WO2022206826A1 - 一种多粘菌素衍生物的硫酸盐及其药物组合物 - Google Patents

一种多粘菌素衍生物的硫酸盐及其药物组合物 Download PDF

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WO2022206826A1
WO2022206826A1 PCT/CN2022/084012 CN2022084012W WO2022206826A1 WO 2022206826 A1 WO2022206826 A1 WO 2022206826A1 CN 2022084012 W CN2022084012 W CN 2022084012W WO 2022206826 A1 WO2022206826 A1 WO 2022206826A1
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dab
thr
sulfate
pharmaceutical composition
phe
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PCT/CN2022/084012
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English (en)
French (fr)
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陈庆财
陈虹宇
陈京顺
宗在伟
卢斌
杨奇珍
李明月
张佳伟
鲁晶晶
周宇智
孙敏
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江苏奥赛康药业有限公司
南京海润医药有限公司
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Priority to CN202280007209.3A priority Critical patent/CN117083288A/zh
Publication of WO2022206826A1 publication Critical patent/WO2022206826A1/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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a sulfate of polymyxin derivatives and a pharmaceutical composition thereof.
  • Polymyxins have good curative effect on this type of negative bacteria, so polymyxins become the "last line of defense" in the treatment of multidrug-resistant Gram-negative bacteria.
  • Polymyxins were first reported in 1947 and are cyclic antimicrobial lipopeptides of different structural types produced by fermentation of different Bacillus polymyxa, including polymyxins B, E, A, C, D, F, K, M. , P, S, and T, etc. Among them, the clinical use is polymyxin B and polymyxin E.
  • polymyxin antibiotics is composed of three parts: cyclic heptapeptide, linear tripeptide and N-fatty acyl chain connected with the linear tripeptide. , ⁇ -diaminobutyric acid) and the 10th amino acid L-Thr (or L-Leu) condensation to form.
  • Polymyxins with the same type of structure generally contain more than two different components depending on the N-substituted fatty acyl chain or the 7-position amino acid.
  • systemic administration often resulted in serious adverse reactions, including significant nephrotoxicity. After the 1970s, clinical use gradually decreased.
  • WO2018108154A1 prepared a new derivative of increasing or decreasing the hydrophobicity of the side chain acyl chain (changing R 0 ) by changing the length and volume of the side chain acyl chain, replacing the amino acid at position 1 and/or position 3 with a basic amino acid or a polar amino acid (changing the amino acid at position 3).
  • New derivatives of R 1 , R 3 ), replacement of amino acids at positions 2 and/or 10 with hydrophobic amino acids or polar amino acids (changed R 2 , R 9 ), replacement of hydrophobic amino acids or basic amino acids or polar amino acids
  • New derivatives of amino acids at positions 5 and/or 8 and/or 9 (change R 4 , R 7 , R 8 ), hydrophobic amino acids or polar amino acids replace amino acids at positions 6 and/or 7 (change R 5 , R 6 ) new derivatives.
  • the inventors After systematic structural study and toxicity study, the inventors obtained detailed structure-activity relationship and structure-toxicity relationship, and surprisingly found that the compound 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10 )[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] has the characteristics of high antibacterial activity and low nephrotoxicity, and is a new type of polymyxin derivative.
  • 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10) [Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] is abbreviated as compound A, and its structure is as follows :
  • the sulfate salt of the compound 6-methylheptanoyl-Dab-Thr-D-Ser-ring(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr], compared to Its acetate salt, its trifluoroacetate salt, has many advantages.
  • the study found that the sulfate of 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] has good stability, It has good solubility and low hygroscopicity, which is very beneficial to the preparation of medicinal preparations.
  • the present invention in a first aspect, provides a 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D -Phe-Thr-Dab-Dab-Thr] sulfate.
  • the present invention relates to a 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D-Phe- Thr-Dab-Dab-Thr] sulfate.
  • the 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] sulfate involved in the present invention has a mass percentage 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.0%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7 %, 13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9, 15.0%, or 15.1% sulfate.
  • a 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10) [Dab-Dab-D-Phe-Thr- Dab-Dab-Thr] sulfate, characterized in that the salt is in an amorphous crystalline form.
  • the present invention in a second aspect, provides a pharmaceutical composition comprising the 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D- Phe-Thr-Dab-Dab-Thr] sulfate and a pharmaceutically acceptable carrier.
  • the present invention in a third aspect, provides the 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D-Phe-Thr-Dab- Use of Dab-Thr] sulfate in the preparation of antibacterial drugs.
  • the present invention relates to the 6-methylheptanoyl-Dab-Thr-D-Ser-ring (4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr ] Use of sulfate in preparing antibacterial drugs against "super bacteria" carrying NDM-1 gene.
  • 6-methylheptanoyl-Dab-Thr-D-Ser-cyclo(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] sulfate can be administered orally or parenterally way of administration.
  • parenteral administration is in the form of injection, including water for injection, powder for injection and small infusion.
  • the present invention in a fourth aspect, provides a method for preparing a pharmaceutical composition, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the 6-methylheptanoyl-Dab-Thr-D- Ser-cyclo(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] sulfate, the method comprising adding the 6-methylheptanoyl-Dab-Thr according to any one of the above -D-Ser-ring(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr] sulfate in combination with a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable” as used herein is within the scope of sound medical judgment and is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications , commensurate with a reasonable benefit/risk ratio. "Pharmaceutically acceptable” is also often referred to as “pharmaceutically acceptable”.
  • the sulfate of Compound A of the present invention is safe, effective and controllable in quality during preparation and use, which is more conducive to preparation into pharmaceutical preparations.
  • a specific embodiment of the pharmaceutical composition provided by the second aspect of the present invention is a composition for injection, which includes a pH adjuster.
  • the invention provides a compound A sulfate injection composition and a preparation method thereof. The method solves the defect that the compound A sulfate preparation solution is unstable, and at the same time, it is controlled by pH and then freeze-dried to obtain a stable preparation product.
  • the present invention discloses a polymyxin derivative sulfate injection composition, characterized in that: the composition comprises Compound A sulfate, a pharmaceutically acceptable carrier, a pH adjuster, and Compound A sulfate
  • the salt has sulfate radicals of 12.1%-15.1% by mass, preferably, the sulfate of Compound A has sulfate radicals of 13.6%-14.6% by mass.
  • the related compound A sulfate has mass percentages of 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.0%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9, 15.0% or 15.1% sulfate.
  • the pH adjuster is a water-soluble adjuster selected from H 2 SO 4 , sulfuric acid solution, HCl, hydrochloric acid solution, HAc, acetic acid solution, H 3 PO 4 , and phosphoric acid solution.
  • the pH adjusting agent is selected from sulfuric acid solution, hydrochloric acid solution, acetic acid solution, and phosphoric acid solution
  • the pH adjusting agent concentration is 5-15 mg/ml, preferably 10-15 mg/ml.
  • the pH adjuster adjusts the pH of the solution to 1.0-5.0, preferably adjusts the pH of the solution to 2.0-4.0, preferably adjusts the pH of the solution to 3.0-4.0.
  • the pharmaceutically acceptable carrier is selected from one or more of mannitol, sucrose, lactose, glucose, sorbitol and trehalose.
  • the content in the prescription, in terms of Compound A sulfate, the content of Compound A sulfate per unit preparation is 5-30mg/ml, preferably 9.5-26.25mg/ml, more preferably 23.75-26.25mg/ml , more specifically, in terms of compound A sulfate, the compound A sulfate unit preparation content is 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml, 20mg/ml
  • the content in the prescription is 10-50 mg/ml, preferably 20-40 mg/ml, preferably 20-30 mg/ml, more specifically, the content of the pharmaceutically acceptable carrier per unit preparation is 10 mg /ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 35mg/ml, 40mg/ml, 45mg/ml, 50mg/ml.
  • composition is water injection, freeze-dried powder and sterile powder.
  • the composition is lyophilized after sterile filtration.
  • the volume of the composition solution before lyophilization is 1-10ml, more specifically, the volume of the composition solution before lyophilization is 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml.
  • the present invention discloses a polymyxin derivative sulfate injection composition, characterized in that: the composition comprises Compound A sulfate, mannitol, and a pH adjuster, and Compound A sulfate has The mass percentage is 13.6%-14.6% sulfate radicals. More specifically, the related compound A sulfate has sulfate radicals of 13.6%, 13.7%, 13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6% by mass.
  • the pH adjusting agent is a sulfuric acid solution with a concentration of 5-15 mg/ml, preferably 10-15 mg/ml.
  • the pH adjuster adjusts the pH of the solution to 3.0-4.0.
  • the content in the prescription, calculated as Compound A sulfate, the content of Compound A sulfate per unit preparation is 5 ⁇ 30mg/ml, preferably 9.5 ⁇ 26.25mg/ml, more preferably 23.75 ⁇ 26.25mg/ml, more specifically, the compound In terms of A sulfate, compound A sulfate unit preparation content is 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg /ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml, 26mg/ml , 27
  • the content in the prescription is 10 ⁇ 50mg/ml, preferably 20 ⁇ 30mg/ml, more specifically, the content of pharmaceutically acceptable carrier unit preparation is 10mg/ml, 15mg/ml, 20mg/ml, 25mg/ml. ml, 30mg/ml, 35mg/ml, 40mg/ml, 45mg/ml, 50mg/ml.
  • the volume of the composition solution before lyophilization is 1-10ml, more specifically, the volume of the composition solution before lyophilization is 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml.
  • the present invention also discloses the preparation process of the compound A sulfate composition for injection, which includes the following steps: adding 60-90% (for example, 80%) injection of the total preparation volume to the compounding container at room temperature Water, add the pharmaceutical carrier in the recipe amount, stir to dissolve it completely, add the compound A sulfate in the recipe amount, stir to dissolve it completely; adjust the pH value with a pH adjuster, add water for injection to the full amount, and after the intermediate is tested qualified , Sterile filtration filled in vials, half-pressed into boxes, freeze-dried.
  • the volume of the filling solution is 1-10ml, and more specifically, the volume of the filling solution is 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml.
  • Figure 1 X-ray powder diffraction pattern (XRD) of Example 1 Compound A sulfate.
  • the dried resin peptide was added to the lysis solution (95% TFA, 2.5% H 2 O, 2.5% Tis) 18 mL (5V/M), and the reaction was stirred at 30° C. for 2 h, and filtered to obtain a filtrate.
  • the resins were washed three times with 2 mL of lysis buffer each, and the filtrates were combined.
  • the filtrate was slowly added to 96 mL of methyl tertiary butyl ether, and stirred while adding, a large amount of white solid was precipitated, stirred for 10 min, suction filtration, the filter cake was washed with 20 mL of methyl tertiary butyl ether for 3 times, respectively, suction filtration, It was dried under reduced pressure at 30°C to obtain 1.0 g of crude product with a yield of about 77%.
  • the crude product was added with 2 mL of water, 2 mL of acetonitrile and 1.0 g of ammonium sulfate, and the solution was filtered and diluted with 20 mL of water.
  • the first 60min is the process of changing to sulfate in the preparative column, and the process of eluting the product from the preparative column after 60min.
  • the main peak was collected to obtain an eluent, which was fully concentrated, acetonitrile was removed as much as possible, and the concentration before lyophilization was increased.
  • the sulfate concentration before lyophilization was about 80 mg/mL.
  • the freeze-drying process was carried out for freeze-drying to obtain 0.30 g of a white solid sulfate product with a total yield of 20%.
  • Test solution take an appropriate amount of compound A sulfate, accurately weigh it, dissolve in water and dilute to make a solution containing about 0.25 mg of compound A sulfate per 1 ml.
  • Reference substance solution Take an appropriate amount of ammonium sulfate reference substance, accurately weigh it, and dilute it with diluent to make a solution containing about 0.06 mg of sulfate per 1 ml.
  • Chromatographic conditions use an anion exchange chromatographic column (Dionex IonPac AS15 column, or a chromatographic column with equivalent efficiency); the detector is a conductivity detector; the detection method is inhibited conductivity detection; the column temperature is 30 °C; Eluent; flow rate 1.0 ml per minute; injection volume 25 ⁇ l.
  • anion exchange chromatographic column Dionex IonPac AS15 column, or a chromatographic column with equivalent efficiency
  • Determination method Precisely measure the test solution and the reference solution, inject them into the ion chromatograph respectively, and record the chromatogram.
  • the X-ray powder diffraction pattern is shown in Figure 1 (copper target), showing that it was in an amorphous crystalline form.
  • Example 2 weigh compound A free base and compound A sulfate of 13.6%-14.6% sulfate group prepared in Example 1 (the target ratio needs to be converted when the molar ratio is), and the weighed sample is dissolved in 50ml per gram and purified In water, the dissolved sample is transferred to a freeze-drying bottle and subjected to freeze-drying treatment to obtain compound A sulfate containing 2.5%-3.5% sulfate and 9.5-10.5% sulfate respectively.
  • acetic acid content was determined with reference to high performance liquid chromatography (Acetic acid determination method in synthetic polypeptides in the 2020 edition of the four general rules of the Chinese Pharmacopoeia 0872):
  • the mobile phase A "1% ammonium sulfate/aqueous solution" in the preparation and purification process was changed to "1% trifluoroacetic acid and 1% aqueous ammonia solution", and other methods remained unchanged to obtain a white solid product 0.35 g of trifluoroacetate, the total yield was 20%.
  • the HPLC purity was 99.408%, and it was characterized as Compound A trifluoroacetate salt by 1 H NMR (500 MHz, D 2 O) and MS.
  • trifluoroacetic acid is determined with reference to high performance liquid chromatography (four general chapters of Chinese Pharmacopoeia 2020 edition 0512 high performance liquid chromatography):
  • Test solution take an appropriate amount of compound A trifluoroacetate, accurately weigh it, add diluent (0.5% phosphoric acid solution) to dissolve and dilute to make about 1 mg of compound A trifluoroacetate per 1 ml.
  • Reference substance solution Take an appropriate amount of sodium trifluoroacetate reference substance, accurately weigh it, add diluent to dissolve and dilute to make a solution containing about 0.35mg per 1ml as the reference substance solution.
  • Chromatographic conditions use octadecylsilane-bonded silica gel as a filler (Waters Atlantis T3, 4.6mm ⁇ 250mm, 5 ⁇ m or a chromatographic column with equivalent performance), with 10mM potassium dihydrogen phosphate solution (adjust pH to 2.5 with phosphoric acid)
  • mobile phase A acetonitrile
  • mobile phase B acetonitrile
  • linear gradient elution according to Table 5 the flow rate is 1.0 ml per minute; the column temperature is 30 °C; the detection wavelength is 210 mn; the injection volume is 10 ⁇ l.
  • Determination method Precisely measure the control solution and the test solution, inject them into a liquid chromatograph, and record the chromatogram.
  • the salt of Compound A was prepared according to Examples 1-3, and samples were taken to investigate the solubility of the samples in methanol, acetonitrile and water, respectively. The results are shown in Table 6.
  • Solubility in water Weigh about 1g, put it in a 10ml graduated test tube with a stopper, add less than 1ml of water;
  • Solubility in methanol Weigh about 10mg, put it in a 100ml test tube with a stopper and add 100ml of water;
  • Solubility in acetonitrile Weigh about 10mg, put it in a 100ml test tube with a stopper and add 100ml of water;
  • the salt of compound A was prepared according to Example 1-3, sampled, and the stability of the salt was investigated under the conditions of alkali destruction, acid destruction, high temperature liquid destruction, oxidative destruction, high temperature solid, light solid, and light liquid:
  • Alkali destruction take about 10mg of the test sample, accurately weigh it, put it in a 10ml measuring bottle, add 1ml of 0.05M NaOH solution, shake to dissolve, leave it at room temperature for 45min, add 1ml of 0.05M HCl solution to neutralize, and dilute to the mark with water, Shake well and get it;
  • Acid destruction Take about 10 mg of the test product, accurately weigh it, put it in a 10 ml measuring flask, add 1 ml of 0.5M HCl solution, shake to dissolve, take a water bath at 60°C for 2 hours, leave at room temperature, add 1 ml of 0.5M NaOH solution to neutralize, and use water Dilute to the mark, shake well, and get it;
  • High temperature liquid destruction take about 10mg of the test sample, accurately weigh it, put it in a 10ml volumetric flask, add 5ml of water, shake to dissolve, take a water bath at 80°C for 2h, leave it at room temperature, dilute to the mark with water, and shake well to get it;
  • Oxidative damage take about 10 mg of the test sample, accurately weigh it, put it in a 10 ml volumetric flask, add 5 ml of 3% hydrogen peroxide, shake to dissolve, take a water bath at 50 °C for 3 hours, dilute to the mark with water, and shake well to get it;
  • High temperature solid destruction take about 10mg of the test sample, accurately weigh it, put it in a 10ml volumetric flask, put it in a water bath at 80°C for 4h, leave it at room temperature, add water to dissolve and dilute to the mark, shake well, and then get it;
  • Illumination solid destruction take about 10mg of the test sample, accurately weigh it, put it in a 10ml measuring bottle, place it at 5000lx ⁇ 500lx for 48h, add water to dissolve and dilute to the mark, shake well, and then get it;
  • Destruction of liquid by light Take about 10mg of the test sample, accurately weigh it, put it in a 10ml volumetric flask, add about 5ml of water, shake to dissolve, place it at 5000lx ⁇ 500lx for 48h, dilute it with water to the mark, and shake well.
  • Chromatographic conditions use octadecylsilane-bonded silica gel as filler (ACE EXCEL 5 C18-AR, 4.6mm ⁇ 250mm, 5 ⁇ m or equivalent chromatographic column), use 0.05% phosphoric acid solution as mobile phase A, and use acetonitrile as Mobile phase B, perform linear gradient elution according to Table 7; the flow rate is 1.0 ml per minute; the column temperature is 30°C; the sample tray temperature is 5°C; the detection wavelength is 214 nm; the injection volume is 10 ⁇ l.
  • Determination method Precisely measure the test solution, inject it into a liquid chromatograph, and record the chromatogram.
  • the three salts of compound A are all sensitive to alkali damage, acid damage and oxidative damage, and are relatively stable to high temperature solids, light solids, and light liquids.
  • the sulfate of compound A has better stability to oxidative damage and high temperature liquid damage. .
  • Chromatographic conditions use octadecylsilane-bonded silica gel as filler (ACE UltraCore 5 SuperC18, 250mm ⁇ 4.6mm, 5 ⁇ m or equivalent chromatographic column), 10mmol/L sodium sulfate solution (adjust pH to 2.3 with phosphoric acid)
  • mobile phase A use acetonitrile as mobile phase B, and perform linear gradient elution according to the following table; the flow rate is 1.0 ml per minute; the column temperature is 30 °C; the sample tray temperature is 10 °C; .
  • Determination method Precisely measure the test solution, inject it into a liquid chromatograph, and record the chromatogram.
  • Embodiment 7 investigates the stability of this product intermediate solution under different pH conditions
  • the detection method is the same as that in Example 6.
  • Embodiment 8 The quality comparison of this product with different dosages of mannitol and different pH finished products
  • Example 8-1 and Example 8-2 the product is stable in the pH range of 3.0 to 4.0, and mannitol can reduce the water content of the product and improve the stability after use.
  • the temperature was raised to -10 °C, and the temperature was kept for 3 to 9 hours;
  • the temperature was raised to 10°C for the second time, and the temperature was kept for 2-4h;
  • the temperature was raised to 35°C for the third time, and the temperature was maintained for 3 to 6 hours.
  • Embodiment 10 Different concentrations of sulfate stability results
  • Samples containing different concentrations of sulfate were prepared respectively, and placed for 3 months at 25°C ⁇ 2°C, RH60% ⁇ 5% before setting out (packaging: medicinal low-density polyethylene bag plus aluminum foil bag packaging) to investigate the sulfate content and For substances, evaluate the stability. Different batches of samples have different results of related substances before stakeout, but the total impurities less than 3% are within the limit requirements. Detection is the same as in Example 6. The results show that the higher the sulfate content (13.6%-14.6%), the smaller the total impurities and the more stable the sample after being placed for 3 months.
  • Samples were prepared according to Example 9-4, and placed for 3 months and 6 months under the conditions of 25°C ⁇ 2°C, RH60% ⁇ 5% (inner packaging: medium borosilicate glass vial for injection and halogenated butyl rubber stopper) (bromination)) to investigate the sulfate content and related substances, and to evaluate the stability. Detection is the same as in Example 6.

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Abstract

一种多粘菌素衍生物的硫酸盐，更具体的，为一种具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，优选的，所述盐为无定型结晶形式，所述硫酸盐与其醋酸盐、三氟乙酸盐等盐相比，具有良好的稳定性、溶解度佳、吸湿性小，极有利于制备成药用制剂；此外，还提供了一种多粘菌素衍生物硫酸盐的注射用组合物，所述组合物包括化合物A硫酸盐、可药用载体、pH调节剂。

Description

一种多粘菌素衍生物的硫酸盐及其药物组合物 技术领域
本发明属于生物医药领域，具体涉及一种多粘菌素衍生物的硫酸盐及其药物组合物。
背景技术
2017年2月，WHO发布全球首份急需研发新抗生素的重点病原体清单，其中1类重点极为重要(critical)包括3种:碳青霉烯类药物耐药鲍曼不动杆菌、碳青霉烯类药物耐药铜绿假单胞菌以及碳青霉烯类药物耐药和三代头孢菌素耐药肠杆菌科细菌，这些多药耐药革兰氏阴性菌对碳青霉烯类抗生素具有耐药性，因此，临床急需新的抗生素来治疗这些严重的耐药菌。
多黏菌素对该类阴性菌具有很好疗效,因此多黏菌素成为治疗多药耐药革兰氏阴性菌的“最后一道防线”。多黏菌素首次报道于1947年，是不同多黏芽孢类杆菌发酵产生的不同结构类型的环状抗菌脂肽,包括多黏菌素B、E、A、C、D、F、K、M、P、S和T等。其中，临床使用的是多黏菌素B和多黏菌素E。
多黏菌素类抗生素的共同结构特征为：由环状七肽、线性三肽和与线性三肽连接的N-脂肪酰基链三部分组成，其中七肽环由4位氨基酸L-Dab(α,γ-二氨基丁酸)与10位氨基酸L-Thr(或L-Leu)缩合形成。根据N-取代脂肪酰基链或7位氨基酸不同，同类型结构的多黏菌素中一般又含有两种以上的不同组分。20世纪50年代初，多黏菌素B应用不久，就发现全身给药常发生严重的不良反应，包括显著的肾毒性，20世纪70年代后临床使用逐渐减少。最近临床研究发现CMS诱导的患者急性肾损伤发生率高达60.4％，多黏菌素B的发生率为41.8％，多黏菌素的肾毒性严重限制了临床使用。因此，急需研发新型高效、低毒的多黏菌素衍生物。
目前已经对其做了许多结构改造，但是总存在抗菌活性与肾毒性、神经毒性不能兼得的问题，而且改造结果往往不能令人满意。
WO2018108154A1制备了通过改变侧链酰基链长度和体积来提高或降低侧链酰基链(改变R 0)疏水性的新衍生物，碱性氨基酸或极性氨基酸替换1位和/或3位氨基酸(改变R 1、R 3)的新衍生物，疏水氨基酸或极性氨基酸替换2位和/或10位氨基酸(改变R 2、R 9)的新衍生物，疏水氨基酸或碱性氨基酸或极性氨基酸替换5位和/或8位和/或9位氨基酸(改变R 4、R 7、R 8)的新衍生物，疏水氨基酸或极性氨基酸替换6位和/或7位氨基酸 (改变R 5、R 6)的新衍生物。通过改变多粘菌素分子的氨基数目或疏水性，扩大其抗菌谱或提高抗菌活性或降低肾毒性。在系统的结构研究、毒性研究后，发明人获得了详细的构效关系、构毒关系，并惊喜的发现，化合物6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]具有抗菌活性高和肾毒性低的特点，是一种新型的多黏菌素衍生物。
本发明中将6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]简称为化合物A，其结构如下：
Figure PCTCN2022084012-appb-000001
我们惊奇的发现，化合物6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]的硫酸盐，比起其醋酸盐、其三氟乙酸盐具有许多优点。研究发现，6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]的硫酸盐具有良好的稳定性、溶解度佳、吸湿性小，极有利于制备成药用制剂。
发明内容
本发明，第一方面，提供了一种具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐。
优选的，本发明涉及一种具有质量百分比为13.6％-14.6％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐。
优选的，本发明涉及的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐具有质量百分比为12.1％、12.2％、12.3％、12.4％、12.5％、12.6％、12.7％、12.8％、12.9％、13.0％、13.1％、13.2％、13.3％、13.4％、13.5％、13.6％、13.7％、13.8％、13.9％、14.0％、14.1％、14.2％、14.3％、14.4％、14.5％、14.6％、14.7％、14.8％、14.9、15.0％或15.1％的硫酸根。
优选的，一种具有质量百分比为12.1％-15.1％的硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，其特征在于，所述盐为无定型结晶形式。
本发明，第二方面，提供一种药物组合物，包括上述任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐及可药用载体。
本发明，第三方面，提供了上述任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐在制备抗菌药物中的用途。
优选的，本发明涉及上述任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐在制备抗携带NDM-1基因的“超级细菌”的抗菌药物中的用途。
优选的，6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐可以口服给药或非肠胃方式给药。
优选的，非肠道给药形式为注射剂给药，包括注射用水针，注射用粉针及小输液。
本发明，第四方面，提供了一种制备药物组合物的方法，该药物组合物包含可药用载体及有效量的上述任一项所述6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，所述方法包括将上述任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐与可药用载体相结合。
本发明所述“可药用”是在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。“可药用”也常被称为“药学上可接受的”。
与现有技术相比，本发明所述具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐；优选的，具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，所述盐为无定型结晶形式，本品效果有：
(1)在酸碱、氧化、高温、光照条件下稳定性相比其他盐更好；
(2)在水中极易溶解；
(3)多粘菌素化合物的盐极易吸湿受潮，但在本品研究中我们发现化合物A的硫酸盐吸湿性与其他盐相比出乎意料的小，也因为此，相同工艺得到的产品酸根含量可变范围更小，该工艺所制备的硫酸盐对盐含量稳定性及相应质量稳定性更具优势；
因此，本发明所述化合物A的硫酸盐在制备和使用过程中安全、有效、质量可控，更有利于制备成药用制剂。
本发明第二方面提供的药物组合物的一种具体的实施方式为注射用组合物，包括了pH调节剂。本发明提供了一种化合物A硫酸盐的注射用组合物及其制备方法，该方法解决了化合物A硫酸盐制剂溶液状态不稳定的缺陷，同时通过pH控制后冻干，得到稳定的制剂成品。
更具体的，本发明公开了一种多粘菌素衍生物硫酸盐的注射用组合物，其特征在于：所述组合物包括化合物A硫酸盐、可药用载体、pH调节剂，化合物A硫酸盐具有质量百分比为12.1％-15.1％的硫酸根，优选的，化合物A硫酸盐具有质量百分比为13.6％-14.6％的硫酸根。更具体的，涉及的化合物A硫酸盐具有质量百分比为12.1％、12.2％、12.3％、12.4％、12.5％、12.6％、12.7％、12.8％、12.9％、13.0％、13.1％、13.2％、13.3％、13.4％、13.5％、13.6％、13.7％、13.8％、13.9％、14.0％、14.1％、14.2％、14.3％、14.4％、14.5％、14.6％、14.7％、14.8％、14.9、15.0％或15.1％的硫酸根。
pH调节剂为水溶性调节剂，选自H 2SO 4、硫酸溶液、HCl、盐酸溶液、HAc、醋酸溶液、H 3PO 4、磷酸溶液。
优选的，当pH调节剂选自硫酸溶液、盐酸溶液、醋酸溶液、磷酸溶液时，pH调节剂浓度为5～15mg/ml，优选为10～15mg/ml。
pH调节剂调节溶液pH值至1.0-5.0，优选调节溶液pH值至2.0-4.0，优选调节溶液pH值至3.0-4.0。
优选的，所述的可药用载体选自：甘露醇、蔗糖、乳糖、葡萄糖、山梨醇、海藻糖中的一种或多种。
优选的，其特征在于，处方中含量，以化合物A硫酸盐计，化合物A硫酸盐单位制剂含量为5～30mg/ml，优选为9.5～26.25mg/ml，更优选为23.75～26.25mg/ml，更具体的，以化合物A硫酸盐计，化合物A硫酸盐单位制剂含量为5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml、21mg/ml、22mg/ml、23mg/ml、24mg/ml、25mg/ml、26mg/ml、27mg/ml、28mg/ml、29mg/ml、30mg/ml。
优选的，处方中含量，可药用载体单位制剂含量为10～50mg/ml，优选为20～40mg/ml，优选为20～30mg/ml，更具体的，可药用载体单位制剂含量为10mg/ml、15mg/ml、20mg/ml、25mg/ml、30mg/ml、35mg/ml、40mg/ml、45mg/ml、50mg/ml。
所述组合物为水针，冻干粉，无菌粉。
优选的，所述组合物通过无菌过滤后冻干。
优选的，冻干前组合物溶液体积为1-10ml，更具体的，冻干前组合物溶液体积为1ml、2ml、3ml、4ml、5ml、6ml、7ml、8ml、9ml、10ml。
更具体的，本发明公开了一种多粘菌素衍生物硫酸盐的注射用组合物，其特征在于：所述组合物包括化合物A硫酸盐、甘露醇、pH调节剂，化合物A硫酸盐具有质量百分比为13.6％-14.6％的硫酸根。更具体的，涉及的化合物A硫酸盐具有质量百分比为13.6％、13.7％、13.8％、13.9％、14.0％、14.1％、14.2％、14.3％、14.4％、14.5％、14.6％的硫酸根。
pH调节剂为硫酸溶液，浓度为5～15mg/ml，优选为10～15mg/ml。
pH调节剂调节溶液pH值至3.0-4.0。
处方中含量，以化合物A硫酸盐计，化合物A硫酸盐单位制剂含量为5～30mg/ml，优选为9.5～26.25mg/ml，更优选为23.75～26.25mg/ml，更具体的，以化合物A硫酸盐计，化合物A硫酸盐单位制剂含量为5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml、21mg/ml、22mg/ml、23mg/ml、24mg/ml、25mg/ml、26mg/ml、27mg/ml、28mg/ml、29mg/ml、30mg/ml。
处方中含量，甘露醇单位制剂含量为10～50mg/ml，优选为20～30mg/ml，更具体的，可药用载体单位制剂含量为10mg/ml、15mg/ml、20mg/ml、25mg/ml、30mg/ml、35mg/ml、40mg/ml、45mg/ml、50mg/ml。
优选的，冻干前组合物溶液体积为1-10ml，更具体的，冻干前组合物溶液体积为1ml、2ml、3ml、4ml、5ml、6ml、7ml、8ml、9ml、10ml。
本发明还公开了所述的化合物A硫酸盐的注射用组合物的制备工艺，包括如下步骤：室温下向配料容器中加入配制总体积60～90％(示例性地例如为80％)的注射用水，加入处方量可药用载体，搅拌使之溶解完全，将处方量化合物A硫酸盐加入，搅拌使之溶解完全；用pH调节剂调节pH值，加注射用水至全量，中间体检测合格后，无菌过滤灌装于西林瓶中，压半塞进箱，冷冻干燥。
优选的，灌装溶液体积为1-10ml，更具体的，灌装溶液体积为1ml、2ml、3ml、4ml、5ml、6ml、7ml、8ml、9ml、10ml。
附图说明
图1：实施例1化合物A硫酸盐的X射线粉末衍射图(XRD)。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
DCM：二氯甲烷
DMF：N,N-二甲基甲酰胺
DIEA：N,N-二异丙基乙胺
OAll:烯丙基
PIP：哌啶
TFA：三氟乙酸
TIS：三异丙基硅烷
实施例1具有质量百分比13.6％-14.6％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐的制备
树脂肽的制备：
取2-氯三苯甲基氯树脂(2-CTC)2g(1.12mmol，0.56mmol/g)，于多肽反应瓶中，加入20mL DCM搅拌溶胀30min抽滤，再用20mL DCM洗涤一次，完成树脂溶胀。
称取Fmoc-Dab(NH 2)-OAll·HCl氨基酸1.40g(3.36mmol，3.0eq)和DIEA 1.30g(10.08mol，9.0eq)，溶于12mLDCM，加入多肽反应瓶中，30℃搅拌反应2h，排干反应液，各用16mL DCM洗涤树脂3次。
加入含15％甲醇及5％DIEA的DCM溶液16mL，于30℃下搅拌反应30min，完成树脂封闭，抽滤，DCM、DMF各16mL交替洗涤各3次，以20％PIP/DMF溶液脱Fmoc保护基5min+15min两次，每次16mL，DCM/DMF各16mL交替洗涤各4次。
依次完成Fmoc-Dab(Boc)-OH、Fmoc-Thr(tBu)-OH、Fmoc-D-Phe-OH、Fmoc-Dab(Boc)-OH、Fmoc-Dab(Dde)-OH、Fmoc-D-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Dab(Boc)-OH、6-甲基庚酸、水合肼脱除烯丙基、Fmoc-Thr(tBu)-OH、四三苯基膦钯脱除Dde保护基及肽链环合等步骤，完成树脂肽的合成，干燥后得3.6g树脂肽。
粗品制备：
将干燥的树脂肽加入裂解液(95％TFA，2.5％H 2O，2.5％Tis)18mL(5V/M)，30℃下搅拌反应2h，过滤，得滤液。树脂各用2mL裂解液洗涤3次，合并滤液。
将滤液缓缓加入96mL甲基叔丁基醚中，边加边搅拌，有大量白色固体析出，搅拌10min，抽滤，滤饼用20mL甲基叔丁基醚分别打浆洗涤3次，抽滤、30℃减压干燥得粗品1.0g，收率约77％。
硫酸盐成品的制备：
将粗品中加入2mL水，2mL乙腈及1.0g硫酸铵，溶清过滤后加入20mL水稀释。
通过DAC-100中高压制备液相进行粗品的纯化、换盐及富集：
一次纯化：经制备型高效液相色谱纯化，填料为10μm的反相C18，流动相A为(0.3％硫酸铵+0.1％硫酸氢钠)/水溶液，流动相B为乙腈，检测波长为214nm，经下表1所示梯度洗脱，收集对应的主峰组分，合并HPLC纯度大于95％的组分，经浓缩去除乙腈，进行二次纯化。
表1：一次纯化洗脱梯度
Figure PCTCN2022084012-appb-000002
二次纯化：经制备型高效液相色谱纯化，填料为10μm的反相C18，流动相A为0.2％乙酸铵/水溶液，流动相B为乙腈，经下表2所示梯度洗脱，在波长214nm下，收集主峰组分，并合并HPLC纯度大于98％的组分，经浓缩去除乙腈，进行换盐。
表2：二次纯化洗脱梯度
Figure PCTCN2022084012-appb-000003
硫酸盐的制备：经制备型高效液相色谱纯化，填料为10μm的反相C18，流动相A为1％硫酸铵/水溶液，流动相B为乙腈，经下表3梯度洗脱：
表3：硫酸盐制备洗脱梯度
Figure PCTCN2022084012-appb-000004
前60min为在制备柱内换为硫酸盐的过程，60min后为将产物从制备柱中洗脱的过程。在波长214nm下，洗脱过程中，收集主峰，获得洗脱液，经充分浓缩，尽可能去除乙腈，提高冻干前浓度，冻干前硫酸盐浓度约为80mg/mL，经下表4所述冻干工艺进行冷冻干燥，获得白色固体的硫酸盐产物0.30g，总收率为20％。HPLC纯度为99.540％， 1H NMR(500MHz,D 2O):δ7.29(t,J＝10Hz,2H),7.24-7.19(m,3H),4.65-4.61(m,2H),4.43-4.40(m,1H),4.38-4.34(m,2H),4.29-4.23(m,3H),4.19-4.10(m,5H),4.07-4.06(m,1H),3.85-3.76(m,2H),3.29-3.23(m,1H),3.10-2.93(m,10H),2.79-2.73(m,1H),2.67-2.61(m,1H),2.23(t,J＝10Hz,3H),2.18-2.09(m,3H),2.07-1.96(m,2H),1.92-1.86(m,2H),1.85-1.75(m,11H),1.52-1.46(m,2H),1.44-1.38(m,1H),1.23-1.17(m,2H),1.13-1.06(m,9H),0.75(d,J＝5Hz,6H),0.70(t,J＝10Hz,3H)，MS(m/z):1164.6689[M+H] +,理论值为1164.6663[M+H] +，结构表征为化合物A的硫酸盐。
表4：冻干工艺
Figure PCTCN2022084012-appb-000005
硫酸根参照中国药典2020年版四部通则0513“离子色谱法”测定：
供试品溶液：取化合物A硫酸盐适量，精密称定，加水溶解并稀释制成每1ml中约含化合物A硫酸盐0.25mg的溶液。
对照品溶液：取硫酸铵对照品适量，精密称定，用稀释液稀释制成每1ml中约含硫酸根0.06mg的溶液。
色谱条件：用阴离子交换色谱柱(Dionex IonPac AS15柱，或效能相当的色谱柱)；检测器为电导检测器；检测方式为抑制电导检测；柱温30℃；以30mmol/L氢氧化钾溶液 为淋洗液；流速为每分钟1.0ml；进样体积25μl。
测定法：精密量取供试品溶液与对照品溶液，分别注入离子色谱仪，记录色谱图。
限度：按外标法以峰面积计算。优化后的实施例1的工艺经多次重复，得到的产品含硫酸根为13.6％-14.6％，可变范围小，质量可控。
X-射线粉末衍射图谱如附图1所示(铜靶)，显示其为无定型结晶形式。
另外，称取化合物A游离碱和实施例1制备的13.6％-14.6％硫酸根的化合物A硫酸盐(目标比例为摩尔比时需换算)，将称量好的样品按每克溶于50ml纯化水中，将溶解好的样品转移至冻干瓶中，冻干处理，分别制得包括硫酸根2.5％～3.5％、硫酸根9.5～10.5％的化合物A硫酸盐。
实施例2具有质量百分比15.0％-25.0％醋酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]醋酸盐的制备
参照实施例1所述方法，将制备纯化过程中的流动相A“1％硫酸铵/水溶液”修改为“1％醋酸铵”，其它方法不变，获得白色固体产物的醋酸盐0.32g，总收率为20％。HPLC纯度为99.638％；经 1H NMR(500MHz,D 2O)和MS表征为化合物A的醋酸盐。
醋酸含量参照高效液相色谱法(中国药典2020年版四部通则0872合成多肽中的醋酸测定法)测定：
取化合物A醋酸盐约20mg，精密称定，置10ml量瓶中，加稀释液[流动相A-甲醇(95:5)]溶解并稀释至刻度，摇匀，作为供试品溶液，参照合成多肽中的醋酸测定法(中国药典2020年版四部通则0872)测定，实施例2的工艺经多次重复，得到的产品含醋酸为15.0％-25.0％，可变范围较大，经X-射线粉末衍射图谱检测为无定型结晶形式。
实施例3具有质量百分比24.0％-31.0％三氟乙酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]三氟乙酸盐的制备
参照实施例1所述方法，将制备纯化过程中的流动相A“1％硫酸铵/水溶液”修改为“1％三氟乙酸及1％氨水的水溶液”，其它方法不变，获得白色固体产物的三氟乙酸盐0.35g，总收率为20％。HPLC纯度为99.408％，经 1H NMR(500MHz,D 2O)和MS表征为化合物A的三氟乙酸盐。
三氟乙酸含量参照高效液相色谱法(中国药典2020年版四部通则0512高效液相色谱法)测定：
供试品溶液：取化合物A三氟乙酸盐适量，精密称定，加稀释液(0.5％磷酸溶液)溶解并稀释制成每1ml中约含1mg化合物A三氟乙酸盐。
对照品溶液：取三氟乙酸钠对照品适量，精密称定，加稀释液溶解并稀释制成每1ml中约含0.35mg的溶液作为对照品溶液。
色谱条件：用十八烷基硅烷键合硅胶为填充剂(Waters Atlantis T3，4.6mm×250mm，5μm或效能相当的色谱柱)，以10mM磷酸二氢钾溶液(用磷酸调节pH值至2.5)为流动相A，乙腈为流动相B，按下表5进行线性梯度洗脱；流速为每分钟1.0ml；柱温为30℃；检测波长为210mn；进样体积10μl。
表5：三氟乙酸含量测定洗脱梯度
Figure PCTCN2022084012-appb-000006
测定法：精密量取对照溶液和供试品溶液，分别注人液相色谱仪，记录色谱图。
限度：按外标法以峰面积计算三氟乙酸的含量，实施例3的工艺经多次重复，得到的产品含三氟乙酸为24.0％-31.0％，可变范围较大，经X-射线粉末衍射图谱检测为无定型结晶形式。
实施例4溶解度实验
按照实施例1-3制备化合物A的盐，取样，分别考察样品在甲醇、乙腈及水中的溶解度，结果如表6所示。
操作方法：
水中溶解度：称取约1g，置10ml具塞刻度试管中，加水不到1ml；
甲醇中溶解度：称取约10mg，置100ml具塞刻度试管中，加水100ml；
乙腈中溶解度：称取约10mg，置100ml具塞刻度试管中，加水100ml；
将上述加入溶剂后的样品于25℃±2℃下每隔5分钟强烈振摇30秒，观察30分钟内的溶解情况，如无目视可见的溶质颗粒，即视为完全溶解。
表6：溶解度测试结果
Figure PCTCN2022084012-appb-000007
Figure PCTCN2022084012-appb-000008
结果显示，化合物A的三种盐溶解度相似，在水中均极易溶解。
实施例5稳定性实验
按照实施例1-3制备化合物A的盐，取样，在碱破坏、酸破坏、高温液体破坏、氧化破坏、高温固体、光照固体、光照液体条件下考察盐的稳定性：
(1)供试品溶液的制备：
未破坏：取供试品约10mg，精密称定，置10ml量瓶中，加水溶解并稀释至刻度，摇匀，即得；
碱破坏：取供试品约10mg，精密称定，置10ml量瓶中，加0.05M NaOH溶液1ml，振摇使溶解，室温放置45min，加0.05M HCl溶液1ml中和，用水稀释至刻度，摇匀，即得；
酸破坏：取供试品约10mg，精密称定，置10ml量瓶中，加0.5M HCl溶液1ml，振摇使溶解，60℃水浴2h，放置室温，加0.5M NaOH溶液1ml中和，用水稀释至刻度，摇匀，即得；
高温液体破坏：取供试品约10mg，精密称定，置10ml量瓶中，加水5ml，振摇使溶解，80℃水浴2h，放置室温，用水稀释至刻度，摇匀，即得；
氧化破坏：取供试品约10mg，精密称定，置10ml量瓶中，加3％双氧水5ml，振摇使溶解，50℃水浴3h，用水稀释至刻度，摇匀，即得；
高温固体破坏：取供试品约10mg，精密称定，置10ml量瓶中，80℃水浴4h，放置室温，加水溶解并稀释至刻度，摇匀，即得；
光照固体破坏：取供试品约10mg，精密称定，置10ml量瓶中，于5000lx±500lx放置48h，加水溶解并稀释至刻度，摇匀，即得；
光照液体破坏：取供试品约10mg，精密称定，置10ml量瓶中，加水约5ml，振摇使溶解，于5000lx±500lx放置48h，用水稀释至刻度，摇匀，即得。
(2)检测方法：
参照高效液相色谱法(中国药典2020年版四部通则0512)测定。
色谱条件：用十八烷基硅烷键合硅胶为填充剂(ACE EXCEL 5 C18-AR，4.6mm×250mm，5μm或效能相当的色谱柱)，以0.05％磷酸溶液为流动相A，以乙腈为流动相B，按下表7进行线性梯度洗脱；流速为每分钟1.0ml；柱温为30℃；样品盘温度为5℃；检测波长为214nm；进样体积10μl。
测定法：精密量取供试品溶液，注入液相色谱仪，记录色谱图。
表7：稳定性检测方法洗脱梯度
Figure PCTCN2022084012-appb-000009
稳定性测试结果如下表8所示：
表8：稳定性测试结果
Figure PCTCN2022084012-appb-000010
化合物A的三种盐均对碱破坏、酸破坏、氧化破坏较敏感，对高温固体、光照固体，光照液体均较稳定，化合物A的硫酸盐对氧化破坏、高温液体破坏有更好的稳定性。
综上可知，化合物A的硫酸盐稳定性优于醋酸盐和三氟乙酸盐。
实施例5引湿性实验
(1)取干燥的具塞玻璃称量瓶，于试验前一天置于适宜的25℃±1℃恒温干燥器(下部放置硫酸铵饱和溶液)，精密称定重量(m 1)。
(2)取供试品适量，平铺于上述称量瓶中，精密称定重量(m 2)。
(3)将称量瓶敞口，并与瓶盖同置于上述恒温恒湿条件下24小时。
(4)盖好称量瓶盖子，精密称定重量(m 3)。
(5)计算公式
Figure PCTCN2022084012-appb-000011
引湿性测试结果如下表9所示：
表9：引湿性测试结果
Figure PCTCN2022084012-appb-000012
化合物A硫酸盐的引湿性结果显著低于醋酸盐和三氟乙酸盐，更有助于样品的储存和运输。
实施例6对比溶液灭菌前后质量变化情况
表10：制剂处方：
Figure PCTCN2022084012-appb-000013
工艺方法：
向配料容器中加入配制总体积80％的注射用水(温度25℃以下)，加入处方量甘露醇，搅拌使之溶解完全，将处方量化合物A硫酸盐原料(根据实施例1方法制备得到的化合物A硫酸盐，具有质量百分比13.6％-14.6％硫酸根)加入，搅拌使之溶解完全；用稀硫酸溶液(10mg/ml)调节pH值，加注射用水至全量，中间体含量合格后，灌装于10ml安瓿瓶内，四个处方各取5瓶在121℃灭菌8分钟、各取5瓶在121℃灭菌15分钟，对比灭菌前后各处方质量差异。
按照实施例6-1～6-4制备化合物A硫酸盐的处方，取样，考察对比溶液灭菌前后质量变 化情况：
(1)供试品溶液的制备：
取本品内容物20mg，精密称定，置10ml量瓶中，加水溶解并稀释至刻度，摇匀，即得；溶液稳定性：10℃放置48小时，溶液稳定。
(2)检测方法：
参照高效液相色谱法(中国药典2020年版四部通则0512)测定。
色谱条件：用十八烷基硅烷键合硅胶为填充剂(ACE UltraCore 5 SuperC18，250mm×4.6mm，5μm或效能相当的色谱柱)，10mmol/L硫酸钠溶液(用磷酸调节pH值至2.3)为流动相A，以乙腈为流动相B，按下表进行线性梯度洗脱；流速为每分钟1.0ml；柱温为30℃；样品盘温度为10℃；检测波长为214nm；进样体积10μl。
表11：线性梯度洗脱条件
Figure PCTCN2022084012-appb-000014
测定法：精密量取供试品溶液，注入液相色谱仪，记录色谱图。
对比溶液灭菌前后质量变化情况如下表12所示：
Figure PCTCN2022084012-appb-000015
结果显示，本品不适宜采用终端灭菌方法，为了提高产品稳定性，通过无菌过滤后冻干。
实施例7考察本品中间体溶液在不同pH条件下的稳定性
表13：不同批号的处方
Figure PCTCN2022084012-appb-000016
向配料容器中加入配制总体积80％的注射用水(温度25℃以下)，加入处方量甘露醇，搅拌使之溶解完全，将处方量化合物A硫酸盐原料(根据实施例1方法制备得到的化合物A硫酸盐，具有质量百分比13.6％-14.6％硫酸根)加入，搅拌使之溶解完全；用稀硫酸溶液(10mg/ml)调节pH值，加注射用水至全量。考察各pH下本品中间体溶液的稳定性。
检测方式同实施例6。
表14：稳定性测试结果
Figure PCTCN2022084012-appb-000017
结果显示，本品在pH为2.0条件下不稳定，pH为3.0～5.0范围内较为稳定。
实施例8本品不同甘露醇用量及不同pH成品的质量对比
表15：处方
Figure PCTCN2022084012-appb-000018
Figure PCTCN2022084012-appb-000019
向配料容器中加入配制总体积80％的注射用水(温度25℃以下)，加入处方量甘露醇，搅拌使之溶解完全，将处方量化合物A硫酸盐原料(根据实施例1方法制备得到的化合物A硫酸盐，具有质量百分比13.6％-14.6％硫酸根)加入，搅拌使之溶解完全；用稀硫酸溶液(10mg/ml)调节pH值，加注射用水至全量。中间体检测合格后，灌装于西林瓶中，压半塞进箱，冻干(冻干条件同实施例6)，得到各处方样品。
检测同实施例6
表16：不同pH值冻干制剂样品稳定性考察结果
Figure PCTCN2022084012-appb-000020
结果显示，实施例8-1、实施例8-2，本品在pH3.0～4.0范围内稳定，且甘露醇使用后能降低产品水分提高稳定性。
实施例9
表17：处方
Figure PCTCN2022084012-appb-000021
按实施例9-1～4的处方分别向配料容器中加入配制总体积80％的注射用水(温度25℃以下)，加入处方量甘露醇，搅拌使之溶解完全，将处方量化合物A硫酸盐原料(根据实施例1方法制备得到的化合物A硫酸盐，具有质量百分比13.6％-14.6％硫酸根)加入，搅拌使之溶解完全；用稀硫酸溶液(10mg/ml)调节pH值，加注射用水至全量。
待各处方中间产品检测合格后，开启相应阀门药液经冗余、终端除菌过滤滤芯，过滤至缓冲接料罐，采用如下条件冻干：
将灌装好半压塞的药液放入冻干箱中，关闭箱门。
开机制冷，设置导热油进口温度1小时降至-45℃，待导热油进口温度达到-45℃后，保温2～4小时。关闭板冷阀，当冷阱温度达-45℃且保温时间达到要求时，开启真空泵抽真空。
第一次升温至-10℃，保温3～9h；
第二次升温升温至10℃，保温2～4h；
第三次升温至35℃，保温3～6h。
压塞，放入滤过无菌空气出箱，关机。
实施例10不同浓度硫酸盐稳定性结果
分别制备含不同浓度硫酸根的样品，于放样前和25℃±2℃、RH60％±5％条件放置3个月(包装：药用低密度聚乙烯袋外加铝箔袋包装)考察硫酸根含量和有关物质，评价稳定性。样品不同批次放样前有关物质结果有区别，但总杂小于3％均在限度要求内。检测同实施例6。结果表明，硫酸根含量越高(13.6％-14.6％)，放置3个月后，总杂越小，样品越稳定。
表18：不同浓度硫酸根的化合物A的硫酸盐样品稳定性测试结果
Figure PCTCN2022084012-appb-000022
实施例11长期试验结果
根据实施例9-4制备样品，于放样前和25℃±2℃、RH60％±5％条件放置3个月、6个月(内包装：中硼硅玻璃管制注射剂瓶和卤化丁基橡胶塞(溴化))考察硫酸根含量和有关物质，评价稳定性。检测同实施例6。
Figure PCTCN2022084012-appb-000023
Figure PCTCN2022084012-appb-000024

Claims (20)

  1. 一种具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐。
  2. 一种具有质量百分比为13.6％-14.6％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐。
  3. 一种具有质量百分比为12.1％-15.1％硫酸根的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，其特征在于，所述盐为无定型结晶形式。
  4. 一种药物组合物，包括权利要求1至3任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐及可药用载体。
  5. 权利要求1至3任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐在制备抗菌药物中的用途。
  6. 权利要求1至3任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐在制备抗携带NDM-1基因的“超级细菌”的抗菌药物中的用途。
  7. 权利要求5所述的用途，其中6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐为口服给药。
  8. 权利要求5所述的用途，其中6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐为非肠胃方式给药。
  9. 权利要求8所述的用途，非肠道给药形式为注射剂给药，包括注射用水针，注射用粉针及小输液。
  10. 一种制备药物组合物的方法，该药物组合物包含可药用载体及有效量的权利要求1至3任一项所述6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐，所述方法包括将权利要求1至3任一项所述的6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐与可药用载体相结合。
  11. 权利要求4所述的药物组合物，其特征在于：所述药物组合物为注射用组合物，还包括pH调节剂。
  12. 根据权利要求11所述的药物组合物，其特征在于，pH调节剂为水溶性调节剂，选自H 2SO 4、硫酸溶液、HCl、盐酸溶液、HAc、醋酸溶液、H 3PO 4、磷酸溶液。
  13. 根据权利要求11所述的药物组合物，其特征在于，当pH调节剂选自硫酸溶液、盐酸溶液、醋酸溶液、磷酸溶液时，pH调节剂浓度为5～15mg/ml，优选为10～15mg/ml。
  14. 根据权利要求11所述的药物组合物，其特征在于，pH调节剂调节溶液pH值至1.0-5.0，优选调节溶液pH值至3.0-4.0。
  15. 根据权利要求11所述的药物组合物，其特征在于，所述可药用载体载体选自甘露醇、蔗糖、乳糖、葡萄糖、山梨醇、海藻糖中的一种或多种。
  16. 根据权利要求11所述的药物组合物，其特征在于，处方中含量，6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐单位制剂含量为5～30mg/ml，优选为9.5～26.25mg/ml，更优选为23.75～26.25mg/ml。
  17. 根据权利要求11所述的药物组合物，其特征在于，处方中含量，可药用载体单位制剂含量为10～50mg/ml，优选为20～30mg/ml。
  18. 根据权利要求11所述的药物组合物，其特征在于，所述组合物为水针，冻干粉，无菌粉。
  19. 根据权利要求11所述的药物组合物，其特征在于，所述组合物通过无菌过滤后冻干。
  20. 根据权利要求11所述的药物组合物的制备工艺，包括如下步骤：室温下向配料容器中加入配制总体积60～90％的注射用水，加入处方量可药用的载体，搅拌使之溶解完全，将处方量6-甲基庚酰基-Dab-Thr-D-Ser-环(4-10)[Dab-Dab-D-Phe-Thr-Dab-Dab-Thr]硫酸盐加入，搅拌使之溶解完全；用pH调节剂调节pH值，加注射用水至全量，中间体检测合格后，无菌过滤灌装于西林瓶中，压半塞进箱，冷冻干燥。
PCT/CN2022/084012 2021-03-31 2022-03-30 一种多粘菌素衍生物的硫酸盐及其药物组合物 WO2022206826A1 (zh)

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