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WO2018024186A1 - 一种人胰岛素或其类似物的酰化衍生物 - Google Patents

一种人胰岛素或其类似物的酰化衍生物 Download PDF

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WO2018024186A1
WO2018024186A1 PCT/CN2017/095377 CN2017095377W WO2018024186A1 WO 2018024186 A1 WO2018024186 A1 WO 2018024186A1 CN 2017095377 W CN2017095377 W CN 2017095377W WO 2018024186 A1 WO2018024186 A1 WO 2018024186A1
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human insulin
acylated derivative
zinc complex
insulin analog
group
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PCT/CN2017/095377
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English (en)
French (fr)
Inventor
刘德玉
王宏伟
赵亮
王菲菲
曾翔
王涛
王亚里
戴雨璐
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江苏恒瑞医药股份有限公司
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Priority to CN202210131921.6A priority Critical patent/CN114478747B/zh
Priority to JP2019503703A priority patent/JP7432361B2/ja
Priority to EP17836368.5A priority patent/EP3495384A4/en
Priority to CN201780005001.7A priority patent/CN108463468B/zh
Priority to US16/320,997 priority patent/US10815287B2/en
Publication of WO2018024186A1 publication Critical patent/WO2018024186A1/zh
Priority to JP2022066071A priority patent/JP2022095872A/ja

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    • 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
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to an acylated derivative of a human insulin analog.
  • Diabetes Mellitus is a common metabolic endocrine disease characterized by the absolute or relative lack of insulin in the body, or the chronic hyperglycemia caused by the insensitivity of the target tissue to insulin, accompanied by disorders of glucose, fat and protein metabolism.
  • Clinical chronic, systemic metabolic syndrome It is caused by the interaction of genetic and environmental factors, involving various systems of the human body, including cardiovascular and cerebral vascular, kidney, eye, nerve and other organ complications, which seriously endanger human health and is a lifelong disease.
  • Diabetes has become a common and frequently-occurring disease. It is the third most threatening human life after cancer and cardiovascular and cerebrovascular diseases. It is a challenge for all human beings, and its harm to people's life and health is irrespective of race and country.
  • IDF International Diabetes Federation
  • the total number of diabetic patients increased four-fold to 120 million in the mid-1980s to mid-1990s.
  • the number of people with diabetes worldwide was 246 million, of which 46% were 40-59 years old. It is estimated that by 2025, the number of diabetic patients worldwide will increase to 380 million, accounting for 7.1% of the world's adult population.
  • WO2005012347 and WO2013086927 disclose acylated derivatives of two different classes of human insulin analogs which are modified by covalently linking an acylation group in the form of a fatty acid to an amino acid of a human insulin analog. After that, their half-life is greatly increased relative to existing insulin analogs.
  • physiological insulin needs include basic and mealtime (fast-acting), the former provides full-day glycemic control, and the latter mainly responds to postprandial blood glucose.
  • the ideal mealtime insulin preparation can quickly reach its peak after eating, and can be reduced to the basic level when blood sugar drops to normal level to avoid hypoglycemia before eating.
  • Ideal basal insulin should have a very long duration of action (>24h in all patients), minimal amplitude of insulin action variability (reduced hypoglycemia) and safety, and can be combined with fast-acting insulin (convenience).
  • the present invention provides an acylated derivative of a human insulin analog or a zinc complex thereof, the acylated derivative of the human insulin analog having a structure of the formula:
  • S is the human insulin of the 30th threonine deletion of the B chain;
  • -W-X-Y-Z is an acylation modifying group of the insulin analog;
  • W is a diacyl structure having -OC(CH 2 ) n CO-, wherein n is an integer between 2 and 10, the structure having one of its acyl groups and the A-chain or B-chain of the parent insulin or analog thereof
  • n is an integer between 2 and 10
  • the ⁇ -amino group of the lysine residue present on the ⁇ -amino group or the B-chain of the N-terminal amino acid residue forms an amide bond;
  • X is a carboxylic acid group-containing diamino compound which is bonded to one of the acyl groups in one of its amino groups to form an amide bond;
  • Y is -A(CH 2 ) m -, wherein m is an integer of 6-32, preferably an integer of 10-16, particularly preferably an integer of 12-14, A is absent or CO-;
  • Z is -COOH.
  • W forms an amide bond with the ⁇ -amino group of the lysine residue present on the B-chain.
  • n is an integer between 2 and 5, preferably 2.
  • Human insulin which is deleted at the 30th threonine of the B chain, is a human insulin analog whose amino acid sequences of the A and B chains are as follows:
  • the carboxylic acid group-containing diamino compound represented by X may be -HN(CH 2 )pCH(COOH)NH-, wherein p is an integer of 2 to 10, preferably an integer of 2 to 6, particularly preferably an integer of 2 to 4. Most preferably 4.
  • -W-X-Y-Z has the following structure:
  • the present invention is particularly preferred acylating derivative of a human insulin analogue to follow customary named N ⁇ - (HOOC (CH 2 ) 14 CO) -N ⁇ - (OCCH 2 CH 2 CO- (N ⁇ B29 -Des (B30 ) human insulin))-Lys-OH, with
  • lysine B29 N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)
  • B30 human insulin having the formula (Ib) The structure shown.
  • the present invention also provides a process for preparing an acylated derivative of the above human insulin analog or a zinc complex thereof, which comprises a compound of the formula (III) and a human insulin in which the 30th threonine of the B chain is deleted. step,
  • R is a leaving group, preferably an activated ester group, more preferably selected from
  • a preferred structure of the compound of formula (III) may be as shown in (IIIa) or (IIIb).
  • the preparation method further comprises the step of removing a carboxy protecting group of the compound of formula (III') to give a compound of formula (III),
  • X' and Z' each represent a form in which all of the carboxyl groups in X and Z are protected by a protecting group, and the protecting group is preferably a C 1 -C 6 alkyl group, preferably a C 1 -C 4 alkyl group, particularly preferably Methyl or tert-butyl; R is as defined in formula (III).
  • the zinc complex of the acylated derivative of the human insulin analog is a 6-mer of an acylated derivative of a human insulin analog, wherein each 6-mer comprises more than Four zinc atoms, i.e., 6 molecules of acylated insulin, contain more than 4 zinc atoms; more preferably 5 to 8 zinc atoms, particularly preferably 5 zinc atoms.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, and further comprising a pharmaceutically acceptable carrier.
  • the present invention also provides the use of the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof or a pharmaceutical composition thereof for the preparation of a medicament for treating diabetes.
  • the present invention also provides a pharmaceutical composition comprising a fast-acting insulin in addition to the acylated derivative of the above human insulin analog or a zinc complex thereof.
  • the present invention also relates to the use of an acylated derivative of the above human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same, for the preparation of a medicament for treating diabetes.
  • the present invention also relates to the use of the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same, in combination with a fast-acting insulin for the preparation of a medicament for treating diabetes.
  • the present invention provides a method for treating diabetes, which comprises administering to a patient in need of treatment the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same.
  • Another method of treating diabetes comprises administering to a patient in need of treatment the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same and a fast-acting insulin.
  • the present invention also relates to the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same as a medicament for treating diabetes.
  • the present invention also relates to the aforementioned acylated derivative of a human insulin analog or a zinc complex thereof, or a pharmaceutical composition containing the same, in combination with a fast-acting insulin as a medicament for treating diabetes.
  • Diabetes in the present invention includes type I diabetes and type II diabetes.
  • Figure 1 shows the change in blood glucose level after administration of STZ rats ( * P ⁇ 0.05, ** P ⁇ 0.01 vs model control group; # P ⁇ 0.05 vs de Guins)
  • Figure 2 shows the change in blood glucose level after STZ rat administration within 0 to 6 hours.
  • Figure 3 shows a comparison of blood glucose AUC in 0-2, 0-4, and 0-6 hours after administration ( * P ⁇ 0.05, ** P ⁇ 0.01 vs INS-C)
  • X04 (22 g, 50.1 mmol) was dissolved in dry THF (250 mL), and stirred at room temperature, lysine derivative (20.5 g, 55 mmol) and triethylamine (21 mL) were added to the reaction system and stirred at room temperature for 24 h. The mixture was filtered through Celite, washed with EtOAc EtOAc.
  • X11 (30.6 g, 46.3 mmol) was dissolved in anhydrous ethanol (200 mL), stirred at room temperature, and then added to a mixture of 6.0 g of 10% Pd/C, followed by hydrogen, and the reaction was vigorously stirred at room temperature overnight. The mixture was filtered with Celite, washed three times with anhydrous ethanol, and the filtrate was dried to give 24.5 g of the crude product of X12, which was directly used for the next reaction.
  • the reaction was started in the 30th threonine-depleted human insulin solution, and the addition was completed in 4 portions while stirring. After the addition was completed, the reaction was started. After 1 h of reaction, the pH of the solution was adjusted to about 7.5 with acetic acid, and the reaction was terminated to obtain a crude solution.
  • the reaction process was controlled by RP-HPLC.
  • the above crude precursor solution was diluted with water to have an organic phase content of about 15% (v:v), filtered through a 0.45 ⁇ m filter, and purified by RP-HPLC to obtain a purified liquid.
  • the above purified solution was replaced with water for injection using an ultrafiltration membrane package system, and lyophilized to obtain 26 mg of the lyophilized product.
  • the resulting molecular structure is as follows.
  • Lysine B29 N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) des(B30) human insulin structure confirmation
  • Lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) des(B30) human insulin measured mass spectrum molecular mass 6203.37Da, and theoretical molecular weight 6203.21Da is consistent.
  • Enzymatic hydrolysis of INS with V8 protease and LC-MS analysis of the hydrolyzed product showed that a total of 4 peptides were produced with molecular weights of 416.23 Da (A1-A4) and 2296.29 Da (A5-A17, B1, respectively).
  • Example 2 Investigating the hypoglycemic effect of test drugs on STZ-induced type 1 diabetes model
  • INS-1 and INS-2 respectively represent lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) des (B30)
  • Human insulin is prepared as two different complexes of each of the insulin derivative hexamers comprising 5 Zn and 8 Zn, the formulation method of which is a conventional method in the art, and the hexamer contains 5 Zn per hexamer.
  • test sample was stored at 4 ° C in the dark, and the appropriate amount of the sample liquid was 600 nmol/mL, diluted 400 times to 1.5 nmol/mL with a solvent, and the dose was 7.5 nmol/kg.
  • the drug is returned to room temperature during use.
  • the SPF-grade rat was kept in the laboratory environment for 7 days, and was fed with standard feed and standard cage at a temperature of 20-25 ° C and a humidity of 40-60%.
  • the rats were fasted for 16 hours, and the STZ (65 mg/kg) was quickly injected into the abdominal cavity. After 1 hour, the rats were fed back. After the model is made, give plenty of water (providing normal 2-3 times of drinking water), food, and change the litter 1-2 times a day to keep it dry.
  • fasting blood glucose was measured (fasting for 6 hours), and rats with a blood glucose value of >16.7 mmol/L were picked. According to the blood sugar level, they were divided into 4 groups.
  • the rats were not fasted, and each drug was injected subcutaneously.
  • the control group was injected with the same volume in the vehicle, and the whole process was fasted after the administration.
  • the blood glucose level of the rats was measured at 1, 2, 4, 6, 8, 10, 12, and 24 hours after administration. After the blood glucose level was measured at 24 hours, blood was collected to prepare serum.
  • the blood glucose level of the STZ vehicle group was 27.3 mmol/L before administration, and the blood glucose level gradually decreased after administration, reaching 15.1 mmol/L after 24 hours, and the blood glucose change value was 12.1 mmol/L.
  • the change of blood glucose level before and after administration of the positive drug deglutamine group was 16.8 mmol/L.
  • the blood glucose levels were significantly different from those of the vehicle-negative control group, indicating the effectiveness of the animal model and experimental method.
  • test drugs INS-1 and INS-2 significantly reduced the blood glucose level of the animals within 24 hours after administration, and were significant at the 6, 8, 10, 12 and 24 hour time points. Differences, and blood glucose levels continue to decline, indicating that it has a good long-acting hypoglycemic effect.
  • test drugs INS-1 and INS-2 showed similar changes in the blood glucose level of STZ rats with the time after administration, indicating that the hypoglycemic effects of the two drugs were similar.
  • the hypoglycemic effect of INS-1 and INS-2 was better than that of the deGlutamine group, indicating that INS-1 and INS-2 have better long-acting effects.
  • Example 3 Investigating the hypoglycemic effect of various long-acting insulins on STZ-induced type 1 diabetes models
  • INS-A stands for HS061 in WO2013086927
  • INS-B stands for HS067 in WO2013086927
  • INS-C stands for lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl))des(B30) human insulin, all tested using the same formulation, and in the form of a zinc complex, each insulin hexamer contains 5 Zn.
  • test sample was stored at 4 ° C in the dark, and the appropriate amount of the test sample mother solution was 600 nmol/mL, and diluted with the solvent 80 times to 7.5 nmol/mL, and the dose was 7.5 nmol/kg.
  • the drug is returned to room temperature during use.
  • the SPF-grade rat was kept in the laboratory environment for 7 days, and was fed with standard feed and standard cage at a temperature of 20-25 ° C and a humidity of 40-60%.
  • the rats were fasted for 16 hours, and the STZ (65 mg/kg) was quickly injected into the abdominal cavity. After 1 hour, the rats were fed back. After the model is made, give plenty of water (providing normal 2-3 times of drinking water), food, and change the litter 1-2 times a day to keep it dry.
  • fasting blood glucose was measured (fasting for 6 hours), and rats with a blood glucose value of >16.7 mmol/L were picked. According to the blood glucose level, they were randomly divided into 5 groups.
  • the rats were not fasted, and each drug was injected subcutaneously.
  • the control group was injected with the same volume in the vehicle, and the whole process was fasted after the administration.
  • the blood glucose level of the rats was measured at 1, 2, 4, 6, 8, 10, 12, and 24 hours after administration.
  • the blood glucose level of the STZ vehicle group was 27.01 mmol/L before administration, and the blood glucose level gradually decreased after administration. After 24 hours, the blood glucose level reached 11.64 mmol/L, and the blood glucose change value was 15.37 mmol/L.
  • the change of blood glucose level before and after administration of the positive drug deglutamine group was 19.78 mmol/L.
  • the blood glucose levels of the positive drug deglutathione group were significantly different from those of the vehicle negative control group at 1, 2, 4, 6, 8, 10 and 12 hours after administration, indicating that the animal model and experimental method were effective. Sex.
  • the test drugs INS-A, INS-B and INS-C can significantly reduce the blood glucose level of the animal within 24 hours after administration. From the blood glucose level, the blood glucose level of the test drug ( Separately 7.03 ⁇ 1.15, 3.57 ⁇ 0.44, and 4.20 ⁇ 0.72 mmol/L were lower than the vehicle negative control group (11.64 ⁇ 3.94 mmol/L). This indicates that all three have a relatively good long-acting hypoglycemic effect. The differences between the groups in reducing blood glucose within 0 to 6 hours were compared. The specific values are shown in Tables 1 and 2:
  • INS-C and DeGol Insulin In STZ rats, the blood glucose level of INS-C group decreased less than that of Deguin after administration, and was significantly higher than that of DeGlutamine group after 1 hour of administration (see Figure 2 and Table 1). .
  • the specific values are shown in Table 1.
  • the area under the blood glucose-time curve (AUC) of INS-C was significantly higher than that of insulin in the 0-2, 0-4, and 0-6 hours after administration.
  • INS-C+ fast-acting insulin has a significantly lower risk of hypoglycemia within 0 to 6 hours after administration (time period when fast-acting insulin exerts hypoglycemic effect) if it is administered in combination with fast-acting insulin or a compound preparation.
  • Degutin + fast-acting insulin has a significantly lower risk of hypoglycemia within 0 to 6 hours after administration (time period when fast-acting insulin exerts hypoglycemic effect.
  • the blood glucose level of INS-C group decreased less than INS-A after administration, and was significantly higher than INS-A group after 1, 2 and 4 hours of administration;
  • the drug was significantly lower than the INS-A group after 10 and 12 hours, and the blood glucose level was also lower than that of the INS-A group after 24 hours of administration.
  • INS-C has a slower blood glucose lowering rate than INS-A, and its long-acting effect is better than INS-A, which is more in line with the characteristics of long-acting insulin.
  • the blood glucose AUC of INS-C was significantly higher after 0-2, 0-4, and 0-6 hours after administration.
  • INS-A This indicates that INS-C+ fast-acting insulin is significantly less likely to develop hypoglycemia within 0 to 6 hours after administration than INS-A+ fast-acting insulin if administered in combination with fast-acting insulin or as a combination preparation.
  • INS-C and INS-B for STZ rats the blood glucose level of INS-C group decreased less than INS-B after administration, and was significantly higher than INS-B group after 1 and 2 hours of administration, indicating that INS-C decreased. Blood sugar is slower than INS-B.
  • the blood glucose AUC of INS-C was significantly higher than that of INS-B within 0-2, 0-4, and 0-6 hours after administration. This indicates that INS-C+ fast-acting insulin is significantly less likely to develop hypoglycemia within 0 to 6 hours after administration than INS-B+ fast-acting insulin if administered in combination with fast-acting insulin or as a compound preparation.
  • INS-C Combined with the effect of lowering blood glucose within 24 hours and 0-6 hours, INS-C has the characteristics of long-lasting hypoglycemic effect and mild hypoglycemic effect. It is the most ideal long-acting insulin, especially suitable for combination with quick-acting insulin or composition. Reduce the number of injections and improve patient compliance.
  • Example 4 Canine PK test (INS-B and INS-C have the same meaning as in Example 3)
  • INS-B Six male beagle dogs (3 in each group) were injected subcutaneously with INS-B or INS-C (3nmol/kg). Blood was collected at different time points and serum was separated to detect the concentration of INS-B or INS-C in serum. . The results showed that INS-B peaked at 4.7 ⁇ 1.2 hours (4 animals for 4, 4, and 6 hours, respectively), while INS-C peaked for 8 hours (all animals were 8 hours). The half-lives of INS-B and INS-C were 6.0 ⁇ 1.2 and 7.8 ⁇ 1.1 hours, respectively. This suggests that the INS-C action is more gradual and has a longer duration of action.
  • INS-A, INS-B, INS-C (various codes have the same meaning as in Example 3), and insulin glutamine were prepared according to the formulation of Degu insulin on the market, and then their stability was examined, and the initiality was determined by HPLC. The purity of the main peak and the impurity content at each time point are shown in Table 3. As can be seen from Table 3, the stability of INS-C is significantly better than that of INS-A and INS-B; slightly better than Degu insulin.

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Abstract

一种人胰岛素或其类似物的酰化衍生物。具体而言，一种人胰岛素类似物的酰化衍生物或其锌复合物，该酰化衍生物相较于已有的胰岛素类似物及其酰化衍生物更加长效，且降血糖更加平稳，储存更加稳定。

Description

一种人胰岛素或其类似物的酰化衍生物 技术领域
本发明涉及一种人胰岛素类似物的酰化衍生物。
背景技术
糖尿病(Diabetes Mellitus，DM)是一种常见的代谢内分泌疾病，是由于体内胰岛素绝对或相对缺乏，或靶组织对胰岛素不敏感而引起的慢性高血糖为特征的伴随糖、脂肪和蛋白质代谢紊乱的临床慢性、全身性代谢综合症。它由遗传和环境因素相互作用而引起，涉及人体各个系统，包括心脑血管、肾、眼、神经等脏器并发症，严重危害人类健康，是一种终生疾病。
糖尿病已成为一种常见病、多发病，是继癌症、心脑血管病之后，第三大威胁人类生命的疾病，是全人类的挑战，其对人们生命健康的危害不分种族和国家。据国际糖尿病联盟(IDF)的统计，上个世纪八十年代中期至九十年代中期十年间，糖尿病患者总量增长4倍，达到1.2亿。2007年全球糖尿病患者人数为2.46亿，其中46％为40-59岁劳动力人口。预计到2025年全世界糖尿病患者将增加到3.8亿，占到世界成年人口的7.1％。
使用人胰岛素是治疗糖尿病的一种主要方法，但是人胰岛素作用时间较短，使得病人必须频繁注射，极为不便。因此，人们致力于获得一些能够更长时间作用于人体的胰岛素类似物及其衍生物。其中，使用酰化基团修饰人胰岛素或其类似物，是一种提高其半衰期的有效方法。WO2005012347和WO2013086927分别公开了两类不同的人胰岛素类似物的酰化衍生物，此类衍生物是通过将脂肪酸形式的酰化基团共价连接到人胰岛素类似物的氨基酸上，经过此种修饰后，它们的半衰期相对于现有的胰岛素类似物大大提高。然而，仍然需要提供半衰期更长的胰岛素类似物或其酰化衍生物，以提高治疗效果，并维持血糖平稳。并且现有的酰化胰岛素还存在不稳定的问题，也需要提供更为稳定的胰岛素类似物或其酰化衍生物。
另一方面，生理性胰岛素需求包括基础和餐时(速效)，前者提供全天血糖控制，后者主要应对餐后血糖升高。根据其生理特点，理想的餐时胰岛素制剂能在进餐后迅速达峰发挥作用，并能在血糖降到正常水平时降至基础水平，以避免下餐前低血糖。理想的基础胰岛素则应有超长作用时间(在所有患者中持续＞24h)、最小幅度胰岛素作用变异性(减少低血糖)和安全性，且能与速效胰岛素结合使用(便利性)。由于速效胰岛素降血糖作用十分迅速，这就要求当基础胰岛素与其合用时应当提供一种较为平缓的降血糖作用，以免二者共同作用时产生低血糖的风险。因此，提供一种具有足够长的作用时间，且初始作用时平缓降血糖的基 础胰岛素具有十分迫切的需求。
发明内容
本发明的目的是提供一种新的人胰岛素类似物的酰化衍生物，其降低血糖的效果更佳持久，且具有更高的稳定性。
本发明提供的一种人胰岛素类似物的酰化衍生物或其锌复合物，所述人胰岛素类似物的酰化衍生物具有如下通式如示的结构：
S-W-X-Y-Z
(I)
其中S为B链第30位苏氨酸缺失的人胰岛素；-W-X-Y-Z为胰岛素类似物的酰化修饰基团；
其中W为具有-OC(CH2)nCO-的二酰基结构，其中n为2～10之间整数，该结构以其酰基之一和母体胰岛素或其类似物的A-链或B-链N-末端氨基酸残基的α-氨基或B-链上存在的赖氨酸残基的ε-氨基形成酰胺键；
X为含羧酸基团的二氨基化合物，该化合物以其氨基之一与W中的一个酰基连接形成酰胺键；
Y为-A(CH2)m-，其中m为6-32的整数，优选10-16的整数，特别优选12-14的整数，A不存在或CO-；
Z为-COOH。
优选地，W与B-链上存在的赖氨酸残基的ε-氨基形成酰胺键。优选地，n为2～5之间的整数，优选2。
B链第30位苏氨酸缺失的人胰岛素，是一种人胰岛素类似物，其A、B链的氨基酸序列如下所示：
A链：GIVEQCCTSICSLYQLENYCN         SEQ ID NO.1
B链：FVNQHLCGSHLVEALYLVCGERGFFYTPK         SEQ ID NO.2。
X代表的含羧酸基团的二氨基化合物可以为-HN(CH2)pCH(COOH)NH-，其中p为2-10的整数，优选2-6的整数，特别优选2-4的整数，最优选4。
在本发明特别优选的实施方式中，-W-X-Y-Z具有如下结构：
Figure PCTCN2017095377-appb-000001
优选
Figure PCTCN2017095377-appb-000002
本发明中特别优选的人胰岛素类似物的酰化衍生物可依习惯命名为Nα-(HOOC(CH2)14CO)-Nε-(OCCH2CH2CO-(NεB29-Des(B30)人胰岛素))-Lys-OH，其具
有如下式(Ia)的具体结构，
Figure PCTCN2017095377-appb-000003
更优选，赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素，其具有式(Ib)所示的结构。
Figure PCTCN2017095377-appb-000004
本发明还提供了一种制备前述的人胰岛素类似物的酰化衍生物或其锌复合物的制备方法，其包括使式(III)化合物与B链第30位苏氨酸缺失的人胰岛素的步骤，
R-W-X-Y-Z
(III)
其中R为离去基团，优选活化酯基团，更优选选自
Figure PCTCN2017095377-appb-000005
Figure PCTCN2017095377-appb-000006
优选的式(III)化合物具体结构可为(IIIa)或(IIIb)所示，
Figure PCTCN2017095377-appb-000007
所述的制备方法优选还包括式(III’)化合物脱除羧基保护基得到式(III)化合物的步骤，
R-W-X’-Y-Z’→R-W-X-Y-Z
(III’)             (III)
其中，X’、Z’分别代表X、Z中所有的羧基被保护基保护的形式，所述的保护基优选C1-C6的烷基，优选C1-C4的烷基，特别优选甲基或特丁基；R如通式(III)中定义。
在本发明特别优选的实施方案中，人胰岛素类似物的酰化衍生物的锌复合物是一种人胰岛素类似物的酰化衍生物的6聚体，其中每个6聚体中包含多于4个锌原子，即6分子酰化胰岛素包含多于4个锌原子；更优选5至8个锌原子，特别优选5个锌原子。
本发明还提供了一种药物组合物，含前述的人胰岛素类似物的酰化衍生物或其锌复合物，还含有药学上可接受的载体。
本发明还提供了前述的人胰岛素类似物的酰化衍生物或其锌复合物或其药物组合物在制备治疗糖尿病的药物中的用途。
本发明还提供一种药物组合物，除含有上述人胰岛素类似物的酰化衍生物或其锌复合物外，还含有速效胰岛素。
本发明还涉及上述人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物在制备治疗糖尿病的药物中的用途。
本发明还涉及前述的人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物与速效胰岛素联合在制备治疗糖尿病的药物中的用途。
另外，本发明提供了一种治疗糖尿病的方法，所述方法包括对需要治疗的患者给予前述的人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物。
本发明提供的另一种治疗糖尿病的方法，所述方法包括对需要治疗的患者给予前述的人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物和速效胰岛素。本发明还涉及前述的人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物作为治疗糖尿病的药物。
本发明还涉及前述的人胰岛素类似物的酰化衍生物或其锌复合物、或含有它们的药物组合物联合速效胰岛素作为治疗糖尿病的药物。
本发明中的糖尿病包括I型糖尿病和II型糖尿病。
附图说明
图1显示STZ大鼠给药后血糖值变化曲线(*P<0.05，**P<0.01vs模型对照组；#P<0.05vs德谷胰岛素)
图2显示0～6小时内STZ大鼠给药后血糖值变化曲线
图3显示给药后0～2、0～4和0～6小时内血糖AUC的比较(*P<0.05，**P<0.01vs INS-C)
具体实施方式
以下结合实施例进一步描述解释本发明。
实施例1：
赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素的制备
1、Nα-(十六烷脂肪二酸)-Nε-(3-酰基丙酸-OSu)赖氨酸的制备
Figure PCTCN2017095377-appb-000008
取X01(150g，524.5mmol)，室温下加入干燥的THF(2.5L)，随后加入催化量的DMF(1.0mL)，将草酰氯(49mL)加入到100mL的恒压滴液漏斗中，将草酰氯缓慢滴加到反应瓶中，期间有气体生成，需不断放气，约滴加两小时。滴加完毕后，室温搅拌1.5h，随后减压旋干THF，向反应瓶中加入DCM(800mL)和叔丁醇(500mL)，室温下搅拌过夜。旋干叔丁醇和二氯甲烷，加入二氯甲烷(1L)，过滤，除去不溶固体。滤液旋干过柱。收集所需成分，旋干，石油醚重结晶，得54g产品X02，同时回收二叔丁酯产物X03。
将X02(28.5g，83.3mmol)溶于DCM(200mL)中，室温下加入N-羟基丁二酰亚胺(5.54g，48.1mmol)和二异丙基碳二酰亚胺(7.6mL)，室温搅拌1天，TLC显示基本反应完全。过滤，除去不溶固体，减压旋干溶剂，柱层析纯化，得28.5g X04。
将X04(22g，50.1mmol)溶于干燥的THF(250mL)中，室温搅拌，赖氨酸衍生物(20.5g，55mmol)和三乙胺(21mL)分别加入到反应体系中，室温搅拌24h。硅藻土过滤，THF洗涤三次，旋干溶剂，柱层析纯化，得31g产物X11。
将X11(30.6g，46.3mmol)溶于无水乙醇(200mL)中，室温搅拌，加入6.0g10％Pd/C,随后通入氢气，室温剧烈搅拌反应过夜。硅藻土抽滤，无水乙醇洗涤三次，滤液旋干，得24.5g X12粗品，直接用于下一步反应。
将X12(24g，45.6mmol)溶于干燥THF(200mL)中，加入三乙胺(12.7mL)，随后降温至0℃。将丁二酸酐(5.2g，52mmol)分批加入到反应体系中，0℃下继续搅拌30min，随后转移至室温搅拌过夜。减压旋干THF，剩余物溶于二氯甲烷(500mL)中，用5％柠檬酸水溶液(500mL*2)洗涤2次，饱和食盐水洗涤1次，无水硫酸钠干燥。减压旋干溶剂，得29.0g X13粗品，直接用于下一步反应。
将X13(28.5g，45.5mmol)溶于DCM(200mL)中，室温下加入N-羟基丁二酰亚胺(5.54g，48.2mmol)和二异丙基碳二酰亚胺(7.6mL)，室温搅拌1天，TLC显示基本反应完全。过滤，除去不溶固体，减压旋干溶剂，柱层析纯化，得28.5g产品X14。
将X14(3.0g，4.1mmol)溶于三氟乙酸(15mL)中，室温搅拌45min，随后低温减压旋干三氟乙酸，加入无水乙醚，有固体析出，过滤，滤饼用无水乙醚洗涤三次，固体干燥后，得1.8g产品X15。
2、赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素的制备
取B链第30位苏氨酸缺失的人胰岛素53mg(8mg/mL，50mM Tris HCl pH8.5缓冲液)，用1.5M Na2CO3调节pH至约10.75，后用缓冲液(50mM Tris HCl pH8.5缓冲液)定容至4mg/mL。将Nα-(十六烷脂肪二酸)-Nε-(3-酰基丙酸-OSu)赖氨酸(18mg)悬浮于7mL乙腈后，每隔15min将1.75mL本溶液加入到上述B链第30位苏氨酸缺失的人胰岛素溶液中开始反应，分4次加完，同时搅拌。加完后开始计时，反应1h后，用乙酸将溶液pH调节至约7.5，终止反应，获得粗品溶液。采用RP-HPLC对反应过 程进行中控。
3、赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素的纯化
将上述前体粗品溶液加入水稀释使有机相含量约15％(v:v)，用0.45μm滤膜过滤后采用RP-HPLC对其进行纯化得纯化液。
4、赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素的超滤及冻干
将上述纯化溶液用超滤膜包系统将样品置换至注射用水中，进行冻干获得冻干产物26mg。所得分子结构式如下。
Figure PCTCN2017095377-appb-000009
5、赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素结构确证
赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素实测质谱分子量为6203.37Da，与理论分子量6203.21Da一致。
用V8蛋白酶对INS进行酶解，并对酶解产物进行LC-MS分析，结果显示共产生4个肽段，肽段分子量分别为416.23Da(A1-A4)、2968.29Da(A5-A17,B1-B13)、1376.57Da(A18-A21,B14-B21)和1510.84Da(B22-B29)，与理论肽段分子量相符，其中肽段B22-B29为脂肪酸链修饰的肽段。证明修饰位点和预期一致。
实施例2：考察受试药物在STZ诱导的I型糖尿病模型上的降血糖作用
1、供试品
Figure PCTCN2017095377-appb-000010
其中，INS-1和INS-2分别代表将赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素制备成每个胰岛素衍生物六聚体包含5个Zn和8个Zn的两种不同复合物，其配制方法为本领域常规方法，德谷胰岛素中每六聚体包含5个Zn。
2、供试品配制
供试品避光保存在4℃条件下，取适量受试样品母液600nmol/mL，用溶媒稀释400倍至1.5nmol/mL，剂量为7.5nmol/kg。使用时药物恢复至室温。
3、实验动物
4、实验方法
SPF级大鼠实验室环境饲养7天，均以标准饲料、标准笼喂养，温度20-25℃，湿度40-60％。造模前一天，大鼠禁食16小时，腹腔快速注射STZ(65mg/kg)，1小时后，恢复给食。造模后每日给予充足饮水(提供正常的2-3倍的饮水量)、食物，每日换垫料1-2次，保持干燥。在第五天测量空腹血糖(禁食6小时)，挑出血糖值>16.7mmol/L的大鼠。根据血糖水平，将其分为4组。
动物分组及给药信息如下：
Figure PCTCN2017095377-appb-000012
给药前不禁食，单次皮下注射各药物，对照组用溶媒注射相同体积，给药后全程禁食。给药后1、2、4、6、8、10、12、24小时测定大鼠血糖值。24小时点测完血糖值后，采血制备血清。
所有的数据被录入到Excel文档中，并以Mean±SEM的方式表示。数据统计分析使用Graphpad Prism 6.0软件，单因素或双因素方差分析比较方法，以P<0.05 作为显著性差异的判断标准。
5、结果：
SD大鼠注射STZ后第五天，禁食6小时，进行血糖测试，造模成功率达80％。
由图1可见，STZ溶媒组基础血糖值给药前为27.3mmol/L，给药后其血糖值逐步降低，24小时后达到15.1mmol/L，血糖变化值为12.1mmol/L。阳性药德谷胰岛素组给药前后血糖值变化为16.8mmol/L。阳性药物德谷胰岛素组给药后6、8、10、12和24小时时间点，其血糖水平与溶媒阴性对照组比较都具有显著性差异，表明该动物模型及实验方法的有效性。
与溶媒阴性对照组比较，受试药物INS-1和INS-2在给药后24小时内均能显著降低动物的血糖水平，在6、8、10、12和24小时时间点都具有显著性差异，并且血糖水平持续下降，表明其具有良好的长效降糖作用。
试药物INS-1和INS-2对于STZ大鼠，其血糖值随给药后时间的变化曲线基本一致，表明两者的降糖效果类似。在24小时时间点，INS-1和INS-2的降糖效果优于德谷胰岛素组，表明INS-1和INS-2具有更好的长效作用。
实施例3：考察各种长效胰岛素在STZ诱导的I型糖尿病模型上的降血糖作用
1、供试品
Figure PCTCN2017095377-appb-000013
其中INS-A代表WO2013086927中的HS061、INS-B代表WO2013086927中的HS067、INS-C代表赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素，所有供试品均使用相同的处方，且使用锌复合物形式，每个胰岛素六聚体包含5个Zn。
2、供试品配制
供试品避光保存在4℃条件下，取适量受试样品母液600nmol/mL，用溶媒稀释80倍至7.5nmol/mL，剂量为7.5nmol/kg。使用时药物恢复至室温。
3、实验动物
Figure PCTCN2017095377-appb-000014
Figure PCTCN2017095377-appb-000015
4、实验方法
SPF级大鼠实验室环境饲养7天，均以标准饲料、标准笼喂养，温度20-25℃，湿度40-60％。造模前一天，大鼠禁食16小时，腹腔快速注射STZ(65mg/kg)，1小时后，恢复给食。造模后每日给予充足饮水(提供正常的2-3倍的饮水量)、食物，每日换垫料1-2次，保持干燥。在第五天测量空腹血糖(禁食6小时)，挑出血糖值>16.7mmol/L的大鼠。根据血糖水平随机分为5组。
动物分组及给药信息如下：
Figure PCTCN2017095377-appb-000016
给药前不禁食，单次皮下注射各药物，对照组用溶媒注射相同体积，给药后全程禁食。给药后1、2、4、6、8、10、12、24小时测定大鼠血糖值。
所有的数据被录入到Excel文档中，并以Mean±SEM的方式表示。数据统计分析使用SPSS软件，单因素或双因素方差分析比较方法，以P<0.05作为显著性差异的判断标准。
5、结果
STZ溶媒组基础血糖值给药前为27.01mmol/L，给药后其血糖值逐步降低，24小时后达到11.64mmol/L，血糖变化值为15.37mmol/L。阳性药德谷胰岛素组给药前后血糖值变化为19.78mmol/L。阳性药物德谷胰岛素组给药后1、2、4、6、8、10和12小时时间点，其血糖水平与溶媒阴性对照组比较都具有显著性差异，表明该动物模型及实验方法的有效性。
与溶媒阴性对照组比较，受试药物INS-A、INS-B和INS-C在给药后24小时内均能显著降低动物的血糖水平，从血糖值来看，受试药物的血糖值(分别为 7.03±1.15、3.57±0.44及4.20±0.72mmol/L)低于溶媒阴性对照组(11.64±3.94mmol/L)。这表明三者均具有较良好的长效降糖作用。重点比较了各组在0～6小时内降低血糖的差异性，具体数值如表1和表2所示：
表1单次给药对STZ诱导的I型糖尿病大鼠血糖的影响(Mean±SEM,n＝8)
Figure PCTCN2017095377-appb-000017
注：*P<0.05,**P<0.01vs空白溶媒组；$P<0.05，$$P<0.01vs INS-C。
表2单次给药后0～2、0～4及0～6小时内的血糖AUC值(Mean±SEM,n＝8)
Figure PCTCN2017095377-appb-000018
注：$P<0.05,$$P<0.01vs INS-C。
INS-C和德谷胰岛素对于STZ大鼠，给药后INS-C组血糖值下降幅度小于德谷胰岛素，且在给药1小时后显著高于德谷胰岛素组(见图2和表1)。具体数值如表1所示。另由图3和表2可见，INS-C在给药后0～2、0～4和0～6小时内血糖-时间曲线下面积(AUC)均显著高于德谷胰岛素。这表明，若与速效胰岛素联合给药或组成复方制剂给药，INS-C+速效胰岛素在给药后0～6小时内(速效胰岛素发挥降血糖作用的时间段)发生低血糖的风险明显低于德谷胰岛素+速效胰岛素。
INS-C和INS-A对于STZ大鼠，给药后INS-C组血糖值下降幅度小于INS-A，且在给药1、2和4小时后显著高于INS-A组；而在给药10和12小时后则显著低于INS-A组，给药24小时后血糖值也有低于INS-A组的趋势。表明INS-C降血糖速度慢于INS-A，且长效作用也优于INS-A，更符合长效胰岛素的作用特点。此外，由图3可见，INS-C在给药后0～2、0～4和0～6小时内血糖AUC均显著高于 INS-A。这表明，若与速效胰岛素联合给药或组成复方制剂给药，INS-C+速效胰岛素在给药后0～6小时内发生低血糖的风险明显低于INS-A+速效胰岛素。
INS-C和INS-B对于STZ大鼠，给药后INS-C组血糖值下降幅度小于INS-B，且在给药1和2小时后显著高于INS-B组，表明INS-C降血糖速度慢于INS-B。此外，由图3可见，INS-C在给药后0～2、0～4和0～6小时内血糖AUC均显著高于INS-B。这表明，若与速效胰岛素联合给药或组成复方制剂给药，INS-C+速效胰岛素在给药后0～6小时内发生低血糖的风险明显低于INS-B+速效胰岛素。
综合24小时和0～6小时内降血糖的效果，INS-C有降糖作用持久且初期降糖平缓的特点，是最为理想的长效胰岛素，特别适合于与速效胰岛素联合或组成复方，从而减少注射次数，提高患者的依从性。
实施例4：犬PK试验(INS-B和INS-C与实施例3中具有相同的含义)
6只雄性比格犬(每组3只)单次皮下注射INS-B或INS-C(3nmol/kg)后，不同时间点采血并分离血清，检测血清中INS-B或INS-C的浓度。结果显示，INS-B在4.7±1.2小时(3只动物分别为4、4和6小时)内达峰，而INS-C则为8小时(三只动物均为8小时)达峰。INS-B和INS-C的半衰期分别为6.0±1.2和7.8±1.1小时。这提示INS-C作用更为平缓且作用时间更长。
实施例5：稳定性比较
INS-A、INS-B、INS-C(各种代号与实施例3含义相同)、德谷胰岛素均按照德谷胰岛素上市处方制备成制剂，然后考察它们的稳定性，使用HPLC测定起始和各时间点主峰纯度以及杂质含量，具体结果如表3所示。从表3可以看出，INS-C的稳定性明显优于INS-A和INS-B；略优于德谷胰岛素。
表3稳定性数据比较
Figure PCTCN2017095377-appb-000019
Figure PCTCN2017095377-appb-000020
注：由于7天时INS-A和INS-B杂质增长已经过快，所以后续未再测14天数据。

Claims (18)

  1. 一种人胰岛素类似物的酰化衍生物或其锌复合物，所述人胰岛素类似物的酰化衍生物具有如下通式如示的结构：
    S-W-X-Y-Z
    (I)
    其中S为B链第30位苏氨酸缺失的人胰岛素；-W-X-Y-Z为胰岛素类似物的酰化修饰基团；
    其中W为具有-OC(CH2)nCO-的二酰基结构，其中n为2～10之间整数，该结构以其酰基之一和母体胰岛素或其类似物的A-链或B-链N-末端氨基酸残基的α-氨基或B-链上存在的赖氨酸残基的ε-氨基形成酰胺键；
    X为含羧酸基团的二氨基化合物，该化合物以其氨基之一与W中的一个酰基连接形成酰胺键；
    Y为-A(CH2)m-，其中m为6-32的整数，优选10-16的整数，特别优选12-14的整数，A不存在或为CO-；
    Z为-COOH。
  2. 根据权利要求1所述的人胰岛素类似物的酰化衍生物或其锌复合物，其中W与B-链上存在的赖氨酸残基的ε-氨基形成酰胺键。
  3. 根据权利要求1所述的人胰岛素类似物的酰化衍生物或其锌复合物，其中n为2～5之间的整数，优选2。
  4. 根据权利要求1所述的人胰岛素类似物的酰化衍生物或其锌复合物，其中X为-HN(CH2)pCH(COOH)NH-，p为2-10的整数，优选2-6的整数，特别优选2-4的整数，最优选4。
  5. 根据权利要求1所述的人胰岛素类似物的酰化衍生物或其锌复合物，其中-W-X-Y-Z具有如下结构：
    Figure PCTCN2017095377-appb-100001
    优选为
    Figure PCTCN2017095377-appb-100002
  6. Nα-(HOOC(CH2)14CO)-Nε-(OCCH2CH2CO-(NεB29-Des(B30)人胰岛素))-Lys-OH或其锌复合物，优选赖氨酸B29(Nε-(Nα-十六烷脂肪二酸-L-赖氨酸-Nε-氧代丁酰基))des(B30)人胰岛素或其锌复合物。
  7. 一种权利要求1至5任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物的制备方法，其包括使式(III)化合物与B链第30位苏氨酸缺失的人胰岛素反应的步骤，
    R-W-X-Y-Z
    (III)
    其中R为离去基团，优选活化酯基团，更优选选自
    Figure PCTCN2017095377-appb-100003
    Figure PCTCN2017095377-appb-100004
  8. 根据权利要求7所述的方法，还包括式(III’)化合物脱除羧基保护基得到式(III)化合物的步骤，
    Figure PCTCN2017095377-appb-100005
    其中，X’、Z’分别代表X、Z中所有的羧基被保护基保护的形式，所述的保护基优选C1-C6的烷基，优选C1-C4的烷基，特别优选甲基或特丁基；R如权利要求7中定义。
  9. 一种式(III)所示化合物，
    R-W-X-Y-Z
    (III)
    其中W、X、Y、Z如权利要求1至5中任意一项所定义，
    R为离去基团，优选活化酯基团，更优选选自
    Figure PCTCN2017095377-appb-100006
  10. 一种通过将权利要求9中式(III)化合物的X和Z中所有羧基经过羧基保护得到的式(III’)所示的化合物，
    R-W-X’-Y-Z’
    (III’)
    其中，X’、Z’分别代表X、Z中所有的羧基被保护基保护的形式，所述的保护基优选C1-C6的烷基，优选C1-C4的烷基，特别优选甲基或特丁基。
  11. 根据权利要求9所述的化合物，其为
    Figure PCTCN2017095377-appb-100007
    优选
    Figure PCTCN2017095377-appb-100008
    其中R如权利要求9中定义。
  12. 根据权利要求1至6任意一项所述的人胰岛素类似物的酰化衍生物的锌复合物，其中每6分子酰化胰岛素包含多于4个锌原子。
  13. 一种药物组合物，含有权利要求1－5任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物或权利要求12所述的人胰岛素类似物的酰化衍生物的锌复合物，还含有药学上可接受的载体。
  14. 根据权利要求13所述的药物组合物，还含有速效胰岛素。
  15. 权利要求1至6任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物、权利要求12所述的人胰岛素类似物的酰化衍生物的锌复合物、或权利要求13或14所述的药物组合物在制备治疗糖尿病的药物中的用途。
  16. 权利要求1至6任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物、权利要求12所述的人胰岛素类似物的酰化衍生物的锌复合物、或权利要求13所述的药物组合物与速效胰岛素联合在制备治疗糖尿病的药物中的用途。
  17. 一种治疗糖尿病的方法，所述方法包括对需要治疗的患者给予权利要求1至6任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物、权利要求12所述的人胰岛素类似物的酰化衍生物的锌复合物、或权利要求13或14所述的药物组合物。
  18. 一种治疗糖尿病的方法，所述方法包括对需要治疗的患者给予权利要求1至6任意一项所述的人胰岛素类似物的酰化衍生物或其锌复合物、权利要求12所述的人胰岛素类似物的酰化衍生物的锌复合物、或权利要求13所述的药物组合物和速效胰岛素。
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WO2019149245A1 (zh) * 2018-02-01 2019-08-08 江苏恒瑞医药股份有限公司 含人胰岛素类似物的酰化衍生物的药物组合物及其制备方法
WO2019154311A1 (zh) 2018-02-09 2019-08-15 江苏恒瑞医药股份有限公司 一种密码子优化的人胰岛素类似物前体基因和信号肽基因
WO2019223752A1 (zh) 2018-05-24 2019-11-28 江苏恒瑞医药股份有限公司 一种重组人胰岛素或其类似物的前体的制备方法
CN114901681A (zh) * 2019-12-30 2022-08-12 甘李药业股份有限公司 胰岛素衍生物
WO2021136302A1 (zh) 2019-12-30 2021-07-08 甘李药业股份有限公司 胰岛素衍生物
WO2021136293A1 (zh) 2019-12-30 2021-07-08 甘李药业股份有限公司 胰岛素衍生物
CN114901683A (zh) * 2019-12-30 2022-08-12 甘李药业股份有限公司 胰岛素衍生物
CN114901682A (zh) * 2019-12-30 2022-08-12 甘李药业股份有限公司 胰岛素衍生物
WO2021136296A1 (zh) 2019-12-30 2021-07-08 甘李药业股份有限公司 胰岛素衍生物
CN114901681B (zh) * 2019-12-30 2024-04-09 甘李药业股份有限公司 胰岛素衍生物
CN114901682B (zh) * 2019-12-30 2024-04-16 甘李药业股份有限公司 胰岛素衍生物
WO2022111642A1 (zh) * 2020-11-27 2022-06-02 江苏恒瑞医药股份有限公司 一种制备胰岛素或其类似物的酰化衍生物的方法
WO2022268208A1 (zh) 2021-06-25 2022-12-29 甘李药业股份有限公司 含酰化胰岛素的药物组合物
WO2023143458A1 (zh) 2022-01-28 2023-08-03 甘李药业股份有限公司 酰化胰岛素
CN117756914A (zh) * 2023-12-15 2024-03-26 瀚晖制药有限公司 依柯胰岛素的制备方法

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