B29K(N ε-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素”、或“B29K(N ε-二十烷二酰基-gGlu-2xOEG)，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N ε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(N ε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N ε)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 PCTCN2020141056-appb-000002
本中，“nxPEG”表示基团-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 Symposiμm(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 PCTCN2020141056-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。
术语"赋形剂"在广义上是指除了活性治疗成分之外的任何组分。赋形剂可以是惰性物质、无活性物质和/或无药用活性物质。
赋形剂可用于各种目的，这取决于药物组合物，例如作为载体、溶媒、稀释剂、片剂助剂，和/或用于改善活性物质的给予和/或吸收。赋形剂的实例包括但不限于稀释剂、缓冲剂、防腐剂、张力调节剂(亦称为张度剂或等渗剂)、螯合剂、表面活性剂、蛋白酶抑制剂、湿润剂、乳化剂、抗氧化剂、填充剂、金属离子、油性溶媒、蛋白质和/或两性离子和稳定剂。
药学活性成分与各种赋形剂的药物组合物是本领域已知的，参见例如Remington:The Science and Practice of Pharmacy(例如第19版(1995)和任何后来的版本)。
为患者的便利，假定患者偏好从给予本发明的酰化胰岛素至下一次给予本发明的酰化胰岛素的时间间隔(时间延迟)具有相同的长度或近似相同的长 度，以天数计。甚至可预期，患者将偏好给予酰化胰岛素每周发生一次，即在一周的同一天，例如每个星期天。在1个月、6个月或1年的时间内按平均计算，这将是每隔6天和不以更高频率给予酰化胰岛素。对于一些患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔5天或近似每隔5天和不以更高频率给予酰化胰岛素。对于其它患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔4天或近似每隔4天和不以更高频率给予酰化胰岛素。对于其它患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔3天或近似每隔3天和不以更高频率给予酰化胰岛素。甚至其它患者可发现有利的是，在1个月、6个月或1年的时间内按平均计算，按每周两次，例如在各次给予之间以大约3-4天的间隔给予酰化胰岛素。对于一些患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔2天或近似每隔2天和不以更高频率给予酰化胰岛素。对于其它患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔1天或近似每隔1天和不以更高频率给予酰化胰岛素。对于一些患者，在1个月、6个月或1年的时间内按平均计算，可能需要每隔7天或近似每隔7天和不以更高频率给予酰化胰岛素。甚至其它患者可能在每周、每月或每年，不以准确相同长度的时间间隔(以天计)给予酰化胰岛素。在1个月、6个月或1年的时间内按平均计算，一些患者有时可以每隔5天至每隔7天的时间间隔和不以更高频率给予酰化胰岛素。在1个月、6个月或1年的时间内按平均计算，其它患者有时可以每隔4天至每隔6天的时间间隔和不以更高频率给予酰化胰岛素。在1个月、6个月或1年的时间内按平均计算，甚至其它患者有时可以每隔3天至每隔7天的时间间隔和不以更高频率给予酰化胰岛素。
作为本发明的主要靶标的疾病和病况是糖尿病(1型或2型)或特征为高血糖症的其它病况，但还是大体上其中胰岛素的代谢作用具有临床相关性或具有益处的代谢疾病和病况，例如前糖尿病、葡萄糖耐量受损、代谢综合征、肥胖、恶病质、体内β-细胞受损/死亡、食欲过盛和炎症。已知或认为所有这些类型的病况获益于患有所述疾病或病况的受试者的稳定代谢状态。无论如何，其中包括给予胰岛素的任何治疗方案可通过实施本发明的教导而改变，意思是这样的疗法将包括给予根据本文提供的延长作用的胰岛素。
实施例
以示例而非限制地提供下列实施例。
本文中所用的缩写如下：
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；
CH 3CN是乙腈；
Gly是甘氨酸；
Arg是精氨酸；
TFA是三氟乙酸；
HbA1c是糖化血红蛋白；
AUC是时间-血糖曲线的曲线下面积；
RU:响应单位(Response Unit)。
以下实施例和通用方法针对说明书和合成方案中确定的中间化合物和最终产物。采用以下实施例对本发明化合物的制备进行详细描述，但描述的化学反应根据它们对本发明化合物制备的一般适用性来公开。有时，所述反应可能不能适用于如所述的本发明范围内的每种化合物。本领域技术人员容易识别会发生这种情况的化合物。在这些情况下，所述反应可成功地通过本领域技术人员已知的常规改进进行，也就是说，通过合适保护干扰基团，通过改变为其它常规试剂，或通过反应条件的常规改进。在所有制备方法中，所有原料都是已知的或能容易地采用已知原料制备。所有温度都以摄氏度给出，除非另外明确指出，当提到产率时，所有份和百分比都以重量计，当提到溶剂和洗脱液时，所有份都以体积计。
实施例1
B29K(N(ε)-二十烷二酰基-γGlu-5xOEG),desB30人胰岛素(化合物1)
Figure PCTCN2020141056-appb-000004
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11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pH10-12.5。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，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、得到标题化合物1。
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)，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) +
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10min，NHS(5.38g，50.17mmo1)，和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) +
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) +
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洗涤，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2：
B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物2)
Figure PCTCN2020141056-appb-000005
以与实施例1第2部分类似的步骤制备化合物2。
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1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1551.90(M+1) +
实施例3：
B29K(N(ε)-二十烷二酰基-γGlu-8xOEG),desB30人胰岛素(化合物3)
Figure PCTCN2020141056-appb-000006
以与实施例1第2部分类似的步骤制备化合物3。
LC-MS(电喷雾)：m/z＝1464.30[M+5H] 5+
中间体 叔丁基二十烷二酰-γGlu-(8xOEG-OSu)-OtBu以与实施例1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1814.02(M+1) +
实施例4：
B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物4)
Figure PCTCN2020141056-appb-000007
以与实施例1第2部分类似的步骤制备化合物4。
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1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1579.94(M+1) +
实施例5：
B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素(化合物5)
Figure PCTCN2020141056-appb-000008
以与实施例1第2部分类似的步骤制备化合物5。
LC-MS(电喷雾)：m/z＝1469.91[M+5H] 5+
中间体 叔丁基二十二烷二酰-γGlu-(8xOEG-OSu)-OtBu的制备以与实施例1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1870.08(M+1) +
对照例1
B29K(N(ε)-十六烷二酰基-γGlu),desB30人胰岛素(德谷胰岛素，对照化合物1)
对照化合物德谷胰岛素，按照专利CN105820233A的实施例4进行制备。
对照例2
B29K(N(ε)-二十烷二酰基-γGlu-2xOEG),desB30人胰岛素(对照化合物2)
Figure PCTCN2020141056-appb-000009
以与实施例1第2部分类似的步骤制备对照化合物2。
LC-MS(电喷雾)：m/z＝1290.22[M+5H] 5+
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。
LC-MS(Scie×100API)：m/z＝971.61(M+1) +
对照例3
B29K(N(ε)-十八烷二酰基-γGlu-2xOEG),desB30人胰岛素(对照化合物3)
Figure PCTCN2020141056-appb-000010
以与实施例1第2部分类似的步骤制备对照化合物3。
LC-MS(电喷雾)：m/z＝1284.61[M+5H] 5+
对照例4
B29K(N(ε)-十八烷二酰基-γGlu-6xOEG),desB30人胰岛素(对照化合物4)
Figure PCTCN2020141056-appb-000011
以与实施例1第2部分类似的步骤制备对照化合物4。
LC-MS(电喷雾)：m/z＝1400.68[M+5H] 5+
实施例6
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酰化胰岛素对血糖(BG)的调节效应。
在肥胖的糖尿病小鼠模型(db/db小鼠)上，在单剂量研究中测试实施例1-5的酰化胰岛素和对照例1-4的对照化合物。以9U/kg或10U/kg的不同剂量测试所述酰化胰岛素的降低血糖效果。
将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-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，或皮下注射酰化胰岛素9U/kg或10U/kg，其中溶媒包含：甘油19.6mg/ml、苯酚1.5mg/ml，间甲酚1.72mg/ml，锌离子浓度55μg/ml，pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至1.8或2U/ml的给药浓度，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药一次。在大约上午10:30(时间0)给予酰化胰岛素，给药期间动物禁食不禁水，评估给药后3、6、9、12、15小时小鼠血糖。为模拟进餐，试验中在检测完15小时点血糖后，开始口服糖耐量实验(OGTT)，于灌胃给予葡萄糖溶液(100mg/mL，10mL/kg)后30min、60min、120min和180min测定血糖，连续开展OGTT实验三次.根据预实验结果，末次OGTT实验时，受试化合物药效接近消失，评估30小时血糖后终止。
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。对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。
为了说明本发明的酰化胰岛素对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
试验结果：
本发明的酰化胰岛素及对照化合物在db/db小鼠中的降糖效果如图1a至图6b、及表1所示，具体地：
图1a和图1b显示了本发明的酰化胰岛素例如化合物1、和化合物2对db/db小鼠的降糖效果明显优于德谷胰岛素，且有效作用时间相比于德谷胰岛素有延长。
图2a和图2b显示了本发明的酰化胰岛素例如化合物1和化合物2对db/db小鼠的降糖效果也明显优于对照化合物2，本发明的化合物1和化合物2相对于对照化合物2药效在给药的0～16.5小时区间内分别增加了39.5％和45.1％，如表1所示：
表1本发明的酰化胰岛素相对于对照化合物2增加的药效
化合物/对照化合物 实施例 相对于对照化合物2的药效增加的百分比
化合物1 实施例1 39.5％
化合物2 实施例2 45.1％
对照化合物2 对照例2 0％
相对于对照化合物2的药效增加的百分比＝[(AUC(测试化合物)-AUC(溶媒))/((AUC(对照化合物2)-AUC(溶媒))-1]*100％，其中，测试化合物是指本发明的酰化胰岛素
图3a-3b显示了本发明的化合物1、化合物2和化合物3均具有非常好的药效，且在db/db小鼠中，监测至30小时时仍然有效，具有明显延长的降糖作用时间。
图4a-5b显示了本发明的酰化胰岛素例如化合物2对db/db小鼠的降糖效果明显优于对照化合物3和对照化合物4。
图6a-6b显示了本发明的化合物4、化合物5和化合物2均具有非常好的药效，且在db/db小鼠中，监测至41小时时仍然有效，具有明显延长的降糖作用时间。
实施例7
在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)大鼠中的药效学研究
8w周龄雄性wistar大鼠，体重180-220g。于屏障环境内饲养于合适规格的饲养盒中(5只/盒)，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℃。4天适应期后，禁食12h，大鼠按照60mg/kg腹腔注射链脲佐菌霉素(sigma)溶液(10mg/mL，于0.1M柠檬酸缓冲液)。给药后防止大鼠突发血糖过低，饮水中适当补充葡萄糖(20％)，12h后撤去补糖。给予链脲佐菌霉素4d后，进行随机血糖检测， 血糖值在20mmol/L以上的选为T1DM模型大鼠用于后续试验。
于当日实验开始前，在时间-1/1h(上午9:30)评估基础血糖，并对大鼠称重。根据随机血糖和体重将大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，或皮下注射酰化胰岛素，3U/kg，其中溶媒包含：甘油19.6mg/ml，苯酚1.5mg/ml，间甲酚1.72mg/ml，锌离子浓度55μg/ml，所述溶媒的pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至1.5U/ml的给药浓度，给药体积为2ml/kg(即0.2ml/100g体重)。采用皮下给药方式，颈背部皮下给药一次。在大约上午9:30(时间0)给予酰化胰岛素，在给药后2、4小时评估大鼠血糖。4小时和7小时分别进行一次口服糖耐量实验(OGTT)实验(具体见下文)。
口服糖耐量实验(OGTT)
检测时间：于指定时间点尾尖采血测定空腹血糖(0min)，之后灌胃给予葡萄糖溶液(100mg/mL或200mg/mL，10mL/kg)，然后于糖负荷后30min、60min、120min和180min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。
对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。
图7a-7b显示了本发明的酰化胰岛素在I型糖尿病(T1DM)大鼠中也具有非常好的降糖效果，即具有非常好的药效。
实施例8
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6)
Figure PCTCN2020141056-appb-000012
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pH10-12.5。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9
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7)
Figure PCTCN2020141056-appb-000013
以与实施例8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8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10
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8)
Figure PCTCN2020141056-appb-000014
以与实施例8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8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11
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9)
Figure PCTCN2020141056-appb-000015
以与实施例8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8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12
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(化合物10)
Figure PCTCN2020141056-appb-000016
以与实施例8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+
实施例13
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(化合物11)
Figure PCTCN2020141056-appb-000017
以与实施例8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8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14：
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12)
Figure PCTCN2020141056-appb-000018
以与实施例8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8第2部分类似的步骤进行制备。
LC-MS(Scie×100API)：m/z＝999.64(M+1) +
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 PCTCN2020141056-appb-000019
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 PCTCN2020141056-appb-000020
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pH10-12.5。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15
A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-6xOEG),desB30人胰岛素(化合物13)
Figure PCTCN2020141056-appb-000021
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16
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30 人胰岛素(化合物14)
Figure PCTCN2020141056-appb-000022
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17
A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物15)
Figure PCTCN2020141056-appb-000023
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18
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物16)
Figure PCTCN2020141056-appb-000024
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19
参照实施例7类似的实验步骤，在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)大鼠中进行药效学研究。
于当日实验开始前，在时间-1h(上午9:30)评估基础血糖，并对大鼠称重。根据随机血糖和体重将大鼠分配到溶媒组或治疗组，接受如下处理：皮下注射溶媒，或皮下分别注射对照例5、实施例15和16的标题化合物(对照化合物5、化合物13和化合物14)，剂量：33.5U/kg，其中溶媒包含：苯酚5.65mg/ml,甘油15mg/ml，磷酸氢二钠0.708mg/ml，氯化钠0.585mg/ml，所述溶媒的pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至33.5U/ml给药浓度，给药体积为1ml/kg(即0.1ml/100g体重)。采用皮下给药方式(s.c.)，颈背部皮下给药一次。在大约上午9:30-10:00(时间0)给予酰化胰岛素，在给药后3h、6h、9h、24h、48h、72h、96h、120h监测大鼠血糖。
对于每个单剂量的酰化胰岛素(对照化合物5、化合物14、化合物13)绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图8a-图8b显示了本发明的酰化胰岛素具有预料不到的增加的药效，例如，相对于对照例5的化合物，实施例15和16的标题化合物化合物13和化合物14对STZ诱导I型糖尿病(T1DM)大鼠均具有更好的降糖效果，即具有更好的药效。
实施例20
参照实施例19的类似的实验步骤，在链脲佐菌霉素(STZ)诱导的I型糖尿病(T1DM)雌性大鼠中进行药效学研究，不同之处在于，所用的酰化胰岛素是实施例2和4的标题化合物(化合物2和化合物4)，给药剂量为67U/kg。
实验结果如图9a-图9b所示，显示了本发明的酰化胰岛素化合物2和化合物4在I型糖尿病(T1DM)雌性大鼠中也均具有非常好的降糖效果，即具有非常好的药效。
实施例21
在db/db小鼠中的药效学研究
参照实施例6的类似的实验步骤，在肥胖的糖尿病小鼠模型(db/db小鼠)上，在单剂量研究中测试对照例5、及实施例15和16的标题化合物(即，对照化合物5、化合物13和化合物14)。以9U/kg的剂量测试所述酰化胰岛素的降低血糖效果。
将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-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，或皮下注射酰化胰岛素9U/kg，其中所用溶媒包含：苯酚5.65mg/ml,甘油15mg/ml，磷酸氢二钠0.708mg/ml，氯化钠0.585mg/ml，所述溶媒的pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至1.8U/ml的给药浓度，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药一次。在大约上午10:30(时间0)给予酰化胰岛素，给药期间动物禁食不禁水，评估给药后3h、6h、9h、21.5h小鼠血糖。为模拟进餐，试验中在检测完21.5h 点血糖后，开始口服糖耐量实验(OGTT)，于灌胃给予葡萄糖溶液(100mg/mL，7.5mL/kg)后30min、60min、120min和360min测定血糖；首次OGTT测完330min血糖后开始第二次OGTT实验，于灌胃给予葡萄糖溶液(50mg/mL，10mL/kg)后30min、90min、210min和360min测定血糖。第二次OGTT测完360min血糖后开始第三次OGTT实验，于灌胃给予葡萄糖溶液(50mg/mL，10mL/kg)后30min、60min、120min测定血糖，末次OGTT实验时，受试化合物药效尚未消失，评估36小时血糖后终止实验。
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。对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图10a-图10b显示，相对于对照化合物5，本发明的酰化胰岛素化合物14、化合物13在Ⅱ型糖尿病db/db小鼠中具有明显提高的降糖效果。
实施例22
在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)大鼠中的药效学研究
8周龄SD大鼠(雌雄各半)，体重180-220g。于屏障环境内饲养于合适规格的饲养盒中(5只/盒)，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℃。4天适应期后，禁食12h，大鼠按照60mg/kg腹腔注射链脲佐菌霉素(sigma)溶液(10mg/mL，于0.1M柠檬酸缓冲液)。给予链脲佐菌霉素3天后，进行随机血糖检测，血糖值在20mmol/L以上的选为T1DM模型大鼠用于后续试验。
造模14天后开始试验，于当日实验开始前，在时间-1/1h(上午9:30)评估基础血糖，并对大鼠称重。根据随机血糖和体重将大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，或皮下分别注射对照例5、实施例17和18的标题化合物(即对照化合物5、化合物15、化合物16)，剂量为25U/kg，其中溶媒包含：苯酚5.65mg/ml,甘油15mg/ml，磷酸氢二钠0.708mg/ml，氯化钠0.585mg/ml，所述溶媒的pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至25U/ml的给药浓度，给药体积为1ml/kg(即0.1ml/100g体重)。采用皮下给药方式，颈背部皮下给药，重复 给药4次，每次间隔4天，试验期间SD大鼠自由饮食。在大约上午9:30-10:00(时间0)给予酰化胰岛素，在首次给药3h、6h、9h、24h、48h、72h、96h后监测大鼠血糖，之后每次给药后6h以及每24h监测一次大鼠血糖。
对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。
表2本发明的酰化胰岛素相对于对照化合物5增加的药效
化合物/对照化合物 实施例 相对于对照化合物5的药效增加的百分比
化合物15 实施例17 125％
化合物16 实施例18 142％
对照化合物5 对照例5 0％
相对于对照化合物5的药效增加的百分比＝[(AUC(测试化合物)-AUC(溶媒))/((AUC(对照化合物5)-AUC(溶媒))-1]*100％，其中，测试化合物是指本发明的酰化胰岛素
如图11a-11b、表2所显示，相对于对照化合物5，本发明的酰化胰岛素给药后在I型糖尿病(T1DM)大鼠中有预料不到的增加的降糖药效，化合物15和化合物16的降糖效果均明显优于对照化合物5。
实施例23
在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)大鼠中的药效学研究
8周龄SD大鼠(雌雄各半)，体重180-220g。于屏障环境内饲养于合适规格的饲养盒中(5只/盒)，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℃。4天适应期后，禁食12h，大鼠按照60mg/kg腹腔注射链脲佐菌霉素(sigma)溶液(10mg/mL，于0.1M柠檬酸缓冲液)。给予链脲佐菌霉素3天后，进行随机血糖检测，血糖值在20mmol/L以上的选为T1DM模型大鼠用于后续试验。
造模14天后开始试验，于当日实验开始前，在时间-1/1h(上午9:30)评估基础血糖，并对大鼠称重。根据随机血糖和体重将大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，或皮下分别注射对照例5、实施例16的标题化合物(即对照化合物5、化合物14)，剂量为25U/kg，其中溶媒包含：苯酚5.65mg/ml,甘油15mg/ml，磷酸氢二钠0.708mg/ml， 氯化钠0.585mg/ml，所述溶媒的pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至25U/ml的给药浓度，给药体积为1ml/kg(即0.1ml/100g体重)。采用皮下给药方式，颈背部皮下给药，重复给药4次，每次间隔4天，试验期间SD大鼠自由饮食。在大约上午9:30-10:00(时间0)给予酰化胰岛素，在首次给药3h、6h、9h、24h、48h、72h、96h后监测大鼠血糖，之后每次给药后6h以及每24h监测一次大鼠血糖。
对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。
如图12a-12b、表2所显示，相对于对照化合物5，本发明的酰化胰岛素给药后在I型糖尿病(T1DM)大鼠中有预料不到的增加的降糖药效，化合物14的降糖效果明显优于对照化合物5。
实施例24
在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)C57/6J小鼠中的药效学研究
本研究的目的在于证实包含本发明的酰化胰岛素与门冬胰岛素的组合物在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)C57/6J小鼠中对血糖(BG)的调节效应。
将4-6周龄雄性C57/6J小鼠(购自维通利华)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40％-60％，温度为22℃-24℃。1-2周的适应期后，开始用于实验。
适应期过后，动物禁食12小时，小鼠按照150mg/kg腹腔注射链脲佐菌霉素(sigma)溶液(10mg/mL，于0.1M柠檬酸缓冲液)。给予链脲佐菌霉素3天后，进行随机血糖检测，血糖值在20mmol/L以上的选为T1DM模型小鼠用于后续试验。
当日实验开始前，检测小鼠随机血糖，并对小鼠称重。根据随机血糖和体重将小鼠分配到溶媒组或治疗组，共5组，每组8只，各组分别接受如下处理：皮下注射溶媒，或皮下注射门冬胰岛素(0.36U/kg)、或皮下注射包含德谷胰岛素和门冬胰岛素的药物组合物，其中德谷胰岛素和门冬胰岛素的注射剂量分别为0.84U/kg和0.36U/kg，或皮下注射包含本发明实施 例4的标题化合物化合物4和门冬胰岛素的两种药物组合物，其中在注射所述两种药物组合物时，化合物4的注射剂量分别为0.82U/kg和0.64U/kg，门冬胰岛素的注射剂量均为0.36U/kg，其中所述溶媒包含：甘油19.6mg/ml、苯酚1.5mg/ml，间甲酚1.72mg/ml，锌离子浓度55μg/ml，上述溶媒的pH值为7.6。
将化合物4与门冬胰岛素的预混液溶解在溶媒中至0.072U/mL给药浓度(以预混液中门冬胰岛素浓度计)，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药一次。在大约下午16:00(时间0)给予药物，给药期间动物禁食不禁水，评估给药后0.5、1、2、3、6和15时小鼠血糖。
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。并绘制血糖对时间的剂量响应曲线。为了说明本发明的预混胰岛素对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图13a-13b显示，包含本发明的酰化胰岛素和门冬胰岛素的药物组合物给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在I型糖尿病(T1DM)小鼠中有预料不到的增加的降糖药效，在化合物4与门冬胰岛素的用量比小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好或相当的降糖效果。
实施例25
以与实施例24相似的步骤，测试包含本发明的酰化胰岛素与门冬胰岛素的组合物在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)C57/6J小鼠中对血糖(BG)的调节效应。
当日实验开始前，检测小鼠随机血糖，并对小鼠称重。根据随机血糖和体重将小鼠分配到溶媒组或治疗组，共7组，每组8只，各组分别接受如下处理：皮下注射溶媒，或皮下注射门冬胰岛素(3U/kg)、或皮下注射包含德谷胰岛素和门冬胰岛素的药物组合物，其中德谷胰岛素和门冬胰岛素的注射剂量分别为7U/kg和3U/kg，或皮下注射包含化合物4和门冬胰岛素的四种药物组合物，其中在注射所述四种药物组合物时，化合物4的注射剂量分别为6.79U/kg、5.34U/kg、3.84U/kg和2.39U/kg，门冬胰岛素 的注射剂量均为3U/kg，其中所述溶媒包含：甘油19.6mg/ml、苯酚1.5mg/ml，间甲酚1.72mg/ml，锌离子浓度55μg/ml，上述溶媒的pH值为7.6。
将化合物4与门冬胰岛素预混液溶解在溶媒中至0.6U/mL给药浓度(以预混液中门冬胰岛素浓度计)，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药。在每天下午大约17:00(时间0)给予药物，连续给药10天，给药期间小鼠自由饮食，评估第四、八、十次给药前(0h)小鼠血糖，以及第四、八、十次给药后1小时小鼠随机血糖，对第八次给药前(0h)血糖及给药后0.5、1、1.5、2、3、4、5、6、16、24小时随机血糖进行检测。最后一次给药后小鼠禁食1h，眼眶取血，检测全血中糖化血红蛋白(Hb1Ac)百分比。
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。并绘制第八次给药后血糖对时间的剂量响应曲线。为了说明本发明的预混胰岛素对血糖的影响，对第八次给药后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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图14a-图17显示了包含本发明的酰化胰岛素和门冬胰岛素的组合物给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在I型糖尿病(T1DM)小鼠中有预料不到的增加的降糖药效，具有更优异的降糖累计效果。
具体地，图14a和图14b显示，包含本发明的酰化胰岛素和门冬胰岛素的组合物，在给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在I型糖尿病(T1DM)小鼠中有预料不到的增加的降糖药效，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好的降糖效果。
图15a-图15c分别显示了在第四、八、十次给药前(0h)各个给药组小鼠血糖情况，表明相对于包含德谷胰岛素和门冬胰岛素的药物组合物，包含本发明的酰化胰岛素和门冬胰岛素的药物组合物具有更好的药效，和更优异的降血糖的累积效果。
图16a-图16c分别显示了在第四、八、十次给药后1小时小鼠的血糖情况，表明相对于包含德谷胰岛素和门冬胰岛素的药物组合物，包含本发 明的酰化胰岛素和门冬胰岛素的药物组合物具有更好的药效，和更优异的降血糖的累积效果。
图17显示包含本发明的酰化胰岛素和门冬胰岛素的组合物，在给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，具有更好地降低Hb1Ac的效果，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好地降低Hb1Ac的效果。
实施例26
本实验的目的在于测量本发明的酰化胰岛素制剂的化学稳定性。
酰化胰岛素制剂
将化合物4溶解于0.1％NaOH溶液中，至4.8mM的终浓度(pH值约为10-11)，根据下表中各个组分的量，依次加入苯酚、间甲酚、醋酸锌、甘油和氯化钠，产生最终胰岛素浓度为1.2mM(200U/ml或8.46mg/ml)的酰化胰岛素制剂，其中Zn的含量以Zn/6摩尔的酰化的胰岛素(简写为“Zn/6ins”)来表示。
本实施例中制剂的化学稳定性能够用在25℃和37℃存储14天和20天后，高分子量蛋白(HMWP)相对于第0天时的变化来显示，同时也能够用在25℃和37℃存储14天和20天后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，在型号和规格为：Waters Xbride BEH 200A(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600ml的0.1％精氨酸溶液、150ml冰醋酸和250ml乙腈。检测波长为276nm，进样量为10ul。表3示出了，在25℃和37℃时，第14天和第20天相对于第0天时HMWP的增加量。
表3
Figure PCTCN2020141056-appb-000025
Figure PCTCN2020141056-appb-000026
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量随着时间的变化增加非常缓慢，表明上述酰化胰岛素制剂均具有优异的化学稳定性。特别是，当Zn含量为6.5Zn/6ins时，相对于5.5Zn/6ins时，HMWP的量增加更为缓慢。
有关物质的量的测定
通过高效液相色谱法(HPLC)测定胰岛素有关物质的含量，在Waters Kromasil 300A-5μm-C8(4.6*250mm)柱上，在柱温为40℃、样品池温度为室温时，用洗脱相以1.0ml/min的流速进行测试。洗脱相由以下组成的流动相进行：
A相包含0.1M无水硫酸钠、0.1M二水合磷酸二氢钠、10％乙腈(v/v)，用NaOH调节pH值至为5.0；
B相为50％乙腈(v/v)。
梯度：0-45min的45％/55％A/B至35％/65％A/B的线性变化，45-50min至20％/80％A/B的线性变化，50-60min至20％/80％A/B的等度梯度，60-60.1min的45％/55％A/B的线性变化，60.01-70min至45％/55％A/B的等度梯度。
表4示出了，在37℃时，第14天和第20天相对于第0天时有关物质的增加量。
表4
Figure PCTCN2020141056-appb-000027
由上表可知，本发明的上述酰化胰岛素制剂中有关物质的量随着时间的变化增加也非常缓慢，表明上述酰化胰岛素制剂非常稳定。
实施例27
本实验的目的是测量本发明的酰化胰岛素制剂的化学稳定性。根据下述 表5-7中各组分的量，按照与实施例26类似地步骤，配制表5-7中的酰化胰岛素制剂。并按照与实施例26类似的步骤测定HMWP和有关物质的变化。下表5-7示出了不同配方的酰化胰岛素制剂的HMWP和有关物质的变化。
表5
Figure PCTCN2020141056-appb-000028
表6
Figure PCTCN2020141056-appb-000029
表7
Figure PCTCN2020141056-appb-000030
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量、及有关物质的量随着时间的变化均增加较缓慢，特别是在Zn离子含量增加或添加Na 2HPO 4时，HMWP的量、及有关物质的量增加更为缓慢，表明本发明所获得的酰化胰岛素制剂均具有良好的化学稳定性。
实施例28
本实验的目的是测量本发明的酰化胰岛素制剂的化学稳定性。根据下述表8中各组分的量，按照与实施例26类似地步骤，配制表8中的酰化胰岛素制剂。并按照与实施例26类似的步骤测定HMWP和有关物质的变化。下表示出了不同配方的酰化胰岛素制剂的HMWP和有关物质的变化。
表8
Figure PCTCN2020141056-appb-000031
Figure PCTCN2020141056-appb-000032
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量、及有关物质的量随着时间的变化均增加较缓慢，表明本发明所获得的酰化胰岛素制剂均具有良好的化学稳定性。
实施例29
在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)大鼠中的药效学研究8周龄SD大鼠(雌雄各半)，体重170-250g。于屏障环境内饲养于合适规格的饲养盒中(4只/盒)，自由获取标准食物和纯化水，环境条件控制在相对湿度40％～70％，温度为22℃～26℃。4天适应期后，禁食12h，大鼠按照60mg/kg腹腔注射链脲佐菌霉素(sigma)溶液(10mg/mL，于0.1M柠檬酸缓冲液)。给予链脲佐菌霉素4天和8天后，进行随机血糖检测，血糖值在20mmol/L以上的选为T1DM模型大鼠用于后续试验。
造模8天后开始试验，于给药前一天，监测基础血糖并对大鼠称重。根据随机血糖和体重将大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，或皮下分别注射德谷胰岛素(50U/kg)、化合物4(25U/kg或40U/kg)，其中溶媒包含：苯酚60mM,甘油15mg/ml，间甲酚10mM，氯化钠0.585mg/ml，所述溶媒的pH值为7.4。
将所述酰化胰岛素溶解在溶媒中至25U/ml或40U/ml的给药浓度，给药体积为1ml/kg(即0.1ml/100g体重)。采用皮下给药方式，颈背部皮下给药，隔天给药一次，重复给药11次，试验期间SD大鼠自由饮食。在大约上午9:30-10:30给予酰化胰岛素，在首次给药3h、4h、5h、6h、24h、48h后监测大鼠血糖，之后每次给药后4h、24h、48h监测一次大鼠血糖。
对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。为了说明酰化胰岛素对血糖的影响，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(AUC)。
如图18a-18b显示，相对于德谷胰岛素，本发明的酰化胰岛素给药后在I型糖尿病(T1DM)大鼠中有预料不到的增加的降糖药效，化合物4的降糖效果明显优于德谷胰岛素。
实施例30
以与实施例24相似的步骤，测试包含本发明的酰化胰岛素与门冬胰岛 素的组合物在链脲佐菌霉素(STZ)诱导I型糖尿病(T1DM)C57/6J小鼠中对血糖(BG)的调节效应。
当日实验开始前，检测小鼠随机血糖，并对小鼠称重。根据随机血糖和体重将小鼠分配到溶媒组或治疗组，共8组，每组9只(雄性5只，雌性4只)，各组分别接受如下处理：皮下注射溶媒，或皮下注射包含德谷胰岛素和门冬胰岛素的药物组合物，其中德谷胰岛素和门冬胰岛素的注射剂量分别为7U/kg和3U/kg，或皮下注射包含本发明实施例4的标题化合物化合物4和门冬胰岛素的六种药物组合物，其中在注射所述六种药物组合物时，化合物4的注射剂量分别为1.49U/kg、1.99U/kg、2.45U/kg、2.85U/kg、3.43U/kg、和3.92U/kg，门冬胰岛素的注射剂量均为3U/kg，其中所述溶媒包含：60mM苯酚，10mM间甲酚，15mg/ml甘油，15mM Na 2HPO 4，上述溶媒的pH值为7.6。
将化合物4与门冬胰岛素预混液溶解在溶媒中至0.6U/mL给药浓度(以预混液中门冬胰岛素浓度计)，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药。在每天下午大约16:00(时间0)给予药物，连续给药15天，给药期间小鼠自由饮食，评估第一、二、五、八、十五次给药前(0h)和给药后1小时小鼠随机血糖，以及对第二、五、八、十五次给药前(0h)血糖及给药后0.5、1、2、4、6、16、20、24小时随机血糖进行检测。最后一次给药后小鼠禁食2h，眼眶取血，检测全血中糖化血红蛋白(Hb1Ac)百分比。
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。并绘制第十五次给药后血糖对时间的剂量响应曲线。为了说明本发明的预混胰岛素对血糖的影响，对第十五次给药后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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图19a和图19b显示了包含本发明的酰化胰岛素和门冬胰岛素的组合物在给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在I型糖尿病(T1DM)小鼠中有预料不到的增加的降糖药效，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好的降糖效果。
图20显示包含本发明的酰化胰岛素和门冬胰岛素的组合物，在给药 后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，具有更好地降低Hb1Ac的效果，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好地降低Hb1Ac的效果。
实施例31
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包含本发明的酰化胰岛素与门冬胰岛素的组合在肥胖的糖尿病小鼠模型(db/db小鼠)中对血糖(BG)的调节效应。
将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，检测小鼠随机血糖，并对小鼠称重。根据随机血糖和体重将小鼠分配到溶媒组或治疗组，共5组，每组8只，各组分别接受如下处理：皮下注射溶媒，或皮下注射包含德谷胰岛素和门冬胰岛素的药物组合物，其中德谷胰岛素和门冬胰岛素的注射剂量分别为7U/kg和3U/kg，或皮下注射包含本发明实施例4的标题化合物化合物4和门冬胰岛素的三种药物组合物，其中在注射所述三种药物组合物时，化合物4的注射剂量分别为2.0U/kg、2.4U/kg、3.84U/kg，门冬胰岛素的注射剂量均为3U/kg，其中溶媒包含：60mM苯酚，10mM间甲酚，15mg/ml甘油，15mM Na 2HPO 4，pH值为7.6。
将所述酰化胰岛素溶解在溶媒中至0.6U/ml的给药浓度，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，颈背部皮下注射给药四次。在大约上午09:30(时间0)给予酰化胰岛素，给药期间动物禁食不禁水，评估给药后0.5、1、2、3、4、6、8、10、12小时小鼠血糖。
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。对于每个单剂量的酰化胰岛素绘制血糖对时间的剂量响应曲线。
为了说明本发明的包含酰化胰岛素和门冬胰岛素的药物组合物对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图21a和图21b显示，包含本发明的酰化胰岛素和门冬胰岛素的组合物，在给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在肥胖的糖尿病小鼠模型(db/db小鼠)中有预料不到的增加的降糖药效，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好的降糖效果。
实施例32
本实验的目的在于测量本发明的酰化胰岛素制剂的化学稳定性。
酰化胰岛素制剂
将实施例4的标题化合物化合物4溶解于0.03％NaOH溶液中，至2.4mM的浓度，然后用4％NaOH溶液调节pH值至7.4，根据下表中各个组分的量，将苯酚、间甲酚、甘油和氯化钠混合好后加入化合物4溶液中，调节pH值至7.4，然后根据下表中醋酸锌的量平均分三次加入到化合物4溶液中，调节pH值至最终值。产生最终胰岛素浓度为1.2mM(200U/ml或8.46mg/ml)的酰化胰岛素制剂，其中Zn的含量以Zn/6摩尔的酰化的胰岛素(简写为“Zn/6ins”)来表示。
本实施例中制剂的化学稳定性能够用在25℃和37℃存储14天和21天后，高分子量蛋白(HMWP)相对于第0天时的变化来显示，同时也能够用在37℃存储21天后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，在型号和规格为：ShodexTM PROTEIN KW-802.5(8.0mm×300mm)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3L的0.1％精氨酸溶液、750ml冰醋酸和1250ml乙腈。检测波长为276nm，进样量为10μl。表9示出了，在25℃和37℃时，第14天和第21天相对于第0天时HMWP的增加量。
表9
Figure PCTCN2020141056-appb-000033
由上表可知，本发明的酰化胰岛素制剂在上述pH值范围内HMWP的量随着时间的变化增加非常缓慢，表明本发明的酰化胰岛素制剂在上述pH值范围内均具有优异的化学稳定性。
有关物质的量的测定
通过高效液相色谱法(HPLC)测定胰岛素有关物质的含量，在Waters Kromasil 100A-3.5μm-C8(4.6*250mm)柱上，在柱温为40℃、样品池温度为10℃时，用洗脱相以1.0ml/min的流速进行测试。洗脱相由以下组成的流动相进行：
A相包含0.1M无水硫酸钠、0.1M二水合磷酸二氢钠、10％乙腈(v/v)，用浓磷酸调节pH值至为3.0；
B相为60％乙腈(v/v)。
梯度：0-40min为41.3％/58.7％A/B的等度梯度，40-50min至0％/100％A/B的线性变化，50-51min至41.3％/58.7％A/B的线性变化，51-65min的41.3％/58.7％A/B的等度梯度。表10示出了，在37℃时，第21天相对于第0天时有关物质的增加量。
表10
Figure PCTCN2020141056-appb-000034
由上表可知，本发明的酰化胰岛素制剂在上述pH值范围内有关物质的量随着时间的变化也非常缓慢，本发明的上述酰化胰岛素制剂均具有优异的化学稳定性。
实施例33
本实验的目的是测量本发明的酰化胰岛素制剂的化学稳定性。根据下述表11和12中各组分的量，按照与实施例32类似地步骤，配制表11和12中的酰化胰岛素制剂。并按照与实施例32类似的步骤测定HMWP和有关物质的变化。下表11和12示出了不同配方的酰化胰岛素制剂的HMWP和有关物质的变化。
表11
Figure PCTCN2020141056-appb-000035
表12
Figure PCTCN2020141056-appb-000036
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量、及有关物质的量随着时间的变化均增加较缓慢，表明本发明所获得的酰化胰岛素制剂均具有良好的化学稳定性。
实施例34
本实验的目的在于测量本发明的酰化胰岛素制剂的化学稳定性。
酰化胰岛素制剂
将实施例18的标题化合物化合物16溶解于0.08％NaOH溶液中，至最终胰岛素浓度的两倍，然后用4％NaOH溶液调节pH值至7.45，根据下表中各个组分的量，将苯酚、间甲酚、甘油和氯化钠混合好后加入化合物16溶液中，调节pH值至7.4，然后根据下表中醋酸锌的量平均分三次加入到化合物16溶液中，调节pH值至7.4。产生最终胰岛素浓度为1.2mM(9.43mg/ml)或1.5mM(11.74mg/ml)的酰化胰岛素制剂。
本实施例中制剂的化学稳定性能够用在25℃和37℃存储14天和21天后，高分子量蛋白(HMWP)相对于第0天时的变化来显示，同时也能够用在25℃和37℃存储14天和21天后有关物质的量的变化来表示。
高分子量蛋白(HMWP)的测定
按照实施例32类似的步骤测定HMWP的量。表13-15示出了，在25℃和37℃时，第14天和第21天相对于第0天时HMWP的增加量。
表13
Figure PCTCN2020141056-appb-000037
表14
Figure PCTCN2020141056-appb-000038
Figure PCTCN2020141056-appb-000039
表15
Figure PCTCN2020141056-appb-000040
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量随着时间的变化增加非常缓慢，表明上述酰化胰岛素制剂均具有优异的化学稳定性。
有关物质的量的测定
通过高效液相色谱法(HPLC)测定胰岛素有关物质的含量，在Waters Kromasil 300A-5μm-C4(4.6*150mm)柱上，在柱温为40℃、样品池温度为10℃时，用洗脱相以1.0ml/min的流速进行测试。洗脱相由以下组成的流动相进行：
A相包含0.18M无水硫酸钠、10％乙腈(v/v)，用85％磷酸调节pH值至为2.3；
B相为75％乙腈(v/v)。
梯度：0-40min的48％/52％A/B的等度梯度，40-51min至0％/100％A/B的线性变化，51-65min至48％/52％A/B的线性变化。
表16-17示出了，在25℃和37℃时，第14天和/或第21天相对于第0天时有关物质的增加量。
表16
Figure PCTCN2020141056-appb-000041
表17
Figure PCTCN2020141056-appb-000042
表18
Figure PCTCN2020141056-appb-000043
Figure PCTCN2020141056-appb-000044
由上表可知，本发明的上述酰化胰岛素制剂中胰岛素有关物质的量随着时间的变化增加也非常缓慢，表明上述酰化胰岛素制剂非常稳定。
实施例35
本实验的目的在于测量本发明的酰化胰岛素制剂的化学稳定性。
酰化胰岛素制剂
将实施例18的标题化合物化合物16溶解于10mM的50％终体积磷酸氢二钠溶液中，至最终胰岛素浓度的两倍，然后用4％NaOH调节pH值至终值，根据下表中各个组分的量，将苯酚、间甲酚、甘油和氯化钠混合好后加入化合物16溶液中，调节pH值至终值，然后根据下表中醋酸锌的量平均分三次加入到化合物16溶液中，调节pH值至终值。产生最终胰岛素浓度为1.5mM(11.74mg/ml)的酰化胰岛素制剂。
并按照与实施例32类似的步骤测定HMWP，按照与实施例34类似的步骤测定有关物质的变化。下表19和20示出了不同配方的酰化胰岛素制剂的HMWP和有关物质的变化。
表19
Figure PCTCN2020141056-appb-000045
Figure PCTCN2020141056-appb-000046
表20
Figure PCTCN2020141056-appb-000047
由上表可知，本发明的上述酰化胰岛素制剂中HMWP的量、及有关物质的量随着时间的变化均增加较缓慢，表明本发明所获得的酰化胰岛素制剂均具有良好的化学稳定性。
实施例36
本实验的目的是测量本发明的酰化胰岛素与门冬胰岛素的组合制剂的化学稳定性。
对于该酰化胰岛素与门冬胰岛素的组合制剂，按表21中所列个组分的量制备组合1-5。其中Zn的含量以Zn/6摩尔的酰化的胰岛素(简写为“Zn/6ins”)来表示。
表21
Figure PCTCN2020141056-appb-000048
Figure PCTCN2020141056-appb-000049
本实施例中制剂的化学稳定性能够用在37℃存储14天和28天后，高分子量蛋白(HMWP)相对于第0天时的变化来显示。
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，在型号和规格为：Tskgel，G2000SWXL，5μm(7.8×300mm)柱上，在柱温为30℃、样品池温度为10℃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400ml异丙醇、300ml冰醋酸和300ml水。检测波长为276nm，进样量为10μl。表22示出了，在37℃时，第14天和第28天相对于第0天时HMWP的增加量。
表22
Figure PCTCN2020141056-appb-000050
由上表可知，本发明的上述酰化胰岛素和门冬胰岛素组合制剂中HMWP的量随着时间的变化增加非常缓慢，表明上述组合物制剂均具有优异的化学稳定性。
实施例37
本实验的目的是测量本发明的酰化胰岛素与门冬胰岛素的组合制剂的化学稳定性。
根据下述表23各组分的量配制组合6-10。并按照与实施例36类似的步骤测定HMWP的变化。下表24示出了不同配方的酰化胰岛素制剂的HMWP的变化。
表23
Figure PCTCN2020141056-appb-000051
表24
Figure PCTCN2020141056-appb-000052
由上表可知，本发明的上述酰化胰岛素和门冬胰岛素组合制剂中HMWP的量随着时间的变化增加非常缓慢，表明上述组合物制剂均具有优异的化学稳定性。
实施例38
本实验的目的是测量本发明的酰化胰岛素与门冬胰岛素的组合制剂的化学稳定性。
根据下述表25中各组分的量配制组合11和12。并按照与实施例36类似的步骤测定HMWP的变化。下表26示出了不同配方的酰化胰岛素制剂的HMWP的变化。
表25
Figure PCTCN2020141056-appb-000053
表26
Figure PCTCN2020141056-appb-000054
由上表可知，本发明的上述酰化胰岛素和门冬胰岛素组合制剂中HMWP的量随着时间的变化增加非常缓慢，表明上述组合物制剂均具有优异的化学稳定性。
实施例39
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包含本发明的酰化胰岛素与门冬胰岛素的组合物在肥胖的糖尿病小鼠模型(db/db小鼠)中对血糖(BG)的调节效应。
参照实施例31类似的实验步骤获得实验用db/db小鼠。于当日实验开始前，检测小鼠随机血糖，并对小鼠称重。根据随机血糖和体重将小鼠分 配到溶媒组或治疗组，共4组，每组5只，各组分别接受如下处理：皮下注射溶媒，或皮下注射包含德谷胰岛素和门冬胰岛素的药物组合物，其中德谷胰岛素和门冬胰岛素的注射剂量分别为9.3U/kg和4U/kg，或皮下注射包含本发明实施例4的标题化合物化合物4和门冬胰岛素的药物组合物，其中在注射所述药物组合物时，化合物4的注射剂量为3.7U/kg，门冬胰岛素的注射剂量为4U/kg；其中溶媒包含：55mM苯酚，10mM间甲酚，8.5mg/ml甘油，60mM NaCl，pH值为7.6。
将所述酰化胰岛素和门冬胰岛素的注射液溶解在溶媒中至相应的的给药浓度，给药体积为5ml/kg(即50μl/10g体重)。采用皮下给药(S.C.)方式，每天给药1次。给药期间动物自由饮食饮水，评估连续给药第21天药后0.5、1、2、3、4、6、8小时小鼠随机血糖和连续给药第18天药后0.5、1、2、3、4、6、8、10小时小鼠药后禁食血糖。
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。对于每个单剂量的酰化胰岛素和门冬胰岛素注射液绘制血糖对时间的剂量响应曲线。
为了说明本发明的包含酰化胰岛素和门冬胰岛素的药物组合物对血糖的影响，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(AUC)。其中，AUC值越小，表明降糖效果越好，药效越好。
图22a至图22d显示，包含本发明的酰化胰岛素和门冬胰岛素的组合物，在给药后，相对于包含德谷胰岛素和门冬胰岛素的药物组合物，在肥胖的糖尿病小鼠模型(db/db小鼠)中有预料不到的增加的降糖药效，在化合物4与门冬胰岛素的用量比远小于德谷胰岛素与门冬胰岛素的用量比时，仍能达到更好的降糖效果，且降糖持续时间更长。
实施例40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24只，每组6只(雌雄各半)，分为实施例4标题化合物化合物4低剂量组、化合物4中剂量组、和化合物4高剂量组，分别皮下注射给药2、6、18U/kg，以及德谷胰岛素组，德谷给药14U/kg。化合物4低中高剂量组、德谷胰岛素组分别于给药前(0min)、给药后0.5、1.5、4、6、8、24、48、72h采血测定血药浓度。使用WinNonLin v8.0软件的非房室模 型计算药代参数C max、T max、T 1/2、AUC 0-t、Vd、Cl、MRT，试验结果总结在表27中。
表27：向SD大鼠皮下注射化合物4和德谷后药代动力学参数
Figure PCTCN2020141056-appb-000055
C max＝峰浓度，T max＝达峰时间，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，AUC INF＝血浆浓度时间曲线下从施用时间至无穷大的面积，Vd＝表观分布容积，Cl＝清除率，MRT＝平均驻留时间
比格犬的药代动力学
犬36只，每组6只，雌雄各半，分为化合物4低、中、高剂量组，分别皮下注射给药0.3、0.6、1.2U/kg，化合物4静脉组，静脉注射给药0.6U/kg，德谷胰岛素组，皮下注射给药0.6U/kg，静脉注射给药0.6U/kg。化合物4静脉组和德谷胰岛素静脉组于给药前、给药后2min、10min、30min、1h、2h、4h、6h、8h、12h、24h、30h、36h、48h采血测定血药浓度，化合物4低剂量、高剂量和德谷胰岛素皮下注射组于给药前、药后0.5h、1h、2h、4h、6h、8h、12h、24h、30h、36h、48h采血测定血药浓度，化合物4中剂量组连续给药7天，于首次给药和末次给药采集给药前、药后0.5h、1h、2h、4h、6h、8h、12h、24h、30h、36h、48h、72h(末次)采血测定血药浓度。使用WinNonLin v8.0软件的非房室模型计算药代参数Cmax、Tmax、T1/2、AUC0-t、MRT，试验结果总结在表28中。
表28：向比格犬皮下注射化合物4和德谷后药代动力学参数
Figure PCTCN2020141056-appb-000056
由上述实验结果可知，本发明的酰化胰岛素衍生物化合物4在大鼠和比格犬体内均表现出较长的半衰期、更平稳的降糖效果。
实施例41
B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素(化合物17)
Figure PCTCN2020141056-appb-000057
以与实施例1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1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝2451.38(M+1) +
实施例42
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素(化合物18)
Figure PCTCN2020141056-appb-000058
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43
A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-24xOEG),desB30人胰岛素(化合物19)
Figure PCTCN2020141056-appb-000059
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44
B29K(N(ε)-二十二烷二酰基-γGlu-OEG),desB30人胰岛素(化合物20)
Figure PCTCN2020141056-appb-000060
以与实施例1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1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝854.57(M+1) +
实施例45
B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素
(化合物21)
Figure PCTCN2020141056-appb-000061
以与实施例1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1第3部分类似的步骤进行。
LC-MS(Scie×100API)：m/z＝1294.83(M+1) +
实施例46
本发明的胰岛素衍生物的受体结合能力
本试验目的在于证明本发明的胰岛素衍生物对胰岛素受体的结合能力。
采用表面等离子共振(SPR)方法对本发明的化合物2和对照化合物2，分别在没有人血清白蛋白(HSA)存在下和2％HSA存在下，进行与带his标签的胰岛素受体A胞外域(IRA，Sino Biological)之间的结合能力试验。
用操作缓冲液(Running buffer)(Cytiva)或含2.0％HSA的操作缓冲液分别稀释样品，使得和对照化合物2样品的进样浓度均为400nM。选用NTA传感芯片(Cytiva)，25℃条件下在Biacore T200(Cytiva)上进行SPR分析。以10μL/min的流速注入0.5M NiCl 2(Cytiva)60s，随后用HBS-EP buffer(Cytiva)清洗。以5μL/min的流速注入3μg/mL IRA受体180s，使IRA受体结合在芯片表面。之后以30μL/min流速注入测试的胰岛素衍生物样品60s，并进行60s解离。每次进样后以10μL/min的流速注入350mM EDTA(Cytiva)60s进行芯片再生，最后HBS-P buffer(Cytiva)清洗之后则可进行下一个样品的检测。选用样品开始解离之前第4s的响应(Response)值作为对受体的结合能力测试结果，针对每个样品重复3次试验。
图23示出了化合物2和对照化合物2在2％HSA存在下(模拟生理条件)，相对于0％HSA时，受体结合能力的情况。由图23可以看出，在2％HSA存在下，化合物2相对于对照化合物2，具有明显提高的受体结合能力，白蛋白对本发明的化合物2的受体结合能力的影响，显著低于对照化合物2。
这表明，在白蛋白的存在下，本发明的胰岛素衍生物例如化合物2相对于对照化合物2，具有预料不到的显著提高的受体结合能力，即，白蛋白对本发明的胰岛素衍生物的受体结合能力的影响，显著低于对照化合物2。
实施例47
本发明的胰岛素衍生物的受体结合能力
本试验目的在于证明本发明的胰岛素衍生物对胰岛素受体的结合能力。
采用与实施例46类似的方法对本发明的胰岛素衍生物化合物15和对照化合物5，分别在没有人血清白蛋白(HSA)存在下和2％HSA存在下，进行与IRA之间的结合能力试验，不同的是化合物15、和对照化合物5样品的进样浓度均为12800nM和25600nM，测定结果如图24a和24b所示。
图24a和24b示出了化合物15和对照化合物5在2％HSA存在下(模拟生理条件)，相对于0％HSA时，受体结合能力的情况。由图24a和24b可以看出，在2％HSA存在下，化合物15相对于对照化合物5，具有预料不到的明显提高的受体结合能力，白蛋白对本发明的胰岛素衍生物化合物15的受体结合能力的影响，显著低于对照化合物5。
实施例48
本发明的胰岛素衍生物的受体结合能力
本试验目的在于证明本发明的胰岛素衍生物对胰岛素受体的结合能力。
采用与实施例46类似的方法对本发明的胰岛素衍生物化合物17和对照化合物2，分别在没有人血清白蛋白(HSA)存在下和2％HSA存在下，进行与IRA之间的结合能力试验，测定结果如图25所示。
图25示出了化合物17和对照化合物2在2％HSA存在下(模拟生理条件)，相对于0％HSA时，受体结合能力的情况。由图25可以看出，在2％HSA存在下，化合物17相对于对照化合物2，具有明显提高的受体结合能力，白蛋白对本发明的胰岛素衍生物化合物17的受体结合能力的影响，显著低于对照化合物2。
这表明，在白蛋白的存在下，对本发明的胰岛素衍生物例如化合物17相对于对照化合物2，具有预料不到的显著提高的受体结合能力，即，白蛋白对本发明的胰岛素衍生物的受体结合能力的影响，显著低于对照化合物2。
实施例49
本发明的胰岛素衍生物的受体结合能力
本试验目的在于证明本发明的胰岛素衍生物对胰岛素受体的结合能力。
采用与实施例46类似的方法对本发明的胰岛素衍生物化合物16、化 合物18和对照化合物5，分别在没有人血清白蛋白(HSA)存在下和2％HSA存在下，进行与IRA之间的结合能力试验，不同的是化合物16、化合物18和对照化合物5样品的进样浓度均为12800nM和25600nM，测定结果如图26a和26b所示。
图26a和26b示出了化合物16、化合物18和对照化合物5在2％HSA存在下(模拟生理条件)，相对于0％HSA时，受体结合能力的情况。由图26a和26b可以看出，在2％HSA存在下，化合物16、化合物18相对于对照化合物5，具有预料不到的明显提高的受体结合能力，白蛋白对本发明的胰岛素衍生物的受体结合能力的影响，显著低于对照化合物5。
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。
序列表
SEQ ID NO.1：
DesB30人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
SEQ ID NO.2：
DesB30人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys
SEQ ID NO.3：
A14E，B16H，B25H，desB30人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn
SEQ ID NO.4：
A14E，B16H，B25H，desB30人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys
SEQ ID NO.5：
A14E，B16E，B25H，desB30人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu Glu Asn Tyr Cys Asn
SEQ ID NO.6：
A14E，B16E，B25H，desB30人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys
SEQ ID NO.7：
人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
SEQ ID NO.8：
人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
SEQ ID NO.9：
A21G人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly
SEQ ID NO.10：
A21G人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
SEQ ID NO.11：
A21G，desB30人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly
SEQ ID NO.12：
A21G，desB30人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys
SEQ ID NO.13：
B28D人胰岛素A链：
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
SEQ ID NO.14：
B28D人胰岛素B链：
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr
SEQ ID NO.15：
GLP-1-(7-37)肽
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
SEQ ID NO.16：
[Gly8，Arg34]GLP-1-(7-37)肽
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
SEQ ID NO.17：
[Arg34]GLP-1-(7-37)肽
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Claims (63)

  1. 胰岛素衍生物，所述胰岛素衍生物包含胰岛素母体、白蛋白结合残基、和连接基团Lin，所述胰岛素母体为天然存在的胰岛素或胰岛素类似物，所述白蛋白结合残基经由连接基团Lin与所述胰岛素母体连接，其中，
    所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；或者所述连接基团Lin包含至少5个中性的、包含亚烷基二醇的氨基酸残基；优选所述连接基团Lin包含至少6个中性的、包含亚烷基二醇的氨基酸残基；优选所述连接基团Lin包含5-20个中性的、包含亚烷基二醇的氨基酸残基；或者，所述连接基团Lin包含具有至少15个、优选至少20个、优选至少24个、优选至少30个、优选至少42个、优选15-120个、优选20-120个、优选30-100个、优选39-100个、优选42-80个碳原子的亚烷基二醇；
    所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和所述脂肪族二酸的羧基之一中去除；和
    当所述连接基团Lin是具有60个碳原子的亲水性连接基团，且所述白蛋白结合残基是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
  2. 如权利要求1所述的胰岛素衍生物，其中，所述胰岛素母体包含至少一个赖氨酸残基，所述白蛋白结合残基经由连接基团Lin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。
  3. 如权利要求1或2所述的胰岛素衍生物，其中，所述胰岛素衍生物还包含一个或多个连接基团II，所述连接基团II是酸性的氨基酸残基，所述连接基团II连接在所述白蛋白结合残基与所述连接基团Lin之间、和/或连接在所述连接基团Lin和所述胰岛素母体之间；优选，所述连接基团II连接在所 述白蛋白结合残基与所述连接基团Lin之间。
  4. 胰岛素衍生物，其是酰化胰岛素，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0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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20-26个(优选20-24个)碳原子的脂肪酸或脂肪族二酸，其中在形式上羟基已从所述脂肪酸的羧基和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；
    II和I在式(A)中出现的顺序可以独立地互换；和
    当m为1，n为10，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素；
    或者
    所述酰基部分如式(A’)所示：
    III-(II) m-(I’) n’-  (A’)，
    其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
    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20-26个(优选20-24个)碳原子的脂肪酸或脂肪族二酸，其中在形式上羟基已从所述脂肪酸的羧基和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；
    II和I’在式(A’)中出现的顺序可以独立地互换；
    (I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、 优选66-180个、优选72-120个；和
    当m为1，(I’) n’的总碳原子数是60个，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
  5. 胰岛素衍生物，其是酰化胰岛素，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0，1，2，3，4，5，6，7，8，9或10，n为5，6，7，8，9，10，11，12，13，14，15，16，17，18，19或20；
    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20-26个(优选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；
    II和I在式(A)中出现的顺序可以独立地互换；和
    当m为1，n为10，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素；
    或者
    所述酰基部分如式(A’)所示：
    III-(II) m-(I’) n’-  (A’)，
    其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
    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20-26个(优选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；
    II和I’在式(A’)中出现的顺序可以独立地互换；
    (I’) n’的总碳原子数为20-200个、优选25-180个、优选30-180个、优选 42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个；和
    当m为1，(I’) n’的总碳原子数是60个，且III是包含20个碳原子的脂肪族二酸时，所述胰岛素母体不是A14E,B16H,B25H,desB30人胰岛素。
  6. 如权利要求4或5所述的胰岛素衍生物，其中，
    n为5-18的整数，优选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20-26个(优选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。
  7. 如权利要求4-6任一项所述的胰岛素衍生物，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-；或者，I’是 HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-、-HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-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，优选地，II选自γGlu或βAsp；和/或
    III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-、或HOOC-(CH 2) 24-CO-，优选，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-。
  8. 如权利要求4-7任一项所述的胰岛素衍生物，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，或者式(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。
  9. 如权利要求4-8任一项所述的胰岛素衍生物，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。
  10. 如权利要求1-9任一项所述的胰岛素衍生物，其中，所述胰岛素母体的赖氨酸残基位于B29位处。
  11. 如权利要求1-10任一项所述的胰岛素衍生物，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；优选地，所述胰岛素母体为desB30人胰岛素；A14E,B16H,B25H,desB30人胰岛素；或A14E,B16E,B25H,desB30人胰岛素。
  12. 如权利要求4或5所述的胰岛素衍生物，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；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；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；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；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；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；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；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；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；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；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；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；或B29K(N(ε)-二十二烷二酰基-γGlu-12xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-13xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-13xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-14xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-14xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-15xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-15xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-16xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-16xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-17xPEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-17xPEG),desB30人胰岛素；B29K(N(ε)-二十二烷二酰基-γGlu-18xPEG),desB30人胰岛素；或B29K(N(ε)-二十烷二酰基-γGlu-18xPEG),desB30人胰岛素；
    优选地，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；或B29K(N(ε)-二十二烷二酰基 -γGlu-12xOEG),desB30人胰岛素；
    更优选地，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；或B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素。
  13. 胰岛素衍生物，其是酰化胰岛素，所述酰化胰岛素的胰岛素母体为A14E,B16H,B25H,desB30人胰岛素或A14E,B16E,B25H,desB30人胰岛素，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(C)所示：
    Y1-(Y2) m1-(Y3) n1-  (C)，
    其中,
    m1为0，1，2，3，4，5，6，7，8，9或10，n1为5、6、7、8、9或10的整数；
    Y3是中性的、包含亚烷基二醇的氨基酸残基；
    Y2是酸性的氨基酸残基；
    Y1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；
    Y1、Y2、和Y3之间以酰胺键连接；
    Y2和Y3在式(C)中出现的顺序可以独立地互换；和
    当Y1是包含20个碳原子的脂肪族二酸，且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时，且m1为1时，则n1不是10；
    或者
    所述酰基部分如式(C’)所示：
    Y1-(Y2) m1-(Y3’) n1’-  (C’)，
    其中,
    m1为0，1，2，3，4，5，6，7，8，9或10，n1’为5、6、7、8、9或10的整数；
    Y3’是中性的、包含亚烷基二醇的氨基酸残基；
    Y2是酸性的氨基酸残基；
    Y1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；
    Y1、Y2、和Y3’之间以酰胺键连接；
    Y2和Y3’在式(C’)中出现的顺序可以独立地互换；
    (Y3’) n1’的总碳原子数为15-100个、优选20-100个、优选25-90个、优选30-80个、优选30-59个、优选30-54个；和
    当Y1是包含20个碳原子的脂肪族二酸，且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时，且m1为1时，则(Y3’) n1’的总碳原子数不是60个。
  14. 如权利要求13所述的胰岛素衍生物，其中，
    n1为5、6、7、8、9、，优选地，n1为5、6、7、或8；和/或
    m1为1-6的整数，优选地，m1为1、2、3、或4，优选地，m1为1或2，优选地，m1为1；和/或
    Y1是包含20-23个碳原子的脂肪族二酸，优选Y1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。
  15. 如权利要求13或14所述的胰岛素衍生物，其中，
    Y3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Y3是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；或者，Y3’是-HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-；和/或
    Y2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，Y2选自γGlu或βAsp；和/或
    Y1是是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-、或HOOC-(CH 2) 24-CO-，优选，Y1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Y1是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
  16. 如权利要求13-15任一项所述的胰岛素衍生物，其中，式(C)通过Y3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，或者式(C’)通过Y3’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。
  17. 如权利要求13-16任一项所述的胰岛素衍生物，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。
  18. 如权利要求4-13任一项所述的胰岛素衍生物，其中，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-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,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,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,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,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,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,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,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,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,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,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)-二十烷二酰基-γGlu-9xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；
    优选地，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-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)-二十二烷二酰基-γGlu-9xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-10xOEG),desB30人胰岛素；
    优选地，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；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素。
  19. 胰岛素衍生物，其是酰化胰岛素，所述酰化胰岛素的胰岛素母体为A14E,B16H,B25H,desB30人胰岛素或A14E,B16E,B25H,desB30人胰岛素，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(D)所示：
    W1-(W2) m2-(W3) n2-  (D)，
    其中,
    m2为0，1，2，3，4，5，6，7，8，9或10，n2为11，12，13，14，15，16，17，18，19或20；
    W3是中性的、包含亚烷基二醇的氨基酸残基；
    W2是酸性的氨基酸残基；
    W1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；
    W1、W2、和W3之间以酰胺键连接；和
    W2和W3在式(D)中出现的顺序可以独立地互换；
    或者
    所述酰基部分如式(D’)所示：
    W1-(W2) m2-(W3’) n2’-  (D’)，
    其中,
    m2为0，1，2，3，4，5，6，7，8，9或10，n2’为11，12，13，14，15，16，17，18，19或20；
    W3’是中性的、包含亚烷基二醇的氨基酸残基；
    W2是酸性的氨基酸残基；
    W1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；
    W1、W2、和W3’之间以酰胺键连接；
    W2和W3’在式(D’)中出现的顺序可以独立地互换；和
    (W3’) n2’的总碳原子数为30-180个、42-180个、优选61-180个、优选66-180个、优选72-120个。
  20. 如权利要求19所述的胰岛素衍生物，其中，
    n2为11、12、13、14、15、16、17、18或19，优选地，n2为11、12、13、14、15、16、17、或18，优选地，n2为11、12、13、14、15、或16，优选地，n2为11、12、13、14、或15；和/或
    m2为1-6的整数，优选地，m2为1、2、3、或4，优选地，m2为1或2，优选地，m2为1；和/或
    W1是包含20-23个碳原子的脂肪族二酸，优选W1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。
  21. 如权利要求19或20所述的胰岛素衍生物，其中，
    W3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W3是-HN-(CH 2) 2-O-(CH 2) 2-O-CH 2-CO-；或者，W3’是-HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-CH 2-CO-；和/或W2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，W2选自γGlu或βAsp；和/或
    W1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W1是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
  22. 如权利要求19-21任一项所述的胰岛素衍生物，其中，式(D)通过W3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或者式(D’)通过W3’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。
  23. 如权利要求19-22任一项所述的胰岛素衍生物，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。
  24. 如权利要求4、5或19述的胰岛素衍生物，其中，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-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,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-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,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-13xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-14xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-15xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-13xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-14xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-15xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-19xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-16xOEG),desB30人胰岛素；A14E,B16E, B25H,B29K(N(ε)-二十二烷二酰基-γGlu-17xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-18xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-19xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十烷二酰基-γGlu-20xOEG),desB30人胰岛素；A14E,B16E,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-20xOEG),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；
    优选地，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-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；
    优选地，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-12xOEG),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。
  25. 如权利要求4、5、13或19所述的胰岛素衍生物，其中，所述酰化胰岛素选自下述胰岛素：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；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；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；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；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；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；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；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；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；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；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,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。
  26. 药物组合物，其包含权利要求1-25任一项所述的胰岛素衍生物或A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-10xOEG),desB30人胰岛素、和一种或多种药学上可接受的赋形剂。
  27. 如权利要求26所述的药物组合物，其包含至少1.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少2.2摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少3.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含至少4.5摩尔锌离子/6摩尔的酰化的胰岛素；优选包含2.2-12摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-10摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含 4.5-8摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-7.5摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-7.0摩尔锌离子/6摩尔的酰化的胰岛素；更优选包含4.5-6.5摩尔锌离子/6摩尔的酰化的胰岛素；和/或
    所述药物组合物具有的pH为6.5-8.5；优选pH为6.8-8.2；优选pH为7.0-8.2；优选pH为7.2-7.6；更优选pH为7.4或7.6。
  28. 如权利要求27所述的药物组合物，其中，所述药物组合物进一步包含甘油、苯酚、间甲酚、NaCl、和/或Na 2HPO 4；优选，所述药物组合物进一步包含甘油、苯酚、和NaCl；优选，所述药物组合物进一步包含甘油、苯酚、间甲酚、和NaCl；优选，所述药物组合物进一步包含甘油、苯酚、NaCl和Na 2HPO 4；更优选，所述药物组合物进一步包含甘油、苯酚、间甲酚、NaCl和Na 2HPO 4。
  29. 如权利要求28所述的药物组合物，其中，所述甘油的含量不超过约2.5％(重量/重量)，优选不超过约2％(重量/重量)，优选为约0.3％至约2％(重量/重量)，优选为约0.5％至约1.8％(重量/重量)，优选为约0.7％至约1.8％(重量/重量)，更优选为约1％至约1.8％(重量/重量)；和/或
    所述苯酚的含量为约16至80mM，优选为约25-75mM，优选为约30-70mM，45-70mM，优选为约45-65mM；优选为约45mM、约46mM、约47mM、约48mM、约49mM、50mM、约51mM、约52mM、约53mM、约54mM、约55mM、约56mM、约57mM、约58mM、约59mM、约60mM、约61mM、约62mM、约63mM、约64mM、或约65mM；和/或
    所述间甲酚的含量为约0-35mM,优选为约0-19mM,优选为约0-15mM，优选为约0mM、约1mM、约2mM、约3mM、约4mM、约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、或约15mM；和/或
    所述NaCl的含量为约0-150mM，优选为约5-120mM，优选为约10-120mM，优选为约10-100mM，更优选为约10-75mM，更优选为约10-50mM，更优选为约10-30mM；和/或
    所述Na 2HPO 4的含量为约0-75mM，优选为约5-60mM，优选为低于约50mM，更优选为低于约25mM，更优选为低于约15mM；和/或
    所述胰岛素衍生物的含量高于约0.3mM、优选高于约0.6mM、优选为约0.3-12mM、优选为约0.6-9.0mM、优选为约0.6-8.4mM、优选为约0.6-7.2mM、 优选为约0.6-6.0mM、优选为约0.6-4.2mM、优选为约0.6-3.6mM、优选为约0.6-3.0mM、优选为约0.6-2.4mM、优选为约0.6-2.1mM、优选为约0.6-1.2mM。
  30. 如权利要求26-29任一项所述的药物组合物，其中，所述胰岛素衍生物为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；或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；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-6xOEG),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-10xOEG),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-12xOEG),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。
  31. 药物组合物，其包含约0.6-4.2mM的权利要求1-25任一项所述的胰岛素衍生物、约1％至约1.8％(重量/重量)的甘油、约45-65mM的苯酚、约4.5-6.5摩尔锌离子/6摩尔的胰岛素衍生物、约10-120mM的氯化钠、约0-15mM间甲酚、和具有约7.0-8.2的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H, B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  32. 药物组合物，其包含约0.6mM或1.2mM的权利要求1-25任一项所述的胰岛素衍生物、1.7％(重量/重量)的甘油、约45mM的苯酚、约10mM的间甲酚、约6.5摩尔锌离子/6摩尔的胰岛素衍生物、约20mM的氯化钠、和具有约7.0-8.0的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  33. 药物组合物，其包含约0.6-4.2mM的权利要求1-25任一项所述的胰岛素衍生物、约1％至约2％(优选约1.5％-1.7％)(重量/重量)的甘油、约15mM-60mM(优选约30mM-60mM、更优选约45mM-60mM)的苯酚、约1.5-7.0(优选约2.2-4.5)摩尔锌离子/6摩尔的胰岛素衍生物、约10-120mM(优选约20-50mM)的氯化钠、约0-25mM(优选约0-15mM、0-10mM)间甲酚、和具有约7.0-8.2的pH值，优选，所述胰岛素衍生物为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；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-6xOEG),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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  34. 药物组合物，其包含约1.2-1.5mM的权利要求1-25任一项所述的胰岛素衍生物、约1.5％-1.7％(重量/重量)的甘油、约45mM-60mM的苯酚、约0-10mM的间甲酚、约2.2-2.5摩尔锌离子/6摩尔的胰岛素衍生物、约20mM的氯化钠、和具有约7.0-8.0的pH值，其中所述胰岛素衍生物为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-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；A14E,B16H,B25H,B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或A14E,B16H,B25H,B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  35. 如权利要求26-34任一项所述的药物组合物，其还包含促胰岛素GLP-1化合物；优选地，所述药物组合物还包含选自下述的促胰岛素GLP-1化合物：N-ε 26-(17-羧基十七烷酰基氨基)-4(S)-羧基丁酰基-[Arg34]GLP-1-(7-37)肽、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、N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Aib8,Arg34]GLP-1-(7-37)肽、N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-羧基十七烷酰氨基)-4(S)-羧基丁酰基氨基]乙氧基)乙氧基]乙酰氨基)乙氧基]乙氧基)乙酰基][Gly8,Arg34]GLP-1-(7-37)肽。
  36. 如权利要求26-35任一项所述的药物组合物，其还包含式(B)所示的促胰岛素GLP-1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：15)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、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。
  37. 如权利要求36所述的药物组合物，其中，
    G1为[Gly8，Arg34]GLP-1-(7-37)肽(SEQ ID NO：16)或[Arg34]GLP-1-(7-37)肽(SEQ ID NO：17)，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，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。
  38. 如权利要求36或37所述的药物组合物，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-。
  39. 如权利要求36-38任一项所述的药物组合物，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。
  40. 如权利要求36所述的药物组合物，所述促胰岛素GLP-1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。
  41. 如权利要求36所述的药物组合物，所述促胰岛素GLP-1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、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；
    优选地，所述促胰岛素GLP-1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。
  42. 如权利要求26-34任一项所述的药物组合物，其还包含快速起效的胰岛素。
  43. 如权利要求42所述的药物组合物，其中所述快速起效的胰岛素选自Asp B28人胰岛素、Lys B28Pro B29人胰岛素、Lys B3Glu B29人胰岛素、人胰岛素和desB30人胰岛素中的一种或多种；优选，所述快速起效的胰岛素为Asp B28人胰岛素、Lys B28Pro B29人胰岛素、Lys B3Glu B29人胰岛素、人胰岛素或desB30人胰岛素。
  44. 如权利要求42或43所述的药物组合物，其中所述胰岛素衍生物与所述快速起效的胰岛素的摩尔比为约60:3至约0.5:3、优选为约57:3至约1:3、优选为约55:3至约1.2:3、优选为约50:3至约1.5:3、优选为约40:3至约1.5:3、优选为约30:3至约1.5:3、优选为约27:3至约1.5:3、优选为约25:3至约1.5:3、优选为约22:3至约1.5:3、优选为约20:3至约1.5:3、优选为约17:3至约1.5:3、优选为约15:3至约1.5:3、优选为约12:3至约1.5:3、优选为约10:3至约1.5:3、优选为约9:3至约1.5:3、优选为约8:3至约1.5:3、优选为约7:3至约1.5:3、优选为约6.9:3至约1.5:3、优选为约6.8:3至约1.5:3、优选为约6.5:3至约1.5:3、优选为约6.3:3至约1.5:3、优选为约6:3至约1.5:3、优选为约5.8:3至约1.5:3、 优选为约5.5:3至约1.5:3、优选为约5.3:3至约1.5:3、优选为约5:3至约1.5:3、优选为约4.8:3至约1.5:3、优选为约4.5:3至约1.5:3、优选为约4.2:3至约1.5:3、优选为约4:3至约1.5:3、优选为约3.9:3至约1.5:3、优选为约3.8:3至约1.5:3、优选为约3.5:3至约1.5:3、优选为约3.2:3至约1.5:3、优选为约3:3至约1.5:3、优选为约2.8:3至约1.5:3、优选为约2.5:3至约1.5:3、优选为约15:3至约2:3、优选为约12:3至约2:3、优选为约10:3至约2:3、优选为约9:3至约2:3、优选为约8:3至约2:3、优选为约7:3至约2:3、优选为约6.9:3至约2:3、优选为约6.8:3至约2:3、优选为约6.5:3至约2:3、优选为约6.3:3至约2:3、优选为约6:3至约2:3、优选为约5.8:3至约2:3、优选为约5.5:3至约2:3、优选为约5.3:3至约2:3、优选为约5:3至约2:3、优选为约4.8:3至约2:3、优选为约4.5:3至约2:3、优选为约4.2:3至约2:3、优选为约4:3至约2:3、优选为约3.9:3至约2:3、优选为约3.8:3至约2:3、优选为约3.5:3至约2:3、优选为约3.2:3至约2:3、优选为约3:3至约2:3、优选为约15:3至约2.4:3、优选为约12:3至约2.4:3、优选为约10:3至约2.4:3、优选为约9:3至约2.4:3、优选为约8:3至约2.4:3、优选为约7:3至约2.4:3、优选为约6.9:3至约2.4:3、优选为约6.8:3至约2.4:3、优选为约6.5:3至约2.4:3、优选为约6.3:3至约2.4:3、优选为约6:3至约2.4:3、优选为约5.8:3至约2.4:3、优选为约5.5:3至约2.4:3、优选为约5.3:3至约2.4:3、优选为约5:3至约2.4:3、优选为约4.8:3至约2.4:3、优选为约4.5:3至约2.4:3、优选为约4.2:3至约2.4:3、优选为约4:3至约2.4:3、优选为约3.9:3至约2.4:3、优选为约3.8:3至约2.4:3、优选为约3.5:3至约2.4:3、优选为约3.2:3至约2.4:3、优选为约3:3至约2.4:3、更优选为约1.5:3、更优选为约2:3、更优选为约2.5:3、更优选为约2.75:3、更优选为约3:3。
  45. 如权利要求42-44任一项所述的药物组合物，其中所述胰岛素衍生物为权利要求12、18、24或25所述的胰岛素衍生物；优选，所述胰岛素衍生物为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；或B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  46. 如权利要求42-45任一项所述的药物组合物，其包含约0.09-0.36mM的胰岛素衍生物、约0.18mM的Asp B28人胰岛素、约0.85％至约2.0％(重量/重量)的甘油、约15-70mM的苯酚、约8-14摩尔锌离子/6摩尔的胰岛素衍生物、约10-120mM的氯化钠、约0-15mM间甲酚、和具有约7.0-8.2的pH值，其中所述胰岛素衍生物为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)-二十二烷二酰基-γGlu-6xOEG),desB30人胰岛素；B29K(N(ε)-二十烷二酰基-γGlu-12xOEG),desB30人胰岛素；或B29K(N(ε)-二十二烷二酰基-γGlu-12xOEG),desB30人胰岛素。
  47. 如权利要求42-44任一项所述的药物组合物，其包含约0.165-0.18mM的胰岛素衍生物、约0.18mM的Asp B28人胰岛素、约1.5％-1.7％(重量/重量)的甘油、约20mM-30mM的苯酚、约9-12摩尔锌离子/6摩尔的胰岛素衍生物、约20mM-75mM的氯化钠、约10mM-15mM间甲酚、和具有约7.0-8.2的pH值，其中所述胰岛素衍生物为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；或B29K(N(ε)-二十二烷二酰基-γGlu-8xOEG),desB30人胰岛素。
  48. 权利要求1-25任一项所述的胰岛素衍生物或权利要求26-47任一项 所述的药物组合物，其用作药物。
  49. 权利要求1-25任一项所述的胰岛素衍生物或权利要求26-47任一项所述的药物组合物，其用作用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱的药物。
  50. 权利要求1-25任一项所述的胰岛素衍生物或权利要求26-47任一项所述的药物组合物，其用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱。
  51. 权利要求1-25任一项所述的胰岛素衍生物或权利要求26-47任一项所述的药物组合物在制备药物中的用途；优选地，所述药物用于治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱。
  52. 如权利要求51所述的用途，其中所述药物用于治疗糖尿病，所述胰岛素衍生物以每隔一天或更低频率给予同一患者，并且平均起来，在至少1个月、6个月或1年的时间期间，所述胰岛素衍生物不以更高频率给予同一患者。
  53. 如权利要求51所述的用途，其中所述药物用于治疗糖尿病，所述胰岛素衍生物以一周两次或更低频率给予，并且平均起来，在至少1个月、6个月或1年的时间期间，所述酰化胰岛素不以更高频率给予同一患者。
  54. 治疗或预防糖尿病、高血糖症、和/或葡萄糖耐量削弱的方法，包括施用治疗有效量的权利要求1-25任一项所述的胰岛素衍生物或权利要求26-47任一项所述的药物组合物。
  55. 一种使胰岛素衍生物在白蛋白存在时，对胰岛素受体结合能力提高的方法，所述方法包括：
    经由连接基团Lin将白蛋白结合残基与天然存在的胰岛素或胰岛素类似物连接而得到所述胰岛素衍生物，其中所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)， 其中在形式上羟基已从所述脂肪酸的羧基或所述脂肪族二酸的羧基之一中去除，
    或者
    用式(A)或式(A’)修饰天然存在的胰岛素或胰岛素类似物而得到所述衍生物，III-(II) m-(I) n-  (A)，其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
    (A’)为III-(II) m-(I’) n’-  (A’)，
    其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
    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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
    II和I’在式(A’)中出现的顺序可以独立地互换；和
    (I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个。
  56. 一种使胰岛素衍生物效力提高的方法，包括：
    经由连接基团Lin将白蛋白结合残基与天然存在的胰岛素或胰岛素类似物连接而得到所述胰岛素衍生物，其中所述连接基团Lin是具有至少10个、优选至少15个、优选至少20个、优选至少25个、优选至少30个、优选至少36个、优选至少40个、优选15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个碳原子的亲水性连接基团；所述白蛋白结合残基包含20-40个碳原子，优选所述白蛋白结合残基包含具有20-40个碳原子的线性或分支的亲脂性基团，优选所述白蛋白结合残基是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基或所述脂肪族二酸的羧基之一中去除，
    或者
    用式(A)或式(A’)修饰天然存在的胰岛素或胰岛素类似物而得到所述衍生物，III-(II) m-(I) n-  (A)，其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n为大于等于5的整数，优选n为5-30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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
    (A’)为III-(II) m-(I’) n’-  (A’)，
    其中,
    m为0，1，2，3，4，5，6，7，8，9，或10，n’为整数；
    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是包含具有20-40个碳原子的线性或分支的亲脂性基团的白蛋白结合残基，优选III是包含20-26个碳原子的脂肪酸或脂肪族二酸(更优选20-24 个碳原子的脂肪酸或脂肪族二酸)，其中在形式上羟基已从所述脂肪酸的羧基和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；
    II和I’在式(A’)中出现的顺序可以独立地互换；和
    (I’) n’的总碳原子数为15-200个、优选20-200个、优选25-180个、优选30-180个、优选42-180个、优选54-180个、优选59-180个、优选61-180个、优选66-180个、优选72-120个。
  57. 如权利要求55或56所述的方法，所述天然存在的胰岛素或胰岛素类似物包含至少一个赖氨酸残基，连接基团Lin、式(A)或式(A’)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。
  58. 如权利要求55或56所述的方法，其中，
    n为5-18的整数，优选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20-26个(优选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。
  59. 如权利要求55或56所述的方法，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-；或者，I’是I-HN-(CH 2-CH 2-O) 10-CH 2-CO-、-HN-(CH 2-CH 2-O) 11-CH 2-CO-、-HN-(CH 2-CH 2-O) 12-CH 2-CO-、-HN-(CH 2-CH 2-CH 2-O) 8-CH 2-CO-、--HN-(CH 2-CH 2-O) 20-CH 2-CO-、-HN-(CH 2-CH 2-O) 22-CH 2-CO-、-HN-(CH 2-CH 2-O) 24-CH 2-CO-、或-HN-(CH 2-CH 2-CH 2-O) 15-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，优选地，II选自γGlu或βAsp；和/或
    III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 19-CO-、HOOC-(CH 2) 20-CO-、HOOC-(CH 2) 21-CO-、HOOC-(CH 2) 22-CO-或HOOC-(CH 2) 24-CO-，优选地III是HOOC-(CH 2) 18-CO-、HOOC-(CH 2) 20-CO-或HOOC-(CH 2) 22-CO-。
  60. 如权利要求55或56所述的方法，其中，将式(A)通过I的C末端与所述天然胰岛素或胰岛素类似物的赖氨酸残基或N-末端氨基酸残基的氨基连接或将式(A’)通过I’的C末端与所述天然胰岛素或胰岛素类似物的赖氨酸残基或N-末端氨基酸残基的氨基连接。
  61. 如权利要求55-60任一项所述的方法，其中，式(A)或式(A’)与所述胰岛素母体的赖氨酸残基的ε氨基相连接。
  62. 如权利要求56-61任一项所述的方法，其中，所述天然胰岛素或胰岛素类似物的赖氨酸残基位于B29位处。
  63. 如权利要求56-62任一项所述的方法，其中，所述天然存在的胰岛素或胰岛素类似物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；优选地，所述胰岛素母体为desB30人胰岛素或A14E,B16H,B25H,desB30人胰岛素。
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