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WO2019149245A1 - 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 - Google Patents

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 Download PDF

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WO2019149245A1
WO2019149245A1 PCT/CN2019/074146 CN2019074146W WO2019149245A1 WO 2019149245 A1 WO2019149245 A1 WO 2019149245A1 CN 2019074146 W CN2019074146 W CN 2019074146W WO 2019149245 A1 WO2019149245 A1 WO 2019149245A1
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pharmaceutical composition
composition according
human insulin
group
lysine
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PCT/CN2019/074146
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English (en)
French (fr)
Inventor
杨晓容
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江苏恒瑞医药股份有限公司
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Priority to CN201980005526.XA priority Critical patent/CN111315766B/zh
Priority to MX2020008129A priority patent/MX2020008129A/es
Priority to KR1020207024706A priority patent/KR20200116130A/ko
Priority to EP19747024.8A priority patent/EP3747896A4/en
Priority to AU2019214159A priority patent/AU2019214159A1/en
Priority to CA3090199A priority patent/CA3090199A1/en
Priority to US16/966,694 priority patent/US20210040171A1/en
Priority to JP2020541970A priority patent/JP2021512124A/ja
Priority to BR112020015457-1A priority patent/BR112020015457A2/pt
Publication of WO2019149245A1 publication Critical patent/WO2019149245A1/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
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—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
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K1/107—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
    • 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 a pharmaceutical composition
  • a pharmaceutical composition comprising an acylated derivative of a human insulin analog and a process for the preparation thereof.
  • Human insulin is a major drug for the treatment of diabetes, but human insulin has a short duration of action, making it necessary for patients to inject frequently, which is extremely inconvenient. Therefore, efforts have been made to obtain insulin analogs and derivatives thereof which have a longer half-life and can act on the human body for a long time.
  • the use of an acylating group to modify human insulin or an analog thereof is an effective method for increasing the half-life of insulin.
  • the Applicant's patent application PCT/CN2017/095377 provides a human insulin analog having a B29 amino acid deletion at position B30 substituted with a long chain fatty acid, and discloses the structure and biological activity of the human insulin analog.
  • WO9507931 discloses an insulin (i.e., Des(30) human insulin) analog having a B30 position linked to a tetradecanoyl side chain and a preparation thereof, and a preparation thereof.
  • WO2005012347 discloses a human insulin analog in which the amino acid B30 is deleted at position B29 by a glutamic acid and a long-chain fatty acid
  • WO2007074133 discloses a preparation of the above-mentioned insulin analog, which discloses LysB29 (N ⁇ -hexadecane II).
  • acyl-gamma-glutamyl de(B30) human insulin (deglutathione) formulation contains sodium chloride, but is published in the FDA (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/ 203313 Orig1s000_203314Orig1s000PharmR.pdf) shows that sodium chloride causes a decrease in systemic exposure to insulin. In order to solve the above problem, sodium chloride is removed from the marketed product formulation of Degu insulin.
  • the decrease in the exposure of the insulin system results in a decrease in the amount of insulin that can actually function, thereby reducing its hypoglycemic effect.
  • the present inventors have surprisingly found that for the insulin acylated derivative of the present invention, the liquid preparation containing sodium chloride can be reduced not only high The production of molecular polymers does not affect the effect of insulin on lowering blood sugar.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an acylated derivative of a human insulin analog, the acylated derivative of the human insulin analog having a structure as shown in the following Formula I:
  • S is the human insulin of the 30th threonine deletion of the B chain (B chain deletion as shown in SEQ ID NO. 2) [ie Des (B30) human insulin]; -WXYZ 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 of the parent insulin or analog thereof (the sequence of which is The ⁇ -amino group of the lysine residue present on the ⁇ -amino group or the B-chain of the B-chain N-terminal amino acid residue or the B-chain 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 -D(CH 2 ) m -, wherein m is an integer of 6-32, preferably an integer of 10-16, particularly preferably an integer of 12-14, D is absent or is CO-;
  • Z is -COOH
  • At least one component selected from the group consisting of a stabilizer, a preservative, a pH adjuster, and an osmotic pressure adjusting agent.
  • 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 or B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin, which has The specific structure of the following formula (Ia),
  • the acylated derivative of a human insulin analog forms a complex with zinc, said complex being a 6-mer of an acylated derivative of a human insulin analog, wherein each The 6-mer contains more than 4 zinc atoms, i.e. 6 molecules of acylated insulin comprise more than 4 zinc atoms; more preferably 5 to 8 zinc atoms, particularly preferably 5 zinc atoms.
  • the stabilizer is selected from, but not limited to, sodium chloride, preferably at a concentration of 5 to 20 mM, more preferably 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM. , 13 mM, 14 mM, 15 mM, 20 mM, most preferably 10 mM.
  • concentration of sodium chloride refers to the concentration of sodium chloride in the final product.
  • the preservative is selected from but not limited to phenol and/or m-cresol;
  • the pH adjusting agent is selected from the group consisting of acids and/or bases, preferably but not limited to hydrochloric acid and/or sodium hydroxide
  • the osmotic pressure adjusting agent is selected from the group consisting of, but not limited to, glycerin and/or mannitol.
  • the zinc may be any zinc salt, preferably zinc acetate.
  • the composition comprises:
  • Lysine B29 N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin, 0.1% to 0.5%, preferably 0.3 % ⁇ 0.4%;
  • a phenol content of 0.05% to 0.5%, preferably 0.1% to 0.2%;
  • m-cresol 0.05% to 0.5% content of m-cresol, preferably 0.1% to 0.2%;
  • glycerin 1% to 3% of glycerin, preferably 1.5% to 2%;
  • sodium chloride preferably 0.05% to 0.06%
  • Each 6 molecules of acylated insulin comprises more than 4 zinc atoms
  • a preferred pH is 6-8, a more preferred pH is 7.4-7.8, and most preferably a pH of 7.6.
  • the content in the present invention means the mass (g) volume (100 mL) fraction of each component in the total volume of the composition, based on the total volume of the composition, for example, the 0.05% content means that 0.05 g of the substance is contained in 100 mL of the solution, and the others The content of the analogy.
  • the composition comprises:
  • Lysine B29 N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin 3.72 mg;
  • a preferred pH is 6-8, a more preferred pH is 7.4-7.8, and most preferably a pH of 7.6.
  • a pharmaceutically acceptable carrier is further included.
  • the composition may contain fast-acting insulin in addition to the acylated derivative of the above human insulin analog.
  • the fast-acting insulin also known as ultra-short-acting insulin, has a rapid onset time after injection, and takes effect 10 to 15 minutes after subcutaneous injection, and the peak time is 1 to 2 hours, and the duration is 4 to 6 hours.
  • Conventional fast-acting insulins such as insulin aspart (such as Novo) and insulin lispro (such as eucalyptus) are routine in the art.
  • the present invention also provides a method for producing the above pharmaceutical composition, which comprises acylated derivative of the human insulin analog and any one selected from the group consisting of a stabilizer, a preservative, a pH adjuster, and an osmotic pressure adjusting agent. One or more mixed steps.
  • the present invention also provides the use of the aforementioned pharmaceutical composition for the preparation of a medicament for treating diabetes.
  • the specific administration form of the pharmaceutical composition of the present invention is not limited.
  • the present invention provides a method of treating diabetes comprising administering a pharmaceutical composition of the aforementioned acylated derivative of a human insulin analog to a patient in need of treatment.
  • the preservative and the bacteriostatic agent in the present invention may mean the same meaning.
  • Figure 1 Degutin insulin injection and INS-C injection accelerated stability 25 ° C ⁇ 2 ° C inspection results - RRT1.05 impurities.
  • Figure 3 Degutin injection and INS-C injection accelerated stability 25 ° C ⁇ 2 ° C results - polymer protein.
  • Figure 7 shows the change in blood glucose level after administration of STZ rats within 0 to 24 hours.
  • Figure 8 shows the plasma concentration-time curve after administration of SD rats.
  • the invention is further illustrated in detail by the following examples and experimental examples.
  • the examples and the examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • the experimental methods in the examples of the present invention which do not specify the specific conditions are generally in accordance with the conventional conditions which are favorable for the production or according to the conditions recommended by the raw material or the manufacturer of the commodity.
  • Reagents without specific source are routine reagents purchased from the market.
  • Example 1 Preparation of lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin
  • 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, and then hydrogen was added thereto, and the reaction was vigorously stirred at room temperature overnight. The mixture was filtered through 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 of 6203.37 Da, and theoretical molecular weight 6203.21Da is consistent.
  • the target product was digested with V8 protease, and LC-MS analysis of the hydrolyzed product showed that a total of 4 peptides were produced.
  • the molecular weights of the peptides were 416.23 Da (A1-A4) and 2296.29 Da (A5-A17, respectively).
  • the MS/MS method was used for the mass spectrometric analysis of the B22-B29 peptide.
  • the mass spectrometry confirmed that the modification site of N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl was B29 Amino acid (see the table below), and when the B chain amino acid was sequenced by Edman degradation method, the B29 lysine was modified, resulting in the 29th cycle not corresponding to the standard amino acid peak.
  • the experimental results were in line with expectations. .
  • INS-C in the examples represents lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin.
  • the INS-C injection is prescribed as follows:
  • the auxiliary solution was prepared separately according to the content of the auxiliary materials in the prescription.
  • Sampling test traits pH, INS-C content, phenol content and m-cresol content.
  • the limits of each index are as follows: traits: colorless clear liquid; pH 7.4 ⁇ 7.8; INS-C content: 97.0% ⁇ 103.0%; phenol content: 1.46 ⁇ 1.55mg / mL; m-cresol content: 1.67 ⁇ 1.77mg / mL.
  • the membrane was filtered through a 0.22 ⁇ m PVDF membrane, and the filtrate was filled in a 2 mL neutral borosilicate glass controlled injection bottle in a volume of 1.1 mL per bottle, and the loading range was 1.05 mL to 1.15 mL, which was stoppered and rolled. During the filling process, the loading is monitored, and the filter is subjected to integrity testing, lamp inspection and packaging before and after filtration.
  • Example 3 Screening of sodium chloride concentration in INS-C injection
  • INS-C in the examples represents lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin.
  • the prescription information is as follows:
  • Table 1 shows that sodium chloride can reduce the formation of high molecular polymers, reduce the formation of other impurities, and improve the stability of the active material.
  • Example 4 Stability comparison between INS-C injection and Degu insulin injection
  • INS-C in the examples represents lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin.
  • the prescription information of INS-C injection and Degu insulin injection is as follows:
  • Example 5 Investigating the hypoglycemic effect of INS-C injection and Degutin injection on STZ-induced type I diabetes model
  • INS-C in the examples represents lysine B29 (N ⁇ -(N ⁇ -hexadecane diacid-L-lysine-N ⁇ -oxobutanoyl)) Des (B30) human insulin.
  • test sample was stored in the dark at 4 ° C and the dose was 7.5 nmol/kg.
  • 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%.
  • One day before the modeling 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. Animal grouping and drug administration information are as follows:
  • the rats were not fasted, and each drug was injected subcutaneously.
  • the control group was injected with the same volume of the solution containing INS-C but containing other components of the INS-C prescription, 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 control group was 26.6 mmol/L before administration, and the blood glucose level gradually decreased after administration. After 24 hours, the blood glucose level reached 10.1 mmol/L, and the blood glucose change value was 15.5 mmol/L. The change in blood glucose level before and after administration of the positive drug deglutamine group was 20.1 mmol/L. At the 1, 2, 4, 6, 8, 10, and 12 hour time points after administration of the positive drug deglutathione group, the blood glucose level was significantly different from that of the control group, indicating the effectiveness of the animal model and experimental method.
  • the test drug INS-C can significantly reduce the blood glucose level of the animal within 24 hours after administration, and the blood glucose value of the test drug is 3.9 ⁇ 0.6 mmol/L from the blood glucose value. 10.1 ⁇ 3.3 mmol / L), but also lower than insulin insulin (6.4 ⁇ 1.4 mmol / L), which indicates that INS-C injection has a good long-acting hypoglycemic effect.
  • the differences in blood glucose levels between 0 and 24 hours were compared between the groups. The specific values are shown in Table 5:
  • Example 6 Pharmacokinetics of a preparation containing and not containing sodium chloride and a preparation containing sodium chloride in a normal rat after standing at room temperature for 20 hours
  • mice were acclimated for 5 days in the animal house environment, and fed with standard feed and standard cage. The temperature was 20-25 °C and the humidity was 40-60%. The patients were randomly divided into three groups according to their body weight before administration. The group information is as follows:
  • Group 1 and Group 2 INS-C preparation (600 nmol/mL) containing no or containing sodium chloride was diluted to 50 nmol/mL with a blank solvent before administration;
  • Group 3 INS-C preparation containing sodium chloride ( 600 nmol/mL) was allowed to stand in the dark and room temperature for 20 hours, and then diluted to 50 nmol/mL with a blank solvent before administration, and the sodium chloride concentration of the sodium chloride group was 0.58 mg/mL.
  • Do not fast before administration a single subcutaneous injection of each test drug.
  • Each animal was collected by jugular vein puncture at a dose of about 0.2 mL/time at 0.5, 1, 2, 4, 8, 12, and 24 hours before administration, placed in a test tube containing a coagulant, and placed after collection.
  • the serum was centrifuged in a labeled centrifuge tube (centrifugation conditions: 5000 rpm, 10 minutes, 2 to 8 ° C).
  • Serum samples were determined by LC-MS/MS method, the main pharmacokinetic parameters were calculated, and statistical differences were tested by Student t test.

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Abstract

一种含人胰岛素类似物的组合物。具体而言，提供了一种含人胰岛素类似物的酰化衍生物的组合物，该酰化衍生物组合物相较于已有的胰岛素类似物及其酰化衍生物组合物更加稳定。

Description

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
本申请要求申请日为2018年2月1日的中国专利申请CN201810099660.8的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种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。
背景技术
人胰岛素是治疗糖尿病的一种主要药物，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半衰期更长，能够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，是一种提高胰岛素半衰期的有效方法。本申请人的专利申请PCT/CN2017/095377提供了一种B29位被长链脂肪酸取代的B30位氨基酸缺失的人胰岛素类似物，公开了该人胰岛素类似物的结构和生物活性。WO9507931公开了一种B29位连接一个十四酰基侧链的B30位氨基酸缺失的胰岛素[即Des(30)人胰岛素]类似物及其制剂。WO2005012347公开了一种B29位被一个谷氨酸和长链脂肪酸取代的B30位氨基酸缺失的人胰岛素类似物，WO2007074133公开了上述胰岛素类似物的制剂，该专利中公开LysB29(Nε-十六烷二酰基-γ-谷氨酰基脱(B30)人胰岛素(德谷胰岛素)制剂中含有氯化钠，但在FDA公开的资料(https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/203313Orig1s000_203314Orig1s000PharmR.pdf)中显示氯化钠会导致德谷胰岛素的系统暴露量降低。为了解决上述问题，在德谷胰岛素的上市产品制剂中去除了氯化钠。
胰岛素系统暴露量的降低导致能够确实发挥作用的胰岛素量减少，进而降低其降血糖效果，本发明惊奇地发现对于本发明中的胰岛素酰化衍生物，含有氯化钠的液体制剂不但可以降低高分子聚合物的产生，而且不影响胰岛素降低血糖的效果。
发明内容
本发明提供了一种含人胰岛素类似物的酰化衍生物的药物组合物，所述人胰岛素类似物的酰化衍生物具有如以下通式I所示的结构：
S-W-X-Y-Z
(I)
其中S为B链第30位苏氨酸缺失(B链缺失如SEQ ID NO.2所示)的人胰岛素[即Des(B30)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(CH 2) 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(其序列如SEQ ID NO.1所示)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-D(CH 2) 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，D不存在或为CO-；
Z为-COOH；
还包含选自稳定剂，防腐剂，pH调节剂，渗透压调节剂的至少一种成分。
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(CH 2)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 PCTCN2019074146-appb-000001
优选
Figure PCTCN2019074146-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N α-(HOOC(CH 2) 14CO)-N ε-(OCCH 2CH 2CO-(N εB29-Des(B30)人胰岛素))-Lys-OH或者B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素，其具有如下式(Ia)的具体结构，
Figure PCTCN2019074146-appb-000003
更优选地，其具有式(Ib)所示的结构。
Figure PCTCN2019074146-appb-000004
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与锌形成复合物，所述的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。
本发明优选的实施方案中，所述稳定剂选自但不限于氯化钠，优选的氯化钠的浓度为5～20mM，更优选5mM，6mM，7mM，8mM，9mM，10mM，11mM，12mM，13mM，14mM，15mM，20mM，最优选10mM。当本发明中提及氯化钠的浓度时，所述浓度是指氯化钠在终产品中的浓度。
本发明的另一个实施方案中，所述防腐剂选自但不限于苯酚和/或间甲酚；所述pH调节剂选自酸和/或碱，优选但不限于盐酸和/或氢氧化钠；所述渗透压调节剂选自但不限于甘油和/或甘露醇。
本发明的一些实施方案中，所述的锌可以是任何一种锌盐，优选醋酸锌。
本发明优选的实施方案中，所述的组合物包含：
0.1％～0.5％含量的赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素，优选0.3％～0.4％；
0.05％～0.5％含量的苯酚，优选0.1％～0.2％；
0.05％～0.5％含量间甲酚，优选0.1％～0.2％；
1％～3％含量的甘油，优选1.5％～2％；
0.01％～0.2％的氯化钠，优选0.05％～0.06％；
每6分子酰化胰岛素包含多于4个锌原子；
pH调节剂和注射用水。
优选的pH值为6～8，更优选的pH值为7.4～7.8，最优选pH值为7.6。
本发明中的含量指以组合物的总体积计算，每种组份占组合物的总体积的质量(g)体积(100mL)分数，例如0.05％含量指100mL溶液中含有0.05g的物质，其他的含量以此类推。
本发明优选的实施方案中，所述的组合物包含：
赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素3.72mg；
Figure PCTCN2019074146-appb-000005
氢氧化钠；盐酸；用注射用水补足至1mL。
优选的pH值为6～8，更优选的pH值为7.4～7.8，最优选pH值为7.6。
本发明的一个实施方案中，进一步还含有药学上可接受的载体。
本发明的一些实施方案中，所述的组合物除含有上述人胰岛素类似物的酰化衍生物外，还可以含有速效胰岛素。所述速效胰岛素，又称作超短效胰岛素，注射后起效时间迅速，皮下注射后10～15分钟起效，达峰时间为1～2小时，持续时间为4～6小时。本领域常规的速效胰岛素如门冬胰岛素(例如诺和锐)、赖脯胰岛素(例如优泌乐)。
本发明还提供上述的药物组合物的制备方法，所述方法包含将所述的人胰岛素类似物的酰化衍生物与选自稳定剂，防腐剂，pH调节剂，渗透压调节剂的任意一种或多种混合的步骤。
本发明还提供前述药物组合物在制备治疗糖尿病的药物中的用途。
本发明中所述药物组合物的具体给药剂型不需进行限定。
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的药物组合物。
本发明中防腐剂与抑菌剂可以表示相同的意义。
附图说明
图1德谷胰岛素注射液与INS-C注射液加速稳定性25℃±2℃考察结果-RRT1.05杂质。
图2德谷胰岛素注射液与INS-C注射液加速稳定性25℃±2℃考察结果-其它总杂质。
图3德谷胰岛素注射液与INS-C注射液加速稳定性25℃±2℃考察结果-高分子蛋白质。
图4德谷胰岛素注射液与INS-C注射液长期稳定性(5℃±3℃)考察结果-RRT1.05杂质。
图5德谷胰岛素注射液与INS-C注射液长期稳定性(5℃±3℃)考察结果-其它总杂质。
图6德谷胰岛素注射液与INS-C注射液长期稳定性(5℃±3℃)考察结果-RRT1.05杂质高分子蛋白质。
图7显示0～24小时内STZ大鼠给药后血糖值变化曲线。
图8显示SD大鼠给药后血药浓度-时间曲线。
具体实施方式
通过以下实施例和实验例进一步详细说明本发明。这些实施例和实验例仅用于说明性目的，而并不用于限制本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照有利于生产的常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
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
Figure PCTCN2019074146-appb-000006
Figure PCTCN2019074146-appb-000007
1、N α-(十六烷脂肪二酸)-N ε-(3-酰基丙酸-OSu)赖氨酸的制备
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6.0g 10％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(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-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 Na 2CO3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N α-(十六烷脂肪二酸)-N ε-(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(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-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(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-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 PCTCN2019074146-appb-000008
5、赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素结构确证
赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-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蛋白酶对目标产物进行酶解，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，采用LC-MS/MS法对B22-B29肽段进行二级质谱分析，质谱结果确证了N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基的修饰位点为B29位赖氨酸(详见下表)，另外采用Edman降解法对B链氨基酸进行序列测定时，B29位赖氨酸由于被修饰，导致第29个循环不能与标准氨基酸峰对应，该实验结果与预期相符。上述实验结果共同确证了修饰位点和预期一致。
Figure PCTCN2019074146-appb-000009
B22-B29肽段的二级质谱b/y离子示意
B22-B29肽段的二级质谱b/y离子表
Figure PCTCN2019074146-appb-000010
Figure PCTCN2019074146-appb-000011
实施例2：INS-C注射液的制备
实施例中的INS-C代表赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素。
INS-C注射液处方如下：
Figure PCTCN2019074146-appb-000012
注：醋酸锌分子式：C 4H 6O 4Zn·2H 2O
按照处方中的辅料含量分别配制辅料溶液备用。
将辅料与处方量的INS-C原料药混合，补充注射用水至目标体积的90％，用NaOH溶液或HCl溶液调节pH至7.4～7.8，目标pH为7.6，注射用水加至全量，混匀。
取样检验性状、pH、INS-C含量、苯酚含量和间甲酚含量。各指标限度如下：性状：无色的澄明液体；pH为7.4～7.8；INS-C含量：97.0％～103.0％；苯酚含量：1.46～1.55mg/mL；间甲酚含量：1.67～1.77mg/mL。
用0.22μm PVDF膜过滤，滤液按每瓶1.1mL装量灌装于2mL中性硼硅玻璃管制注射剂瓶中，装量范围1.05mL～1.15mL，加塞，轧盖。灌装过程中对装量进行监测，过滤前后对滤芯进行完整性测试，灯检，包装。
实施例3：INS-C注射液中氯化钠浓度的筛选
实施例中的INS-C代表赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素。
处方信息如下：
Figure PCTCN2019074146-appb-000013
Figure PCTCN2019074146-appb-000014
评价氯化钠对组合物稳定性的影响试验，结果如下。
表1 氯化钠对组合物稳定性的影响
Figure PCTCN2019074146-appb-000015
表1中显示，氯化钠可减少高分子聚合物的形成，也可减少其它杂质的形成，提高活性物质的稳定性。
实施例4：INS-C注射液与德谷胰岛素注射液稳定性对比
实施例中的INS-C代表赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素。
INS-C注射液与德谷胰岛素注射液处方信息如下：
Figure PCTCN2019074146-appb-000016
A、加速试验
将德谷胰岛素注射液与INS-C注射液中试3批样品，于加速试验条件25℃±2℃下放置，分别于第0、1、2、3、6个月取样进行测定，考察各指标。
B、长期试验
将德谷胰岛素注射液与INS-C注射液中试3批样品，于低温5℃±3℃条件下长期放置，于第3、6、9、12、18、24、36个月取样进行测定，定时取样测定，德谷胰岛素注射液的考察起始点是以其出厂时间计算。
表2 德谷胰岛素注射液与INS-C注射液起始检验结果
Figure PCTCN2019074146-appb-000017
备注：外观均是无色澄明液体。
表3 德谷胰岛素注射液与INS-C注射液加速稳定性25℃±2℃考察结果
Figure PCTCN2019074146-appb-000018
备注：INS-C注射液正置样品稳定性考察结果与倒置样品稳定性考察结果无明显差异，此处只列出倒置(相对剧烈)条件考察结果。其中，外观均是无色澄明液体。
表4 德谷胰岛素注射液与INS-C注射液长期稳定性(5℃±3℃)考察结果
Figure PCTCN2019074146-appb-000019
Figure PCTCN2019074146-appb-000020
备注：INS-C注射液正置样品稳定性考察结果与倒置样品稳定性考察结果无明显差异，此处只列出倒置(相对剧烈)条件考察结果。其中，外观均是无色澄明液体。
实施例5：考察INS-C注射液与德谷胰岛素注射液在STZ诱导的I型糖尿病模型上的降血糖作用
实施例中的INS-C代表赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素。
1、供试品
Figure PCTCN2019074146-appb-000021
2、供试品配制
供试品避光保存在4℃条件下，剂量为7.5nmol/kg。使用时药物恢复至室温。
3、实验动物
Figure PCTCN2019074146-appb-000022
Figure PCTCN2019074146-appb-000023
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 PCTCN2019074146-appb-000024
给药前不禁食，单次皮下注射各药物，对照组用去除INS-C但包含INS-C处方其它成分的溶液，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
对照组基础血糖值给药前为26.6mmol/L，给药后其血糖值逐步降低，24小时后达到10.1mmol/L，血糖变化值为15.5mmol/L。阳性药德谷胰岛素组给药前后血糖值变化为20.1mmol/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。
与对照组比较，受试药物INS-C在给药后24小时内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3.9±0.6mmol/L)低于对照组(10.1±3.3mmol/L)，同时也低于德谷胰岛素(6.4±1.4mmol/L)，这表明INS-C注射剂具有较良好的长效降糖作用。比较了各组在0～24小时内降低血糖的差异性，具体数值如表5所示：
表5单次给药对STZ诱导的I型糖尿病大鼠血糖的影响
Figure PCTCN2019074146-appb-000025
注：对照组、德谷胰岛素组：N＝10，INS-C组：N＝9；**P<0.01vs对照组
按照同样的实验方法，对比不含氯化钠的INS-C注射液和德谷胰岛素注射液对大鼠降糖作用的影响，结果显示其与上述实验呈现基本相同的降糖曲线，证明有无氯化钠对INS-C的暴露量几乎没有影响。
实施例6：考察含及不含氯化钠的制剂及含氯化钠的制剂室温放置20小时后在正常大鼠中的药代动力学特性
实验动物
Figure PCTCN2019074146-appb-000026
1、实验方法：SD大鼠在动物房环境适应5天，均以标准饲料、标准笼喂养，温度20～25℃，湿度40～60％。给药前按体重随机分为三组，分组信息如下：
Figure PCTCN2019074146-appb-000027
组1和组2：给药前将不含或含氯化钠的INS-C制剂(600nmol/mL)用空白溶媒稀释至50nmol/mL；组3：将含氯化钠的INS-C制剂(600nmol/mL)在避光、室温环境下放置20小时后，给药前再用空白溶媒稀释至50nmol/mL，其中含氯化钠组的氯化钠浓度为0.58mg/mL。给药前不禁食，单次皮下注射各受试药物。每只动物在给药前及给药后0.5、1、2、4、8、12和24小时经颈静脉穿刺采血约0.2mL/时间点，置于含促凝剂的试管中，采集后放置于标记好的离心管中，离心分离血清(离心条件：5000转/分钟，10分钟，2～8℃)。
血清样品采用LC-MS/MS法测定药物浓度，计算主要的药代参数，并采用student t test检验统计学差异。
2、实验结果：以各时间点平均血药浓度(个体血药浓度详见表7)为纵坐标、时间点为横坐标作图，得血药浓度-时间曲线(图8)。结果表明，受试药物在3组动物体内的浓度随时间变化的趋势基本一致。各主要药代参数详见表6，结果显示各组间主要药代参数基本一致，无统计学差异。
实验结果表明制剂中加入氯化钠后对其在大鼠体内的药代性质无明显影响；室温放置20小时对受试药物的药代性质也无明显影响。
表6 SD大鼠给药后主要药代参数(Mean±SD)
组别 t max(h) C max(ng/mL) AUC 0-t(ng*h/mL) t 1/2(h) MRT(h)
1 5.6±2.2 712.8±181.4 8209±1149 5.3±0.6 10.2±0.9
2 4.8±1.8 622.3±146.6 7585±478 5.3±0.7 10.2±1.6
3 4.0±0.0 614.5±102.2 7767±474 5.8±0.8 10.8±1.4
表7 SD大鼠给药后个体血药浓度(ng/mL)
Figure PCTCN2019074146-appb-000028
Figure PCTCN2019074146-appb-000029
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (14)

  1. 含人胰岛素类似物的酰化衍生物的药物组合物，所述人胰岛素类似物的酰化衍生物具有如下通式I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(CH 2) 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-链的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-D(CH 2) 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，D不存在或为CO-；
    Z为-COOH；
    所述的组合物还包含选自稳定剂，防腐剂，pH调节剂，渗透压调节剂的至少一种成分。
  2. 如权利要求1所述的药物组合物，所述人胰岛素类似物的酰化衍生物，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. 如权利要求1或2所述的药物组合物，所述人胰岛素类似物的酰化衍生物，其中n为2～5之间的整数，优选2。
  4. 如权利要求1至3中任意一项所述的药物组合物，所述人胰岛素类似物的酰化衍生物，其中X为-HN(CH 2) 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. 如权利要求1至4中任意一项所述的药物组合物，所述人胰岛素类似物的酰化衍生物，其中-W-X-Y-Z具有如下结构：
    Figure PCTCN2019074146-appb-100001
    优选为
    Figure PCTCN2019074146-appb-100002
  6. 如权利要求1至5中任意一项所述的药物组合物，所述人胰岛素类似物的酰化衍生物与锌形成复合物；优选地，其中每6分子酰化胰岛素包含多于4个锌原子。
  7. 如权利要求1至6中任意一项所述的药物组合物，所述稳定剂选自氯化钠，优选的氯化钠的浓度为5～20mM，更优选10mM。
  8. 如权利要求1至7中任意一项所述的药物组合物，所述防腐剂选自苯酚和/或间甲酚；所述pH调节剂选自酸和/或碱；所述渗透压调节剂选自甘油和/或甘露醇。
  9. 如权利要求8所述的药物组合物，包含如下组分：
    0.1％～0.5％含量的赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素，优选0.3％～0.4％；
    0.05％～0.5％含量的苯酚，优选0.1％～0.2％；
    0.05％～0.5％含量间甲酚，优选0.1％～0.2％；
    1％～3％含量的甘油，优选1.5％～2％；
    0.01％～0.2％的氯化钠，优选0.05％～0.06％；
    每6分子所述胰岛素包含多于4个锌原子；
    pH调节剂和注射用水，优选的pH值为6～8，更优选的pH值为7.4～7.8。
  10. 如权利要求8所述的药物组合物，其包含如下组分：
    赖氨酸B29(N ε-(N α-十六烷脂肪二酸-L-赖氨酸-N ε-氧代丁酰基))Des(B30)人胰岛素3.72mg；
    Figure PCTCN2019074146-appb-100003
    氢氧化钠；盐酸；用注射用水补足至1ml，优选pH值为6～8，更优选pH值为7.4～7.8，最优选pH值为7.6。
  11. 如权利要求1至10中任意一项所述的药物组合物，还含有速效胰岛素。
  12. 一种如权利要求1至10中任意一项所述的药物组合物的制备方法，所述方法包 含将胰岛素类似物的酰化衍生物与选自稳定剂，防腐剂，pH调节剂，渗透压调节剂中任意一种或多种混合的步骤。
  13. 如权利要求1至11中任意一项所述的药物组合物在制备治疗糖尿病的药物中的用途。
  14. 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11中任意一项所述的药物组合物。
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