WO2024051787A1 - 一种长效酰化胰岛素衍生物及其应用 - Google Patents

一种长效酰化胰岛素衍生物及其应用 Download PDF

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WO2024051787A1
WO2024051787A1 PCT/CN2023/117567 CN2023117567W WO2024051787A1 WO 2024051787 A1 WO2024051787 A1 WO 2024051787A1 CN 2023117567 W CN2023117567 W CN 2023117567W WO 2024051787 A1 WO2024051787 A1 WO 2024051787A1
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chain
insulin
moles
recombinant
long
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PCT/CN2023/117567
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English (en)
French (fr)
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曹海燕
林兆生
朱志伟
张海波
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北京惠之衡生物科技有限公司
惠升生物制药股份有限公司
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Publication of WO2024051787A1 publication Critical patent/WO2024051787A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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

Definitions

  • the present invention relates to the field of insulin derivatives and their applications, in particular to a long-acting acylated insulin derivative and its applications and preparation methods, and to insulin preparations containing the insulin derivatives of the present invention.
  • Diabetes is a group of metabolic disorders of carbohydrates, proteins, fats, etc. caused by absolute or relative insufficient secretion of insulin and/or impairment of insulin utilization.
  • Hyperglycemia is the main sign, and it can be caused by various factors such as genetics and environment. Diabetes is one of the three leading causes of death in humans, with its mortality rate second only to cardiovascular and cerebrovascular diseases and cancer. Diabetes is mainly divided into type 1 diabetes and type 2 diabetes, with most patients suffering from type 2 diabetes (according to statistics, about 90%).
  • Type 2 diabetes diabetes (diabetes mellitus type 2, T2DM) is characterized by hyperglycemia, relative lack of insulin, and insulin resistance.
  • the drugs used clinically to treat type 2 diabetes mainly include biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors and GLP-1 derivatives.
  • GLP-1 derivatives are gradually becoming the main treatment drugs and research hotspots for type 2 diabetes because they have similar hypoglycemic effects as insulin, but at the same time have almost no risk of hypoglycemia, and have both weight loss effects and cardiovascular protective functions. .
  • Insulin is the only hormone in the body that lowers blood sugar and also promotes the synthesis of glycogen, fat, and protein. Exogenous insulin and insulin derivatives are mainly used to treat diabetes. Insulin is composed of two peptide chains, A and B.
  • the A chain of human insulin (Insulin Human) has 11 kinds of 21 amino acids, and the B chain has 15 kinds of 30 amino acids, a total of 51 amino acids; among them, A7 (Cys)-B7 (Cys) , A20(Cys)-B19(Cys)
  • the sulfhydryl groups in the four cysteines form two disulfide bonds to connect the two peptide chains A and B.
  • A6(Cys) and A11(Cys) in the A chain There is also a disulfide bond between them.
  • Insulin is secreted by islet beta cells in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc.
  • endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc.
  • the biological effects of insulin at the cellular level are initiated by binding to specific receptors on the target cell membrane; pancreas
  • the insulin receptor is a specific site on the target cell membrane where insulin acts. It can only bind to insulin or proinsulin containing insulin molecules and is highly specific.
  • Icodec insulin is a long-acting basal insulin derivative under development. Its molecule is designed to remove B30 of insulin and introduce several amino acid mutations: A14E, B16H, and B25H. And connect a C20 fatty acid side chain to B29K. Compared with insulin detemir and insulin degludec, Icodec has a longer half-life. The purpose of A14E, B16H, and B25H mutations is to reduce enzymatic degradation, while weakening the affinity with the insulin receptor (IR), reducing IR-mediated clearance, and further extending the half-life. After injection into the human body, Icodec insulin binds tightly but reversibly to albumin.
  • IR insulin receptor
  • the dosage of Icodec insulin for once-weekly injection is equivalent to that of insulin glargine U100 for once-daily injection, allowing once-weekly dosing.
  • the present invention provides a new long-acting acylated insulin derivative with significantly prolonged action and its application, which can be administered at least once a week.
  • insulin derivative in the present invention refers to a chemically modified insulin analog in which one or more fatty acid side chains are covalently linked to the insulin peptide chain backbone.
  • human insulin and “parent insulin” refer to the natural human insulin hormone without structural changes, and its structure and properties are well known. Human insulin has two polypeptide chains, named A chain (ie, parent insulin A chain) and B chain (ie, parent insulin B chain).
  • amino acid includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) as well as non-proteinogenic (or unnatural) amino acids.
  • Proteinogenic amino acids are amino acids naturally present in proteins, and proteinogenic amino acids are amino acids encoded by the genetic code.
  • Non-proteinogenic amino acids either are not found in proteins or are not produced by standard cellular mechanisms (e.g., they can may have undergone post-translational modification).
  • diabetes includes type 1 diabetes and type 2 diabetes.
  • Type 1 diabetes also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by the destruction of B cells, often resulting in absolute insulin deficiency.
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with primary insulin resistance and therefore relative insulin deficiency, and/or with a primary defect in insulin secretion with insulin resistance.
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with primary insulin resistance and therefore relative insulin deficiency, and/or with a primary defect in insulin secretion with insulin resistance.
  • a first aspect of the present invention provides a novel long-acting acylated insulin derivative, which is composed of a fatty acid side chain and an acylated insulin peptide chain, and the fatty acid side chain is connected to the epsilon amino group of amino acid K on the insulin peptide chain. Acylation linkage.
  • the fatty acid side chain of the present invention is HOOC(CH 2 ) a CO- ⁇ -Glu-(AEEA) 2
  • a is any integer from 14 to 20
  • the side chain is HOOC(CH 2 ) 14 CO- ⁇ -Glu-(AEEA) 2 or HOOC(CH 2 ) 16 CO- ⁇ -Glu-(AEEA) 2
  • the AEEA refers to 2-[2-(2-amino-ethoxy)-ethoxy]-acetic acid, so the ⁇ -Glu-AEEA-AEEA or ⁇ -Glu-(AEEA) 2 represents The chemical formula structure is as follows (s and n are both 1):
  • the peptide chain of the insulin derivative of the present invention consists of an A chain and a B chain, wherein the amino acid sequence of the A chain is as follows:
  • GQAP GIVEQCCTSICSLX 1 QLENYCN(GQAP) m , where n is selected from any integer from 0 to 6, m is selected from any integer from 0 to 6, and X 1 is Y or E;
  • amino acid sequence of the B chain is shown in the following formula:
  • GQAP GQAP r FVNQHLCGSHLVEALX 2 LVCGERGFX 3 YTP(GQAP) t K, where r is selected from any integer from 0 to 6, t is selected from any integer from 0 to 6, X 2 is Y, H or E, and X 3 is F or H;
  • the fatty acid side chain is HOOC(CH 2 ) a CO- ⁇ -Glu-(AEEA) 2 , a is any integer from 14 to 20.
  • n is selected from 0, 1, 2, 3, 4, 5 or 6, m is selected from 0, 1, 2, 3, 4, 5 or 6, and X 1 is Y or E;
  • r is selected from 0, 1, 2, 3, 4, 5 or 6, t is selected from 0, 1, 2, 3, 4, 5 or 6, X 2 is Y, H or E, X 3 is F or H.
  • n is selected from 0, 1, 2 or 3
  • m is selected from 0, 1, 2 or 3
  • r is selected from 0, 1, 2 or 3
  • t is selected from 0, 1, 2 or 3.
  • n is selected from 0, 1, 2 or 3, m is selected from 0; or n is selected from 0, m is selected from 0, 1, 2 or 3; in the B chain, r Selected from 0, 1, 2 or 3, t is selected from 0; or r is selected from 0, t is selected from 0, 1, 2 or 3.
  • the peptide chain of the insulin derivative of the present invention consists of A chain and B chain, wherein the exemplary A chain is selected from:
  • Exemplary said B chain is selected from:
  • the insulin peptide chain of the insulin derivative of the present invention is a specific combination of each of the above-mentioned A chains and each of the B chains. Examples are as follows (only examples are given below. Due to the large number of combinations, not all are listed):
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN and the B chain is FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN
  • the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK;
  • a chain is GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN
  • B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK;
  • the A chain is GIVEQCCTSICSLEQLENYCN
  • the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK, or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK; or,
  • the A chain is GIVEQCCTSICSLEQLENYCN and the B chain is FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK.
  • the present invention provides a long-acting acylated insulin derivative, which is composed of a fatty acid side chain and an acylated insulin peptide chain, and the fatty acid side chain is acylated with the epsilon amino acid of amino acid K on the insulin peptide chain.
  • the fatty acid side chain is HOOC(CH 2 ) 18 CO- ⁇ -Glu-(AEEA) 2
  • the insulin peptide chain of the derivative is composed of A chain and B chain.
  • the insulin peptide chain of the insulin derivative is as follows:
  • a chain is:
  • a chain is:
  • a chain is:
  • a chain is:
  • the second aspect of the present invention provides an injection preparation containing the novel long-acting acylated insulin derivative of the present invention.
  • the injection preparation of the present invention contains the long-acting acylated insulin derivative of the present invention and 1.5-12 moles of Zinc ions/6 moles of insulin derivatives; preferably, 1.5-8 moles of zinc ions/6 moles of insulin derivatives, or 1.5-6 moles of zinc ions/6 moles of insulin derivatives, or 1.5-3.5 moles of zinc ions/ 6 moles of insulin derivative, or 1.5-2.5 moles of zinc ions/6 moles of insulin derivatives, or 3.5-5.5 moles of zinc ions/6 moles of insulin derivatives.
  • the content of the injection insulin derivative of the present invention is 1-9mM; more preferably, the content is 3-8mM, or 3.5-7mM, or 4-6mM.
  • the injection preparation of the present invention further contains glycerol, phenol and/or m-cresol, and sodium chloride.
  • the injection preparation of the present invention contains 1-2% (weight/weight) glycerol, 15-35mM phenol, 15-35mM m-cresol and 0-75mM sodium chloride; or the injection preparation of the present invention contains 1-2% (w/w) glycerol, 45-75mM phenol, 0-20mM m-cresol and 0-75mM sodium chloride.
  • the injection preparation of the present invention further contains 1-9mM of the insulin derivative of the present invention, 1-2% (weight/weight) glycerol, 15-35mM phenol, 15-35mM m-cresol, 0- 75mM sodium chloride and 1.5-12 moles of zinc ions/6 moles of insulin derivative.
  • the content of the insulin derivative is 3-8mM, or 3.5-7mM, or 4-6mM; preferably, the content of the phenol is 16-30mM, or 20-30mM; preferably, the m-cresol is The content is 16-30mM, or 20-30mM; preferably, the content of zinc ions is 1.5-8 moles of zinc ions/6 moles of insulin derivatives, or 1.5-6 moles of zinc ions/6 moles of insulin derivatives, or 1.5 -3.5 moles of zinc ions/6 moles of insulin derivatives, or 1.5-2.5 moles of zinc ions/6 moles of insulin derivatives, or 3.5-5.5 moles of zinc ions/6 moles of insulin derivatives; preferably, the chlorination
  • the content of sodium is 5-50mM, or 5-30mM, or 10-30mM, or 15-25mM, or 20mM.
  • the injection preparation of the present invention further contains 1-9mM of the insulin derivative of the present invention, 1-2% (weight/weight) glycerol, 15-75mM phenol, 0-20mM m-cresol, 0- 75mM sodium chloride and 1.5-12 moles of zinc ions/6 moles of insulin derivative.
  • the content of the insulin derivative is 3-8mM, or 3.5-7mM, or 4-6mM; preferably, the content of the phenol is 45-75mM, or 55-65mM; preferably, the m-cresol The content is 0-15mM, or 0mM; preferably, the content of the zinc ions is 1.5-8 moles of zinc ions/6 moles of insulin derivatives, or 1.5-6 moles of zinc ions/6 moles of insulin derivatives, or 1.5-3.5 moles of zinc ions/6 moles of insulin derivatives, or 1.5-2.5 moles of zinc ions/6 moles of insulin derivatives, or 3.5-5.5 moles of zinc ions/6 moles of insulin derivatives; preferably, the chlorine The content of sodium chloride is 5-50mM, or 5-30mM, or 10-30mM, or 15-25mM, or 20mM.
  • the injection preparation of the present invention has a pH value in the range of 7.0 to 8.5, preferably, the pH value range is in the range of 7.2-8.2.
  • the third aspect of the present invention provides an insulin expressing the long-acting acylated insulin derivative of the present invention.
  • Recombinant engineering bacteria of the peptide chain The recombinant engineering bacteria of the present invention are transfected with a recombinant plasmid.
  • the recombinant plasmid is capable of expressing a recombinant fusion protein containing the insulin peptide chain of the present invention.
  • the recombinant fusion protein is composed of an inclusion body promoting sequence and lysine. It consists of endonuclease cleavage sequence, B chain, C peptide and A chain.
  • the inclusion body promoting sequence is preferably FKFEFKFE (SEQ ID NQ.49), HQHQHQHQHQ (SEQ ID NQ.50) or HQHQHQHQHQ (SEQ ID NQ.51); the lysine endonuclease digestion sequence is K ;
  • the C peptide is preferably GGGPGRK (SEQ ID NQ.52). That is to say, taking FKFEFKFE as an example, the fusion protein structure is: FKFEFKFEK-human B chain-GGGPGRK-A chain.
  • the recombinant engineering bacterium of the present invention is a recombinant E.
  • coli engineering bacterium more preferably, it is a recombinant BL21 (DE3) E. coli engineering bacterium, and the recombinant plasmid is preferably pET-28a(+), pET-30a(+) Or pET-32a(+) recombinant plasmid.
  • a fourth aspect of the present invention provides a method for preparing recombinant engineering bacteria.
  • the recombinant engineering bacteria of the present invention are recombinant Escherichia coli engineering bacteria, and the recombinant engineering bacteria are obtained by the following preparation method:
  • a gene expression fragment encoding a fusion protein consisting of an inclusion-promoting sequence, a lysine endonuclease cleavage sequence, a B chain, a C peptide and an A chain will be constructed;
  • the inclusion body promoting sequence is preferably FKFEFKFE, HQHQHQHQHQ or HQHQHQHQHQ; the lysine endonuclease digestion sequence is K; and the C peptide is preferably GGGPGRK.
  • the recombinant engineering bacterium of the present invention is a recombinant E. coli engineering bacterium, more preferably, it is a recombinant BL21 (DE3) E. coli engineering bacterium, and the recombinant plasmid is preferably pET-28a(+), pET-30a(+) Or pET-32a(+) recombinant plasmid.
  • a fifth aspect of the present invention provides the use of the long-acting acylated insulin derivative described in the first aspect in preparing a pharmaceutical composition for treating diabetes.
  • Diabetes is a group of carbohydrate, protein, fat and other metabolic disorders caused by absolute or relative insufficient secretion of insulin and/or impairment of insulin utilization.
  • Hyperglycemia is the main sign and can be caused by various factors such as genetics and environment.
  • the diabetes can be type 1 diabetes and type 2 diabetes.
  • Type 1 diabetes is also called insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes. It is caused by the destruction of B cells and often leads to terminal illness. Deficiency of insulin.
  • Type 2 diabetes also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with primary insulin resistance and therefore relative insulin deficiency, and/or with a primary defect in insulin secretion with insulin resistance.
  • the long-acting acylated insulin provided by the embodiments of the present invention has excellent blood sugar-lowering ability, and the action time is significantly extended. It can achieve a once-a-week dosing frequency, significantly improve the patient's compliance and willingness to control sugar, and has broader potential. market expectation.
  • Figure 1 is a diagram showing the hypoglycemic effect of insulin derivative HSP002-018 in Example 2 of the present invention
  • Figure 2 is a diagram showing the hypoglycemic effect of the insulin derivative HSP002-070 in Example 2 of the present invention
  • Figure 3 is a diagram showing the hypoglycemic effect of insulin derivatives HSP002-018-3 and HSP002-029 in Example 2 of the present invention
  • Figure 4 is a diagram showing the hypoglycemic effect of insulin derivative HSP002-018-3 in Example 4 of the present invention.
  • Figure 5 is a diagram showing the hypoglycemic effect of insulin derivative HSP002-029 in Example 5 of the present invention.
  • This embodiment provides a method for preparing long-acting acylated insulin derivatives.
  • the preparation method includes the following steps:
  • the peptide chain includes modified insulin A chain (as shown in SEQ ID NQ.1) and modified The insulin B chain (as shown in SEQ ID NQ.4); sequence the peptide chain prepared by expression for later use.
  • step (1) Take the insulin derivative peptide chain prepared in step (1), prepare about 6 mg/mL, adjust the pH to about 11.0, and mix the peptide chain with eicosanedioic acid monotert-butyl ester-glutamic acid (1-tert-butyl ester)-
  • the AEEA-AEEA-OSU molar ratio is 1:4.
  • Continue to add 2 times the volume of the acid solution leave it at room temperature for 1 hour for deprotection, and then add NaOH dropwise to adjust the pH to 7.5-8.5 to terminate the reaction.
  • the reaction solution was diluted 5 times with water, loaded onto UniPS10-300 (purchased from Suzhou Nanowei Technology Co., Ltd.), and eluted with 0 to 100% eluent (10mM TFA, 80% acetonitrile). The purity of the elution peak reached 95% by HPLC. Above, the insulin derivative HSP002-018 was obtained, and the eluent was freeze-dried and stored at 20°C for later use.
  • insulin derivatives HSP002-018-3, HSP002-029, HSP002-070 and control Icodec were prepared.
  • This example demonstrates the hypoglycemic effect of the insulin derivative of the present invention through a hypoglycemic experiment in hyperglycemic mice induced by STZ+HFD.
  • Modeling Select healthy SPF grade male C57 mice aged 6 to 8 weeks, weighing 18 to 20g. After one week of adaptive feeding, the feed is replaced with 60% high-fat feed and fed for 8 to 12 weeks. After the body weight reaches the expected level, the mice are fasted for 16 hours and intraperitoneally injected with STZ (80mpk) to induce a hyperglycemia model. Blood glucose is measured 5 days after induction. A random blood glucose value above 16.8mmol/L indicates that the model is successfully established, such as the first STZ induction. The molding rate is relatively If it is low, then conduct the second induction one week after the first induction using the same method as the first. The non-model mice were eliminated and randomly divided into groups according to blood sugar and body weight.
  • the above administered insulin derivatives and Icodec are administered in equimolar concentrations and equimolar amounts.
  • Blood glucose value Measure blood glucose at 0h before administration, conduct blood glucose testing at 2h, 4h, 6h, 24h and 48h after administration, and draw a blood glucose change curve.
  • hypoglycemic data of insulin derivative HSP002-018 are shown in Table 2 and Figure 1:
  • the insulin derivative HSP002-018 of the present invention has a significantly better blood sugar-lowering effect than Novo Nordisk's once-a-week long-acting acylated insulin derivative Icodec within 48 hours of administration, and the duration of blood sugar reduction is basically the same as that of Icodec. Same or even better than Icodec.
  • hypoglycemic data of insulin derivative HSP002-070 are shown in Table 3 and Figure 2:
  • the insulin derivative HSP002-070 of the present invention has a slightly lower blood sugar-lowering effect within 48 hours of administration than Novo Nordisk's once-a-week long-acting acylated insulin derivative Icodec, but it still has a significant long-acting blood sugar-lowering effect. , especially the duration of blood sugar reduction is basically the same as that of Icodec. At the 48th hour, it basically reaches the same blood sugar value as the positive control group, and at the 48th hour it is still lower than the blood sugar value of the model control group.
  • hypoglycemic data of insulin derivatives HSP002-018-3 and HSP002-029 are shown in Table 4 and Figure 3:
  • the insulin derivatives HSP002-018-3 and HSP002-029 of the present invention have basically the same hypoglycemic effect as Novo Nordisk's once-a-week long-acting acylated insulin derivative Icodec within 48 hours of administration, and The duration of blood sugar reduction is basically the same as that of Icodec. It is still lower than the blood sugar value of the model control group at 48 hours. The blood sugar lowering effect of insulin derivatives HSP002-018-3 and HSP002-029 after 48 hours is even better than that of Icodec.
  • This example demonstrates the half-life of the insulin derivatives HSP002-018, HSP002-018-3, HSP002-029, and HSP002-070 of the present invention through subcutaneous injection PK test in beagle dogs.
  • the insulin derivatives HSP002-018, HSP002-018-3, HSP002-029, HSP002-070 and Icodec were administered in equimolar concentrations and equimolar amounts.
  • Blood collection time points All experimental groups collected blood before and 1h, 3h, 6h, 12h, 24h, 30h, 48h, 54h, 60h, 72h, 78h, 96h, and 102h after drug administration, and separated plasma (EDTA anticoagulant tube, 150 ⁇ L of plasma) was stored in a -80°C refrigerator until testing.
  • the half-lives of the insulin derivatives HSP002-070 and HSP002-018-3 of the present invention are 61.11h and 60.29h respectively, which are both higher than the 56.19h of the positive control group Icodec.
  • the half-life of insulin derivative HSP002-029 is 56.63h, which is basically equivalent to the 56.19h of the positive control group Icodec. It can be seen from this that the half-life of the insulin derivative HSP002-070 and the derivative HSP002-018-3 of the present invention is longer in beagle dogs.
  • This example explores the hypoglycemic effect of the insulin derivative HSP002-018-3 prepared by the present invention.
  • Modeling 45 healthy SPF male C57 mice aged 6 to 8 weeks, weighing 18 to 20 g, were selected. After one week of adaptive feeding, the feed was changed to 60% high-fat feed and fed for 8 to 12 weeks. After the body weight reaches the expected level, the mice are fasted for 16 hours and intraperitoneally injected with STZ (80mpk) to induce a hyperglycemia model. Blood glucose is measured 5 days after induction. A random blood glucose value above 16.8mmol/L indicates that the model is successfully established, such as the first STZ induction. If the mold formation rate is low, the second induction will be carried out one week after the first induction using the same method as the first. The non-model mice were eliminated and randomly divided into groups according to blood sugar and body weight, with 5 mice in each group.
  • the above administered insulin derivatives and Icodec are administered in equimolar concentrations and equimolar amounts.
  • Blood glucose level For the first dose, measure the blood glucose level 0h before dosing and 2h, 4h, 6h, 24h, 48h, and 72h after dosing; measure the blood glucose level 2h, 4h, 6h, 24h, 48h, and 72h after dosing for the second time. Blood glucose, and blood glucose at 2h, 4h, 6h, 24h, 48h, and 72h after the third dose. Draw the blood glucose change curve and calculate the AUC area.
  • hypoglycemic experimental data of insulin derivative HSP002-018-3 are shown in Table 8, Table 9, Table 10 and Figure 4:
  • This example explores the hypoglycemic effect of the insulin derivative HSP002-029 prepared by the present invention.
  • Modeling 45 healthy SPF male C57 mice aged 6 to 8 weeks, weighing 18 to 20 g, were selected. After one week of adaptive feeding, the feed was changed to 60% high-fat feed and fed for 8 to 12 weeks. After the body weight reaches the expected level, the mice are fasted for 16 hours and intraperitoneally injected with STZ (80mpk) to induce a hyperglycemia model. Blood glucose is measured 5 days after induction. A random blood glucose value above 16.8mmol/L indicates that the model is successfully established, such as the first STZ induction. If the mold formation rate is low, the second induction will be carried out one week after the first induction using the same method as the first. The non-model mice were eliminated and randomly divided into groups according to blood sugar and body weight, with 5 mice in each group.
  • the above administered insulin derivatives and Icodec are administered in equimolar concentrations and equimolar amounts.
  • Blood glucose level For the first dose, measure the blood glucose level 0h before dosing and 2h, 4h, 6h, 24h, 48h, and 72h after dosing; measure the blood glucose level 2h, 4h, 6h, 24h, 48h, and 72h after dosing for the second time. Blood glucose, and blood glucose at 2h, 4h, 6h, 24h, 48h, and 72h after the third dose. Draw the blood glucose change curve and calculate the AUC area.
  • hypoglycemic experimental data of insulin derivative HSP002-029 are shown in Table 12, Table 13, Table 14 and Figure 5:
  • the hypoglycemic effect and effective glucose control time of three doses of the insulin derivative HSP002-029 of the present invention have obvious dose correlation during the entire administration cycle.
  • the higher the dose the greater the hypoglycemic effect and the effective time.
  • doses of 94 nmol/kg and 188 nmol/kg the insulin derivative HSP002-029 has the same hypoglycemic effect as the positive control group administered with equal molar amounts, and the effective glucose control time exceeds 72 hours.

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Abstract

提供一种长效酰化胰岛素衍生物及其应用和制备方法。所述长效酰化胰岛素衍生物由脂肪酸侧链与胰岛素肽链酰化连接而成，所述脂肪酸侧链与胰岛素肽链上氨基酸K的ε氨基酰化连接；胰岛素衍生物的肽链由经修饰的胰岛素A链和经修饰的胰岛素B链组成。所述长效酰化胰岛素衍生物具备显著延长的作用时间，可用于治疗糖尿病，能够显著提高患者的依从度和控糖意愿，具备广阔的市场前景。

Description

一种长效酰化胰岛素衍生物及其应用
本发明要求于2022年09月09日提交中国专利局、申请号为202211104924.7、发明名称为“一种长效酰化胰岛素衍生物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及胰岛素衍生物及其应用领域，尤其涉及一种长效酰化胰岛素衍生物及其应用和制备方法等，并涉及包含本发明胰岛素衍生物的胰岛素制剂。
背景技术
糖尿病是一组因胰岛素绝对或相对分泌不足和/或胰岛素利用障碍引起的碳水化合物、蛋白质、脂肪等代谢紊乱性疾病，以高血糖为主要标志，可由遗传和环境等多种因素引起。糖尿病是人类三大致死疾病之一，它的死亡率仅次于心脑血管疾病和癌症。糖尿病主要分为1型糖尿病和2型糖尿病，其中大多数患者为2型糖尿病患者(据统计，约占90％)。2型糖尿病(diabetes mellitus type2，T2DM)，患者特征为高血糖、相对缺乏胰岛素、胰岛素抵抗等。目前，临床上使用的治疗2型糖尿病的药物主要有双胍类、磺酰脲类、噻唑烷二酮类、DPP-4受体抑制剂、SGLT-2受体抑制剂和GLP-1衍生物。而其中，GLP-1衍生物由于具有与胰岛素类似的降糖效果，但同时几乎无低血糖风险、兼具减重效果和心血管保护功能，正逐渐成为2型糖尿病的主要治疗药物和研究热点。
胰岛素是机体内唯一降低血糖的激素，同时促进糖原、脂肪、蛋白质合成，外源性胰岛素及胰岛素衍生物主要用来治疗糖尿病。胰岛素由A、B两个肽链组成，人胰岛素(Insulin Human)A链有11种21个氨基酸，B链有15种30个氨基酸，共51个氨基酸；其中A7(Cys)-B7(Cys)、A20(Cys)-B19(Cys)四个半胱氨酸中的巯基形成两个二硫键，使A、B两条肽链连接起来，此外A链中A6(Cys)与A11(Cys)之间也存在一个二硫键。胰岛素由胰脏内的胰岛β细胞受内源性或外源性物质如葡萄糖、乳糖、核糖、精氨酸、胰高血糖素等的刺激而分泌。胰岛素在细胞水平的生物作用是通过与靶细胞膜上的特异受体结合而启动的；胰 岛素受体为胰岛素起作用的靶细胞膜上特定部位，仅可与胰岛素或含有胰岛素分子的胰岛素原结合，具有高度的特异性。
胰岛素虽然是治疗糖尿病最有效的手段，但目前上市胰岛素产品最长为一天一次使用，因此，患者需要频繁注射，以便满足身体对胰岛素的需求，但是频繁的注射会给患者带来很大痛苦，因此，人们希望可以通过延长胰岛素的降糖效果，以减少注射的次数，减少患者的痛苦。
Icodec胰岛素是一种在研的长效基础胰岛素衍生物，其分子设计为去除胰岛素的B30，同时引入若干个氨基酸突变：A14E、B16H、B25H。并在B29K连接一个C20的脂肪酸侧链。相对于地特胰岛素和德谷胰岛素，Icodec具备更长半衰期。A14E、B16H、B25H突变目的在于减少酶切降解，同时减弱与胰岛素受体(IR)的亲和力，减少IR介导的清除，进一步延长半衰期。在注射进人体后，Icodec胰岛素会与白蛋白紧密但可逆的结合在一起。这一结果可在一周时间内连续、缓慢且稳定地降低血糖。基于其浓缩配方，每周注射一次的Icodec胰岛素用量与每日注射一次的甘精胰岛素U100相当，从而可以实现一周一次给药。
尽管如此，为达到更好的改性效果，以获得更好的药效，延长胰岛素衍生物的半衰期，提高其降糖活性，提供更多的长效的达到一周甚至一周以上注射一次的胰岛素衍生物的需求仍然存在。
发明内容
为了解决上述技术问题，本发明提供了一种具备显著延长作用时效的新型长效酰化胰岛素衍生物及其应用，可实现至少每周给药一次。
本发明中术语“胰岛素衍生物”是指经化学修饰的胰岛素类似物，其中一个或多个脂肪酸侧链共价连接至胰岛素肽链骨架。
术语“人胰岛素”、“母体胰岛素”指未经结构变化的、天然的人胰岛素激素，其结构和性质是众所周知的。人胰岛素具有两条多肽链，被命名为A链(即母体胰岛素A链)和B链(即母体胰岛素B链)。
术语“氨基酸”包括蛋白型(proteinogenic)(或天然)氨基酸(其中有20种标准氨基酸)以及非蛋白型(或非天然)氨基酸。蛋白型氨基酸是天然地存在于蛋白质中的氨基酸，蛋白型氨基酸是由遗传密码编码的氨基酸。非蛋白型氨基酸或者不存在于蛋白质中，或者不通过标准细胞机制产生(例如，它们可 能已经经历翻译后修饰)。
术语“糖尿病”包括1型糖尿病、2型糖尿病。“1型糖尿病”也称为胰岛素依赖型糖尿病(IDDM)和幼年型糖尿病，是由B细胞破坏引起的，通常导致绝对胰岛素缺乏。“2型糖尿病”也称为非胰岛素依赖型糖尿病(NIDDM)和成年型糖尿病，与主要的胰岛素抵抗相关，因此与相对胰岛素缺乏相关，并且/或者与主要的胰岛素分泌缺陷伴胰岛素抵抗相关。
本发明第一方面，提供一种新型长效酰化胰岛素衍生物，该衍生物由脂肪酸侧链与胰岛素肽链酰化连接而成，所述脂肪酸侧链与胰岛素肽链上氨基酸K的ε氨基酰化连接。
优选地，本发明的脂肪酸侧链为HOOC(CH2)aCO-γ-Glu-(AEEA)2，a为14-20的任意整数，优选所述侧链为HOOC(CH2)14CO-γ-Glu-(AEEA)2或HOOC(CH2)16CO-γ-Glu-(AEEA)2，HOOC(CH2)18CO-γ-Glu-(AEEA)2或HOOC(CH2)20CO-γ-Glu-(AEEA)2。
其中，所述AEEA是指2-[2-(2-氨基-乙氧基)-乙氧基]-乙酸，故所述γ-Glu-AEEA-AEEA或γ-Glu-(AEEA)2表示的化学式结构如下所示(s和n均为1)：
本发明的胰岛素衍生物的肽链由A链和B链组成，其中，A链的氨基酸序列如下式所示：
(GQAP)nGIVEQCCTSICSLX1QLENYCN(GQAP)m，其中，n选自0-6的任意整数，m选自0-6的任意整数，X1为Y或E；
B链氨基酸序列如下式所示：
(GQAP)rFVNQHLCGSHLVEALX2LVCGERGFX3YTP(GQAP)tK，其中，r选自0-6的任意整数，t选自0-6的任意整数，X2为Y、H或E，X3为F或H；
所述脂肪酸侧链为HOOC(CH2)aCO-γ-Glu-(AEEA)2，a为14-20的任意整数。
优选地，所述A链中，n选自0、1、2、3、4、5或6，m选自0、1、2、3、4、5或6，X1为Y或E；所述B链中，r选自0、1、2、3、4、5或6，t选自0、1、2、3、4、5或6，X2为Y、H或E，X3为F或H。
更优选地，所述A链中，n选自0、1、2或3，m选自0、1、2或3；所述 B链中，r选自0、1、2或3，t选自0、1、2或3。
更优选地，所述A链中，n选自0、1、2或3，m选自0；或n选自0，m选自0、1、2或3；所述B链中，r选自0、1、2或3，t选自0；或r选自0，t选自0、1、2或3。
优选地，本发明的胰岛素衍生物的肽链由A链和B链组成，其中，示例性的所述A链选自：
GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN(SEQ ID NQ.1)，
GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.2)，
GQAPGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.3)，
GQAPGQAPGIVEQCCTSICSLYQLENYCN(SEQ ID NQ.7)，
GQAPGIVEQCCTSICSLYQLENYCN(SEQ ID NQ.8)，
GQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.9)，
GIVEQCCTSICSLEQLYNYCN(SEQ ID NQ.10)，或
GIVEQCCTSICSLEQLENYCN(SEQ ID NQ.11)；
示例性的所述B链选自：
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.4)，
GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.5)，
FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.6)，
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPGQAPK(SEQ ID NQ.12)，
GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.13)，
GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.14)，
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NQ.15)，
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK(SEQ ID  NQ.16)，
GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK(SEQ ID NQ.17)，
GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK(SEQ ID NQ.18)，
FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPGQAPK(SEQ ID NQ.19)，
FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.20)，
FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.21)，
GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPK(SEQ ID NQ.22)，
GQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPK
(SEQ ID NQ.23)，
GQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.24)，
GQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPK(SEQ ID NQ.25)，
GQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPK(SEQ ID NQ.26)，
GQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPK(SEQ ID NQ.27)，
GQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPK(SEQ ID NQ.28)，
GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NQ.29)，
GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK(SEQ ID NQ.30)，
GQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK(SEQ ID NQ.31)，
GQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK(SEQ ID NQ.32)，
GQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NQ.33)，
GQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK(SEQ ID NQ.34)，
GQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK(SEQ ID NQ.35)，
GQAPFVNQHLCGSHLVEALELVCGERGFHYTPK(SEQ ID NQ.36)，
FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPK(SEQ ID NQ.37)，
FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.38)，
FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.39)，
FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.40)，
FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPK(SEQ ID NQ.41)，
FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPK(SEQ ID NQ.42)，
FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPK(SEQ ID NQ.43)，
FVNQHLCGSHLVEALELVCGERGFHYTPGQAPK(SEQ ID NQ.44)，
FVNQHLCGSHLVEALYLVCGERGFFYTPK(SEQ ID NQ.45)，
FVNQHLCGSHLVEALYLVCGERGFHYTPK(SEQ ID NQ.46)，
FVNQHLCGSHLVEALHLVCGERGFHYTPK(SEQ ID NQ.47)，或
FVNQHLCGSHLVEALELVCGERGFHYTPK(SEQ ID NQ.48)。
优选地，本发明胰岛素衍生物的胰岛素肽链中，分别为上述每一条A链分别与每一条B链的具体组合，举例如下(仅例举如下，由于组合数量众多，未全部列举)：
A链为GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN，B链为FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GQAPGQAPGIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK；或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK；，或，
A链为GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK；，或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK，或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK；或，
A链为GIVEQCCTSICSLEQLENYCN，B链为FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK。
优选地，本发明提供一种长效酰化胰岛素衍生物，该衍生物由脂肪酸侧链与胰岛素肽链酰化连接而成，所述脂肪酸侧链与胰岛素肽链上氨基酸K的ε氨基酰化连接，所述脂肪酸侧链为HOOC(CH2)18CO-γ-Glu-(AEEA)2，所述胰岛素 衍生物的肽链由A链和B链组成，具体的，所述胰岛素衍生物的胰岛素肽链如下所示：
(1)胰岛素衍生物HSP002-018
A链为：
GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN(SEQ ID NQ.1)；
B链为：
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.4)；
(2)胰岛素衍生物HSP002-018-3
A链为：
GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.2)；
B链为：
FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.6)；
(3)胰岛素衍生物HSP002-029
A链为：
GQAPGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.3)；
B链为：
GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK(SEQ ID NQ.5)；
(4)胰岛素衍生物HSP002-070
A链为：
GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN(SEQ ID NQ.2)；
B链为：
GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK(SEQ ID NQ.4)。
本发明的第二方面，提供一种包含本发明新型长效酰化胰岛素衍生物的注射液制剂，本发明的注射液制剂包含本发明所述长效酰化胰岛素衍生物和1.5-12摩尔的锌离子/6摩尔胰岛素衍生物；优选地，1.5-8摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-6摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-3.5摩尔的锌离子/6 摩尔胰岛素衍生物，或1.5-2.5摩尔的锌离子/6摩尔胰岛素衍生物，或3.5-5.5摩尔的锌离子/6摩尔胰岛素衍生物。优选地，本发明的注射液胰岛素衍生物的含量为1-9mM；更优选地，含量为3-8mM，或3.5-7mM，或4-6mM。
进一步地，本发明的注射液制剂进一步包含甘油、苯酚和/或间甲酚、氯化钠。优选地，本发明注射液制剂含有1-2％(重量/重量)的甘油、15-35mM的苯酚、15-35mM的间甲酚和0-75mM的氯化钠；或者本发明注射液制剂含有1-2％(重量/重量)的甘油、45-75mM的苯酚、0-20mM的间甲酚和0-75mM的氯化钠。
进一步地，本发明的注射液制剂进一步包含1-9mM的本发明的胰岛素衍生物、1-2％(重量/重量)的甘油、15-35mM的苯酚、15-35mM的间甲酚、0-75mM的氯化钠和1.5-12摩尔的锌离子/6摩尔胰岛素衍生物。优选地，胰岛素衍生物的含量为3-8mM，或3.5-7mM，或4-6mM；优选地，所述苯酚的含量为16-30mM，或20-30mM；优选地，所述间甲酚的含量为16-30mM，或20-30mM；优选地，锌离子的含量为1.5-8摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-6摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-3.5摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-2.5摩尔的锌离子/6摩尔胰岛素衍生物，或3.5-5.5摩尔的锌离子/6摩尔胰岛素衍生物；优选地，所述氯化钠的含量为5-50mM，或5-30mM，或10-30mM，或15-25mM，或20mM。
进一步地，本发明的注射液制剂进一步包含1-9mM的本发明的胰岛素衍生物、1-2％(重量/重量)的甘油、15-75mM的苯酚、0-20mM的间甲酚、0-75mM的氯化钠和1.5-12摩尔的锌离子/6摩尔胰岛素衍生物。优选地，胰岛素衍生物的含量为为3-8mM，或3.5-7mM，或4-6mM；优选地，所述苯酚的含量为45-75mM，或55-65mM；优选地，所述间甲酚的含量为0-15mM，或0mM；优选地，所述锌离子的含量为1.5-8摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-6摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-3.5摩尔的锌离子/6摩尔胰岛素衍生物，或1.5-2.5摩尔的锌离子/6摩尔胰岛素衍生物，或3.5-5.5摩尔的锌离子/6摩尔胰岛素衍生物；优选地，所述氯化钠的含量为5-50mM，或5-30mM，或10-30mM，或15-25mM，或20mM。
进一步地，本发明的注射液制剂具有在7.0至8.5范围内的pH值，优选地，所述pH值范围在7.2-8.2。
本发明的第三方面，提供一种表达本发明长效酰化胰岛素衍生物的胰岛素 肽链的重组工程菌，本发明的重组工程菌转染重组质粒，所述重组质粒能够表达包含本发明胰岛素肽链的重组融合蛋白，所述重组融合蛋白是由促包涵体序列、赖氨酸内切酶酶切序列、B链、C肽和A链组成。
其中，所述促包涵体序列优选为FKFEFKFE(SEQ ID NQ.49)、HQHQHQHQHQ(SEQ ID NQ.50)或HQHQHQHQHQHQ(SEQ ID NQ.51)；所述赖氨酸内切酶酶切序列为K；所述C肽优选为GGGPGRK(SEQ ID NQ.52)。也就是说，以FKFEFKFE为例，所述融合蛋白结构为：FKFEFKFEK-人B链-GGGPGRK-A链。优选地，本发明的重组工程菌为重组大肠杆菌工程菌，更优选地，为重组BL21(DE3)大肠杆菌工程菌，所述重组质粒优选为pET-28a(+)、pET-30a(+)或pET-32a(+)重组质粒。
本发明的第四方面，提供一种重组工程菌的制备方法。
本发明所述重组工程菌为重组大肠杆菌工程菌，所述重组工程菌由如下制备方法得到：
(1)将构建编码由促包涵体序列、赖氨酸内切酶酶切序列、B链、C肽和A链组成的融合蛋白的基因表达片段；
(2)将所述基因表达片段插入原核表达质粒，得到相应融合蛋白的表达质粒；
(3)将所述表达质粒转入大肠杆菌，得到表达所述融合蛋白的重组工程菌。
所述促包涵体序列优选为FKFEFKFE、HQHQHQHQHQ或HQHQHQHQHQHQ；所述赖氨酸内切酶酶切序列为K；所述C肽优选为GGGPGRK。优选地，本发明的重组工程菌为重组大肠杆菌工程菌，更优选地，为重组BL21(DE3)大肠杆菌工程菌，所述重组质粒优选为pET-28a(+)、pET-30a(+)或pET-32a(+)重组质粒。
本发明的第五方面，提供第一方面所述的长效酰化胰岛素衍生物在制备用于治疗糖尿病的药物组合物中的应用。
所述糖尿病是一组因胰岛素绝对或相对分泌不足和/或胰岛素利用障碍引起的碳水化合物、蛋白质、脂肪等代谢紊乱性疾病，以高血糖为主要标志，可由遗传和环境等多种因素引起。
所述糖尿病可以为1型糖尿病和2型糖尿病，“1型糖尿病”也称为胰岛素依赖型糖尿病(IDDM)和幼年型糖尿病，是由B细胞破坏引起的，通常导致绝 对胰岛素缺乏。“2型糖尿病”也称为非胰岛素依赖型糖尿病(NIDDM)和成年型糖尿病，与主要的胰岛素抵抗相关，因此与相对胰岛素缺乏相关，并且/或者与主要的胰岛素分泌缺陷伴胰岛素抵抗相关。
本发明实施例提供的上述技术方案与现有技术相比具有如下优点：
本发明实施例提供的长效酰化胰岛素具备优良的降血糖能力，并且作用时间显著延长，可以实现一周一次的给药频率，可显著提高患者的依从度和控糖意愿，具备更为广阔的市场前景。
应当理解的是，以上的一般描述和后文的细节描述仅是示例性和解释性的，并不能限制本发明。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本发明的实施例，并与说明书一起用于解释本发明的原理。
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例2的胰岛素衍生物HSP002-018的降糖效果图；
图2为本发明实施例2的胰岛素衍生物HSP002-070的降糖效果图；
图3为本发明实施例2的胰岛素衍生物HSP002-018-3和HSP002-029的降糖效果图；
图4为本发明实施例4的胰岛素衍生物HSP002-018-3的降糖效果图；
图5为本发明实施例5的胰岛素衍生物HSP002-029的降糖效果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1胰岛素衍生物的制备
本实施例提供了长效酰化胰岛素衍生物的制备方法，以胰岛素衍生物HSP002-018为例，所述制备方法包括如下步骤：
(1)按照专利CN94193852.2中实施例11所述方法制备本发明胰岛素衍生物HSP002-018的肽链，肽链包括经修饰的胰岛素A链(如SEQ ID NQ.1所示)和经修饰的胰岛素B链(如SEQ ID NQ.4所示)；对表达制备的肽链测序后备用。
(2)脂肪酸侧链连接
取步骤(1)制备的胰岛素衍生物肽链，配制6mg/mL左右，调pH至11.0左右，按照肽链与二十烷二酸单叔丁酯-谷氨酸(1-叔丁酯)-AEEA-AEEA-OSU摩尔比1:4称取脂肪酸粉末于乙腈中，两者混合，室温静置1h，加酸调pH为4.8终止反应。继续加2倍体积的酸溶液，置于室温静置脱保护1h，然后滴入NaOH调节pH至7.5-8.5终止反应。
反应液用水稀释5倍，上样UniPS10-300(购自苏州纳微科技有限公司)，0～100％洗脱液(10mM TFA，80％乙腈)洗脱，洗脱峰HPLC检测纯度达到95％以上，获得胰岛素衍生物HSP002-018，洗脱液冻干20℃保存备用。
按照相同的方法，制备得到胰岛素衍生物HSP002-018-3、HSP002-029、HSP002-070及对照Icodec。
实施例2 STZ+HFD诱导高血糖小鼠单剂量单次给药降糖实验
本实施例通过STZ+HFD诱导高血糖小鼠降糖实验，证明本发明胰岛素衍生物的降糖效果。
(1)实验材料
实验动物：STZ+HFD诱导的C57小鼠，6～8周龄，雄性；
实验药物制剂：甘油19.6mg/mL，苯酚1.5mg/mL，间甲酚1.72mg/mL，二水乙酸锌110.43μg/mL，胰岛素衍生物20.0nmol/mL。
(2)实验方法
a.造模与分组：
造模：选取健康SPF级雄性6～8周龄的C57小鼠，体重18～20g，在适应性饲养一周后将饲料更换为60％高脂饲料，饲喂8～12周。在体重达到预期后，小鼠禁食16h后使用STZ(80mpk)腹腔注射诱导高血糖模型，诱导5天后检测血糖，随机血糖值在16.8mmol/L以上为造模成功，如第一次STZ诱导成模率较 低，则在第一次诱导的一周后采用与第一次相同方法进行2次诱导。将未成模小鼠淘汰，按照血糖和体重随机分组。
b.给药方式：按照表1皮下给药：
表1：给药方式
由于分子量差异，上述给药胰岛素衍生物与Icodec为等摩尔浓度及等摩尔量给药。
c.检测指标
血糖值：给药前测量0h血糖，在给药后2h、4h、6h、24h和48h进行血糖检测，绘制血糖变化曲线。
(3)实验结果
胰岛素衍生物HSP002-018的降糖数据如表2和图1所示：
表2
由表2可知，本发明胰岛素衍生物HSP002-018在给药48h内，有显著优于诺和诺德一周一次长效酰化胰岛素衍生物Icodec的降糖效果，且降糖持续时间与Icodec基本相同，甚至优于Icodec。
胰岛素衍生物HSP002-070的降糖数据如表3和图2所示：
表3
由表3可知，本发明胰岛素衍生物HSP002-070在给药48h内，降糖效果略低于诺和诺德一周一次长效酰化胰岛素衍生物Icodec，但仍具备显著的长效降糖效果，尤其降糖持续时间与Icodec基本相同，在第48h基本达到与阳性对照组相同血糖值，在第48h仍低于模型对照组的血糖值。
胰岛素衍生物HSP002-018-3和HSP002-029的降糖数据如表4和图3所示：
表4
由表4可知，本发明的胰岛素衍生物HSP002-018-3和HSP002-029在给药48h内，具有与诺和诺德一周一次长效酰化胰岛素衍生物Icodec基本相同的降糖效果，且降糖持续时间与Icodec同样基本相同，在第48h仍低于模型对照组的血糖值，胰岛素衍生物HSP002-018-3和HSP002-029在48h后的降糖效果甚至优于Icodec。
实施例3比格犬皮下注射PK实验
本实施例通过比格犬皮下注射PK试验，证明本发明胰岛素衍生物HSP002-018、HSP002-018-3、HSP002-029、HSP002-070的半衰期。
(1)实验材料
实验动物：比格犬，17-19月龄，雄性；体重15kg
实验药物制剂：甘油19.6mg/mL，苯酚1.5mg/mL，间甲酚1.72mg/mL，二水乙酸锌110.43μg/mL，胰岛素衍生物HSP002-018、HSP002-018-3、HSP002-029、HSP002-070的浓度均为80.0nmol/mL。
(2)实验方法
a.给药方式：按照表5皮下给药：
表5给药方式
由于分子量差异，上述给药胰岛素衍生物HSP002-018、HSP002-018-3、HSP002-029、HSP002-070与Icodec为等摩尔浓度及等摩尔量给药。
b.检测指标
采血时间点：所有实验组在给药前和给药后1h、3h、6h、12h、24h、30h、48h、54h、60h、72h、78h、96h、102h采血，分离血浆(EDTA抗凝管，150μL血浆)放入-80℃冰箱保存待测。
(3)实验结果
胰岛素衍生物HSP002-018、HSP002-018-3、HSP002-029、HSP002-070半衰期数据如表6所示：
表6
由表6可知，与Icodec相比，本发明胰岛素衍生物HSP002-070和HSP002-018-3的半衰期分别为61.11h和60.29h，均高于阳性对照组Icodec的56.19h。胰岛素衍生物HSP002-029的半衰期为56.63h，与阳性对照组Icodec的56.19h基本相当。由此可知，在比格犬体内本发明胰岛素衍生物HSP002-070和衍生物HSP002-018-3的半衰期更长。
实施例4 STZ+HFD诱导高血糖小鼠多剂量多次给药降糖实验(HSP002-018-3)
本实施例探究本发明制备得到的胰岛素衍生物HSP002-018-3的降糖效果。
(1)实验材料
实验动物：STZ+HFD诱导的C57小鼠，6～8周龄，雄性；
实验药物制剂：甘油19.6mg/mL，苯酚1.5mg/mL，间甲酚1.72mg/mL，二水乙酸锌110.43μg/mL，胰岛素衍生物20.0nmol/mL。
(2)实验方法
a.造模与分组：
造模：选取健康SPF级雄性6～8周龄的C57小鼠45只，体重18～20g，在适应性饲养一周后将饲料更换为60％高脂饲料，饲喂8～12周。在体重达到预期后，小鼠禁食16h后使用STZ(80mpk)腹腔注射诱导高血糖模型，诱导5天后检测血糖，随机血糖值在16.8mmol/L以上为造模成功，如第一次STZ诱导成模率较低，则在第一次诱导的一周后采用与第一次相同方法进行2次诱导。将未成模小鼠淘汰，按照血糖和体重随机分组，每组5只。
b.给药方式：按照表7皮下给药
表7给药方式
由于分子量差异，上述给药胰岛素衍生物与Icodec为等摩尔浓度及等摩尔量给药。
c.检测指标
血糖值：第一次给药时测定给药前0h血糖值及给药后2h、4h、6h、24h、48h、72h血糖，第二次给药后2h、4h、6h、24h、48h、72h血糖，以及第三次给药后2h、4h、6h、24h、48h、72h血糖。绘制血糖变化曲线，计算AUC面积。
(3)实验结果
胰岛素衍生物HSP002-018-3的降糖实验数据如表8、表9、表10和图4所示：
表8
表9

表10
由表8-10可知，本发明胰岛素衍生物HSP002-018-3三个剂量在整个给药周期内，降血糖作用和有效控糖时间均有明显的剂量相关性，剂量越高，降血糖作用越大，有效控糖时间越长。HSP002-018-3在94nmol/kg和188nmol/kg剂量下，降血糖作用均优于等摩尔给药的阳性对照组，且有效控糖时间超过72h。
实施例5 STZ+HFD诱导高血糖小鼠多剂量多次给药降糖实验(HSP002-029)
本实施例探究本发明制备得到的胰岛素衍生物HSP002-029的降糖效果。
(1)实验材料
实验动物：STZ+HFD诱导的C57小鼠，6～8周龄，雄性；
实验药物制剂：甘油19.6mg/mL，苯酚1.5mg/mL，间甲酚1.72mg/mL，二水乙酸锌110.43μg/mL，胰岛素衍生物20.0nmol/mL。
(2)实验方法
a.造模与分组：
造模：选取健康SPF级雄性6～8周龄的C57小鼠45只，体重18～20g，在适应性饲养一周后将饲料更换为60％高脂饲料，饲喂8～12周。在体重达到预期后，小鼠禁食16h后使用STZ(80mpk)腹腔注射诱导高血糖模型，诱导5天后检测血糖，随机血糖值在16.8mmol/L以上为造模成功，如第一次STZ诱导成模率较低，则在第一次诱导的一周后采用与第一次相同方法进行2次诱导。将未成模小鼠淘汰，按照血糖和体重随机分组，每组5只。
b.给药方式：按照表11皮下给药
表11给药方式
由于分子量差异，上述给药胰岛素衍生物与Icodec为等摩尔浓度及等摩尔量给药。
c.检测指标
血糖值：第一次给药时测定给药前0h血糖值及给药后2h、4h、6h、24h、48h、72h血糖，第二次给药后2h、4h、6h、24h、48h、72h血糖，以及第三次给药后2h、4h、6h、24h、48h、72h血糖。绘制血糖变化曲线，计算AUC面积。
(3)实验结果
胰岛素衍生物HSP002-029的降糖实验数据如表12、表13、表14和图5所示：
表12

表13
表14

由表14可知，本发明胰岛素衍生物HSP002-029三个剂量在整个给药周期内，降血糖作用和有效控糖时间均有明显的剂量相关性，剂量越高，降血糖作用越大，有效控糖时间越长。胰岛素衍生物HSP002-029在94nmol/kg和188nmol/kg剂量下，降血糖作用与等摩尔给药的阳性对照组相同，且有效控糖时间超过72h。
需要说明的是，在本文中，诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上所述仅是本发明的具体实施方式，使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (11)

  1. 一种长效酰化胰岛素衍生物，所述衍生物是由脂肪酸侧链酰化连接至胰岛素肽链的氨基酸K上的ε氨基而成；
    其特征在于，所述胰岛素肽链由A链和B链组成，其中，所述A链的序列如下式所示：
    (GQAP)nGIVEQCCTSICSLX1QLENYCN(GQAP)m，其中，n选自0-6的任意整数，m选自0-6的任意整数，X1为Y或E；
    所述B链的序列如下式所示：
    (GQAP)rFVNQHLCGSHLVEALX2LVCGERGFX3YTP(GQAP)tK，其中，r选自0-6的任意整数，t选自0-6的任意整数，X2为Y、H或E，X3为F或H；
    所述脂肪酸侧链为HOOC(CH2)aCO-γ-Glu-(AEEA)2，a为14-20的任意整数；
    优选地，所述A链中，n选自0、1、2、3、4、5或6，m选自0、1、2、3、4、5或6，X1为Y或E；所述B链中，r选自0、1、2、3、4、5或6，t选自0、1、2、3、4、5或6，X2为Y、H或E，X3为F或H；
    优选地，所述A链中，n选自0、1、2或3，m选自0、1、2或3；所述B链中，r选自0、1、2或3，t选自0、1、2或3。
  2. 根据权利要求1所述的长效酰化胰岛素衍生物，其特征在于：
    所述A链中，n选自0、1、2或3，m选自0；或n选自0，m选自0、1、2或3；
    和/或，所述B链中，r选自0、1、2或3，t选自0；或r选自0，t选自0、1、2或3。
  3. 根据权利要求1或2所述的长效酰化胰岛素衍生物，其特征在于：
    所述A链选自：
    GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN，
    GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN，
    GQAPGQAPGIVEQCCTSICSLYQLENYCN，
    GQAPGQAPGIVEQCCTSICSLEQLENYCN，
    GQAPGIVEQCCTSICSLYQLENYCN，
    GQAPGIVEQCCTSICSLEQLENYCN，
    GIVEQCCTSICSLEQLYNYCN，或
    GIVEQCCTSICSLEQLENYCN；
    和/或，所述B链选自：
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPGQAPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK，
    GQAPGQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK，
    FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPGQAPK，
    FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK，
    FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPGQAPK，
    FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPGQAPK，
    GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPK，
    GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK，
    GQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPK，
    GQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQ APK，
    GQAPFVNQHLCGSHLVEALYLVCGERGFFYTPGQAPK，
    GQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPK，
    GQAPFVNQHLCGSHLVEALHLVCGERGFHYTPGQAPK，
    GQAPFVNQHLCGSHLVEALELVCGERGFHYTPGQAPK，
    GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK，
    GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK，
    GQAPGQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK，
    GQAPGQAPFVNQHLCGSHLVEALELVCGERGFHYTPK，
    GQAPFVNQHLCGSHLVEALYLVCGERGFFYTPK，
    GQAPFVNQHLCGSHLVEALYLVCGERGFHYTPK，
    GQAPFVNQHLCGSHLVEALHLVCGERGFHYTPK，
    GQAPFVNQHLCGSHLVEALELVCGERGFHYTPK，
    FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPGQAPK，
    FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK，
    FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPGQAPK，
    FVNQHLCGSHLVEALELVCGERGFHYTPGQAPGQAPK，
    FVNQHLCGSHLVEALYLVCGERGFFYTPGQAPK，
    FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPK，
    FVNQHLCGSHLVEALHLVCGERGFHYTPGQAPK，
    FVNQHLCGSHLVEALELVCGERGFHYTPGQAPK，
    FVNQHLCGSHLVEALYLVCGERGFFYTPK，
    FVNQHLCGSHLVEALYLVCGERGFHYTPK，
    FVNQHLCGSHLVEALHLVCGERGFHYTPK，或
    FVNQHLCGSHLVEALELVCGERGFHYTPK；
    和/或，所述侧链为HOOC(CH2)14CO-γ-Glu-(AEEA)2，HOOC(CH2)16CO-γ-Glu-(AEEA)2，HOOC(CH2)18CO-γ-Glu-(AEEA)2或HOOC(CH2)20CO-γ-Glu-(AEEA)2。
  4. 根据权利要求1-3任一项所述的长效酰化胰岛素衍生物，其特征在于：所述衍生物由脂肪酸侧链酰化连接至胰岛素肽链的氨基酸K 上的ε氨基而成，所述脂肪酸侧链为HOOC(CH2)18CO-γ-Glu-(AEEA)2，所述胰岛素肽链由A链和B链组成；
    其中，
    A链为：GQAPGQAPGQAPGIVEQCCTSICSLYQLENYCN；
    B链为：
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；
    或，
    A链为：GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN；
    B链为：
    FVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK；
    或，
    A链为：GQAPGQAPGIVEQCCTSICSLEQLENYCN；
    B链为：
    GQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPK；
    或，
    A链为：GQAPGQAPGQAPGIVEQCCTSICSLEQLENYCN；
    B链为：
    GQAPGQAPGQAPFVNQHLCGSHLVEALYLVCGERGFHYTPGQAPGQAPGQAPK。
  5. 一种长效酰化胰岛素衍生物的注射液制剂，其特征在于：所述注射液制剂包含权利要求1-4任一项所述的长效酰化胰岛素衍生物和1.5-12摩尔的锌离子/6摩尔胰岛素衍生物；优选地，锌离子的含量为1.5-8摩尔的锌离子/6摩尔胰岛素衍生物，1.5-6摩尔的锌离子/6摩尔胰岛素衍生物，1.5-3.5摩尔的锌离子/6摩尔胰岛素衍生物，1.5-2.5摩尔的锌离子/6摩尔胰岛素衍生物，或3.5-5.5摩尔的锌离子/6摩尔胰岛素衍生物；优选地，胰岛素衍生物的含量为1-9mM，3-8mM，3.5-7mM，或4-6mM。
  6. 根据权利要求5所述的注射液制剂，其特征在于：所述注射液 制剂进一步包含甘油、苯酚和/或间甲酚、氯化钠；优选地，所述注射液制剂含有1-2％(重量/重量)的甘油、15-35mM的苯酚、15-35mM的间甲酚和0-75mM的氯化钠；更优选地，含有1-2％(重量/重量)的甘油、45-75mM的苯酚、0-20mM的间甲酚和0-75mM的氯化钠。
  7. 根据权利要求5所述的注射液制剂，其特征在于：所述注射液制剂进一步包含1-2％(重量/重量)的甘油、15-35mM的苯酚、15-35mM的间甲酚、0-75mM的氯化钠；优选地，所述苯酚的含量为16-30mM或20-30mM；优选地，所述间甲酚的含量为16-30mM或20-30mM；优选地，所述氯化钠的含量为5-50mM，5-30mM，10-30mM，15-25mM或20mM。
  8. 根据权利要求5-7任一项所述的注射液制剂，其特征在于：注射液制剂具有在7.0至8.5范围内的pH值，优选地，所述pH值范围在7.2-8.2。
  9. 一种表达权利要求1-4任一项所述长效酰化胰岛素衍生物的胰岛素肽链的重组工程菌，所述重组工程菌转染有重组质粒，所述重组质粒能够表达包含胰岛素肽链的重组融合蛋白，所述重组融合蛋白由促包涵体序列、赖氨酸内切酶酶切序列、B链、C肽和A链组成；
    其中，所述促包涵体序列优选为FKFEFKFE、HQHQHQHQHQ或HQHQHQHQHQHQ；所述赖氨酸内切酶酶切序列为K；所述C肽优选为GGGPGRK；
    优选地，所述重组工程菌为重组大肠杆菌工程菌，更优选为重组BL21(DE3)大肠杆菌工程菌；
    优选地，所述重组质粒为pET-28a(+)、pET-30a(+)或pET-32a(+)重组质粒。
  10. 权利要求9所述的重组工程菌的制备方法，所述重组工程菌由如下制备方法得到：
    (1)构建编码由促包涵体序列、赖氨酸内切酶酶切序列、B链、C肽和A链组成的融合蛋白的基因表达片段；
    (2)将所述基因表达片段插入原核表达质粒，得到相应融合蛋白的表达质粒；
    (3)将所述表达质粒转入大肠杆菌，得到表达所述融合蛋白的重组工程菌。
  11. 权利要求1-4任一项所述的长效酰化胰岛素衍生物在制备用于治疗糖尿病的药物组合物中的应用。
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