WO2018103758A1 - 高效价的多黏菌素e2甲磺酸钠 - Google Patents

高效价的多黏菌素e2甲磺酸钠 Download PDF

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WO2018103758A1
WO2018103758A1 PCT/CN2017/115467 CN2017115467W WO2018103758A1 WO 2018103758 A1 WO2018103758 A1 WO 2018103758A1 CN 2017115467 W CN2017115467 W CN 2017115467W WO 2018103758 A1 WO2018103758 A1 WO 2018103758A1
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polymyxin
methanesulfonate
modified products
modified
sodium
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PCT/CN2017/115467
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French (fr)
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冯军
张喜全
吴勇
郭猛
薛春佳
胡明通
朱裕辉
周杰
王猛力
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上海医药工业研究院
正大天晴药业集团股份有限公司
上海多米瑞生物技术有限公司
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Priority to CN201780074766.6A priority Critical patent/CN109996806B/zh
Publication of WO2018103758A1 publication Critical patent/WO2018103758A1/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
    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
    • C07K7/62—Polymyxins; Related 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of medicine, and relates to polymyxin E 2 methanesulfonate having a titer of not less than 600 ⁇ g/mg, a preparation method thereof and use thereof for preparing a medicament for treating Gram-negative infection.
  • Colistin is a polypeptide antibiotic consisting of multiple components, consisting mainly of E 1 and E 2 (or A or B).
  • polymyxin E was used clinically, mainly for infections caused by Gram-negative bacteria, especially by multi-drug resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae Treatment of infections such as bacteria.
  • polymyxin E products for clinical use, one is oral or topical polymyxin E (also known as colistin sulfate), and the other is polymyxin E for injection.
  • Sodium methanesulfonate Coldistimethate Sodium).
  • Both drugs are multi-component drugs, in the polymyxin E, containing at least 5 components such as E 1 , E 2 , E 3 (see European Pharmacopoeia 7.0), also in the polymyxin E
  • the sodium sulfonate also contains at least five components including the corresponding polymyxin E 1 methanesulfonate and polymyxin E 2 methanesulfonate.
  • different components may bring pharmacodynamics. Differences in pharmacokinetics and toxicology, in order to ensure the inter-batch quality stability of the polymyxin E product, a single component polymyxin E composition needs to be prepared.
  • Cisoka patent application CN102190710A discloses a polymyxin E 2 composition in which the structure of polymyxin E 2 is as shown in Formula I, and Example 4 discloses a composition of polymyxin E 2 sodium methanesulfonate.
  • the composition has a potency of 560 ⁇ g/mg, and although its potency is higher than that of a commercially available product, it has been determined that the composition has a large variety of modified products, and it is difficult to ensure batch-to-batch quality stability of the product.
  • Chinese patent application CN104918951A discloses a composition containing at least 3 amino groups modified by sodium methanesulfonate, wherein Example 2 discloses the preparation of 10 modified sodium polymyxin E 2 methanesulfonate.
  • the CMS control generally has lower activity.
  • the present invention provides a polymyxin E 2 methanesulfonate having a titer of not less than 600 ⁇ g/mg.
  • the potency is not less than 620 ⁇ g/mg.
  • the potency is not less than 640 ⁇ g/mg.
  • the potency is not less than 660 ⁇ g/mg.
  • polymyxin E of the present invention comprising sodium 2 A modified products, wherein the modification means, the second structure in polymyxin E, the ⁇ -amino-Dbu sodium methoxy group (-CH 2 SO 3 Na) or methanol based (-CH 2 OH) substitution.
  • the gamma amino group of Dbu is substituted with sodium methanesulfonate (-CH 2 SO 3 Na).
  • the polymyxin E 2 methanesulfonate of the present invention comprises 4 modified products, that is, in the polymyxin E 2 structure, the ⁇ -amino group of Dbu is substituted with 4 sodium methanesulfonate groups. (-CH 2 SO 3 Na) substitution, wherein 4 modified products account for 25%-50% in the HPLC profile by area normalization method. Further preferably, the 4 modified product accounts for 30% to 43%.
  • the polymyxin E 2 methanesulfonate of the present invention comprises 6 modified products, that is, in the polymyxin E 2 structure, the ⁇ amino group of Dbu is 6 sodium methanesulfonate groups. (-CH 2 SO 3 Na) substitution wherein 6 modified products account for 35% to 65% by area normalization method in the HPLC profile. Further preferably, the 6 modified product accounts for 45% to 55%.
  • the polymyxin E 2 methanesulfonate of the present invention comprises 8 modified products, that is, in the polymyxin E 2 structure, the ⁇ -amino group of Dbu is 8 sodium methanesulfonate groups. (-CH 2 SO 3 Na) substitution, wherein 8 modified products account for 5%-22% in the HPLC profile by area normalization method. Further preferably, the 8 modified product accounts for 7% to 17%.
  • the polymyxin E 2 methanesulfonate of the present invention comprises 4 modified products, 6 modified products, that is, in the structure of polymyxin E 2 , the ⁇ -amino group of Dbu is 4 Sodium methanesulfonate (-CH 2 SO 3 Na) or 6 sodium methanesulfonate (-CH 2 SO 3 Na), wherein in the HPLC map by area normalization method, 4 modified products accounted for 25 %-50%, 6 modified products accounted for 35%-65%. Further preferably, the 4 modified product accounts for 30% to 43%, and the 6 modified product accounts for 45% to 55%.
  • the polymyxin E 2 methanesulfonate of the present invention comprises 4 modified products, 8 modified products, that is, in the structure of polymyxin E 2 , the ⁇ -amino group of Dbu is 4 Sodium methanesulfonate (-CH 2 SO 3 Na) or 8 sodium methanesulfonate (-CH 2 SO 3 Na), wherein in the HPLC map by area normalization method, 4 modified products accounted for 25 %-50%, 8 modified products accounted for 5%-22%. Further preferably, 4 modified products account for 30%-43%, and 8 modified products account for 7%-17%.
  • the polymyxin E 2 methanesulfonate of the present invention comprises 4 modified products, 6 modified products, 8 modified products, ie, polymyxin E 2 structure, Dbu
  • the ⁇ -amino group is composed of 4 sodium methanesulfonate groups (-CH 2 SO 3 Na), 6 sodium methanesulfonate groups (-CH 2 SO 3 Na) or 8 sodium methanesulfonate groups (-CH 2 SO 3 Na).
  • Substitution, wherein in the HPLC map, by area normalization method 4 modified products account for 25%-50%, 6 modified products account for 35%-65%, and 8 modified products account for 5%-22%. Further preferably, 4 modified products account for 30%-43%, 6 modified products account for 45%-55%, and 8 modified products account for 7%-17%.
  • a method for preparing a polymyxin E 2 methanesulfonate having a titer of not less than 600 ⁇ g/mg comprising:
  • step (1) obtained polymyxin E 2 is reacted with a formaldehyde solution, wherein the molar ratio of polymyxin E 2 to formaldehyde is 1:10-20;
  • step (3) adding sodium bisulfite solution to the reaction product of step (2), the reaction is continued, wherein the molar ratio of polymyxin E 2 and sodium bisulfite is 1:10-20;
  • the molecular weight cut-off of the ultrafiltration membrane is between 1000 and 4000, and adding not more than 5 times the volume of the ultrafiltration retentate to the obtained ultrafiltration retentate. Then, ultrafiltration is performed, and 5 times of volume of water which does not exceed the ultrafiltration retentate is added to the obtained ultrafiltration retentate, and this is repeated several times, and when the conductivity is lowered to 2.0 mS/cm-3.0 mS/cm, the super-stop is stopped. filter;
  • the titer of polymyxin E 2 methanesulfonate is not less than 620 ⁇ g/mg; preferably not less than 640 ⁇ g/mg; more preferably not less than 660 ⁇ g/mg.
  • step (1) purification is carried out using reverse phase chromatography selected from a reverse phase silica gel matrix (eg, C18 filler) or a reverse phase polymer chromatography medium (eg, in polystyrene/two a reversed-phase polymer chromatography medium having a vinylbenzene or polyacrylate as a skeleton, preferably Amberchrom XT30 produced by The Dow Chemical Company or NM-100 produced by Suzhou Namicrobiology Co., Ltd.; the mobile phase of the chromatography is selected from the group consisting of ethanol A dilute solution of hydrochloric acid or a dilute solution of ethanol, the pH is controlled at 2.0-3.0; the elution mode is selected from isocratic fractional elution or gradient elution, preferably isocratic fractional elution.
  • a reverse phase silica gel matrix eg, C18 filler
  • a reverse phase polymer chromatography medium eg, in polystyrene/two a
  • the molar ratio of polymyxin E 2 to formaldehyde in step (2) is preferably 1:12-18.
  • the reaction temperature is controlled to be between 20 and 25 °C.
  • step (2) the pH is controlled between 6.5 and 7.5.
  • the reaction time is controlled to be from 1.5 to 2.5 hours, preferably 2 hours.
  • the molar ratio of polymyxin E 2 to sodium bisulfite in step (3) is preferably 1:12-18.
  • step (3) the pH is controlled between 6.8 and 7.2.
  • the reaction time is controlled to be 15-20 hours, preferably 18 hours.
  • the ultrafiltration membrane in step (4) preferably has a molecular weight cut off of from 1000 to 3000.
  • step (4) it is preferred to add 3-5 times the volume of ultrafiltration retentate to the ultrafiltration retentate, more preferably to super To the filtrate, the ultrafiltration retentate is added with 3.5-4.5 volumes of water, and it is particularly preferred to add 4 volumes of water to the ultrafiltration retentate.
  • the temperature at which ultrafiltration is controlled is between 20 and 25 °C.
  • step (4) it is preferred to stop ultrafiltration when the conductivity drops to 2.2 mS/cm to 2.8 mS/cm, more preferably to stop ultrafiltration when the conductivity drops to 2.4 mS/cm to 2.6 mS/cm. It is particularly preferred to stop ultrafiltration when the conductivity is lowered to 2.5 mS/cm.
  • E 2 colistin sodium methanesulfonate manufacture of a medicament the treatment of gram-negative bacterial infections.
  • the Gram-negative bacteria are selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae or NDM-1 bacteria.
  • the polymyxin E 2 methanesulfonate of the present invention can be used for the treatment of acute or chronic infections caused by sensitive Gram-negative bacilli, particularly against Enterobacter cloacae such as Klebsiella pneumoniae resistant to carbapenem antibiotics.
  • Enterobacter cloacae such as Klebsiella pneumoniae resistant to carbapenem antibiotics.
  • Bacterial resistant Enterobacteriaceae (CRE) and Pseudomonas aeruginosa, Acinetobacter baumannii, etc. including pulmonary infection, urinary tract infection, digestive tract infection or blood infection, including but not limited to sepsis, pneumonia, cystitis , nephritis and so on.
  • the preparation method of the invention prepares high-purity polymyxin E 2 sulfate and ensures high concentration of polymyxin E 2 methanesulfonate in the ultrafiltration retentate during ultrafiltration (ie, ultrafiltration retentate Adding 5 times by volume of water to the ultrafiltration retentate for ultrafiltration), unexpectedly obtaining a polymyxin E 2 methanesulfonate having a high titer (not less than 600 ⁇ g/mg) and a modified product, While ensuring the high efficiency of the product, it also ensures the stable quality of the batch between the products, which is conducive to ensuring the consistency of clinical efficacy and drug safety.
  • Figure 1 HPLC chromatogram of polymyxin E 2 prepared in Example 1.
  • Figure 2 HPLC chromatogram of polymyxin E 2 methanesulfonate prepared in Example 3.
  • Figure 3 HPLC chromatogram of polymyxin E 2 methanesulfonate prepared in Example 4.
  • Figure 4 HPLC chromatogram of polymyxin E 2 methanesulfonate prepared in Example 5.
  • Figure 5 HPLC chromatogram of polymyxin E 2 methanesulfonate prepared in Example 6.
  • Chromatography packing reversed microspheres with polystyrene/divinylbenzene as the skeleton, model XT-30, produced by Dow Chemical Co., Ltd.
  • Elution conditions first elute 5 CV with a dilute sulfuric acid solution containing 3% ethanol (pH of 2.4 with 0.05 mol/L sulfuric acid solution), and then dilute sulfuric acid solution with 8% ethanol (using 0.05 mol/L sulfuric acid) The pH of the solution was adjusted to 2.4) to elute 30 CV, and finally 5 CV was eluted with a dilute sulfuric acid solution containing 60% ethanol (pH of 2.4 with a 0.05 mol/L sulfuric acid solution).
  • Analytical HPLC analysis (analytical conditions refer to the European Pharmacopoeia 7.0 of polymyxin E), the collections with a purity greater than 96% were combined, and the pH of the solution was adjusted with 2 mol/L sodium hydroxide solution. After the value was 5.0 and concentrated under reduced pressure at 55 ° C, 140 ml of a concentrate containing 5.6 g of polymyxin E 2 sulfate was obtained, and the HPLC spectrum thereof is shown in Fig. 1.
  • the degree of modification of the polymyxin E 2 methanesulfonate prepared in Example 1 was analyzed by LC-MS.
  • HPLC conditions are as follows:
  • Phase B 10 mM ammonium formate-acetonitrile (50:50)
  • the mass spectrometry conditions are as follows:
  • Atomizing gas GS1 (Gas 1): 60psi
  • Auxiliary heating gas GS2 (Gas 2): 60 psi
  • 1155 is the molecular weight of polymyxin E 2
  • 82 is the molecular weight of H 2 SO 3
  • 12 is the molecular weight of C.
  • the polymyxin E 2 methanesulfonate obtained by 1.8 min, 2.5 min, 3.0 min, 3.4 min, and 4.7 min is an 8-modified product
  • the polymyxin E 2 methanesulfonate obtained by 8.8 min, 9.1 min, 11.0 min, 11.4 min, 11.9 min, 13.0 min, 13.7 min, and 15.1 min was a modified product of 18.6 min, 19.6 min, 23.7 min, 24.7.
  • the polymyxin E 2 methanesulfonate obtained by min, 25.7 min, 26.5 min, 28.9 min, and 30.0 min was a modified product of 4.
  • the polymyxin E 2 sulfate was purified by the method of the step (1) of Example 1, to obtain a concentrate of 140 ml containing 5.6 g of polymyxin E 2 .
  • the degree of modification of the polymyxin E 2 methanesulfonate prepared in Example 3 was analyzed by HPLC as follows.
  • HPLC conditions are as follows:
  • Phase A Ammonium formate solution (weigh 3.15 g of ammonium formate, dissolve in water and dilute to 1 L.)
  • the obtained HPLC spectrum is shown in Fig. 2.
  • the retention time is 10-20 min, and the chromatographic peak is 8 modified product of polymyxin E 2 methanesulfonate; the peak of 22-50 min is the polymyxin E 2 sodium methanesulfonate. 6 modified product; 51-65min to obtain a chromatographic peak of polymyxin E 2 methanesulfonate 4 modified product.
  • the polymyxin E 2 sulfate was purified by the method of the step (1) of Example 1, to obtain a concentrate of 140 ml containing 5.6 g of polymyxin E 2 .
  • the degree of modification of the polymyxin E 2 methanesulfonate prepared in Example 4 was analyzed by HPLC.
  • HPLC conditions were the same as in Example 3.
  • the obtained HPLC spectrum is shown in Fig. 3.
  • the retention time is 10-20 min, and the chromatographic peak is 8 modified product of polymyxin E 2 methanesulfonate; the peak of 22-50 min is the polymyxin E 2 sodium methanesulfonate. 6 modified product; 51-65min to obtain a chromatographic peak of polymyxin E 2 methanesulfonate 4 modified product.
  • the polymyxin E 2 sulfate was purified by the method of the step (1) of Example 1, to obtain a concentrate of 140 ml containing 5.6 g of polymyxin E 2 .
  • the degree of modification of the polymyxin E 2 methanesulfonate prepared in Example 5 was analyzed by HPLC.
  • HPLC conditions were the same as in Example 3.
  • the obtained HPLC spectrum is shown in Fig. 4.
  • the retention time is 10-20 min, and the chromatographic peak is 8 modified product of polymyxin E 2 methanesulfonate; the peak of 22-50 min is the polymyxin E 2 sodium methanesulfonate. 6 modified product; 51-65min to obtain a chromatographic peak of polymyxin E 2 methanesulfonate 4 modified product.
  • the polymyxin E 2 sulfate was purified by the method of the step (1) of Example 1, to obtain a concentrate of 140 ml containing 5.6 g of polymyxin E 2 .
  • the degree of modification of the polymyxin E 2 methanesulfonate prepared in Example 6 was analyzed by HPLC.
  • HPLC conditions were the same as in Example 3.
  • the obtained HPLC spectrum is shown in Fig. 5.
  • the retention time is 10-20 min, and the chromatographic peak is 8 modified product of polymyxin E 2 methanesulfonate; the peak of 22-50 min is the polymyxin E 2 sodium methanesulfonate. 6 modified product; 51-65min to obtain a chromatographic peak of polymyxin E 2 methanesulfonate 4 modified product.
  • Escherichia coli and Pseudomonas aeruginosa were used as indicator bacteria to compare the antibacterial activity of these polymyxin E methanesulfonate. See the table below.
  • sample name E. coli (ATCC8739) Pseudomonas aeruginosa (ATCC9027)
  • the toxicity symptoms of the reference preparation and sodium polymyxin E 2 mesylate were: soft and weak, and the ears, limbs, and mouth were red and swollen. Before the death, the whole body was soft and weak, and the breathing was weak. After death, the body is edematous. Anatomy: No obvious organ abnormalities were observed. The severity of the toxic reaction is related to the concentration of the drug, and the higher the concentration, the more severe.
  • the animals in the reference preparation group were more severely reacted than the animals in the polymyxin E 2 methanesulfonate group at the same dose.
  • the mortality of the polymyxin E 2 methanesulfonate group was lower than that of the reference group at the same dose, and the reference preparation had two dose groups (80, 40 mg/kg) at 2 or 3 doses.
  • the animal died, and the same dose of polymyxin E 2 mesylate did not show death or a small amount of death after 4 administrations.

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Abstract

提供一种高效价的多黏菌素E 2甲磺酸钠，其制备方法以及在制备治疗革兰氏阴性菌感染的药物中的用途。

Description

高效价的多黏菌素E2甲磺酸钠 技术领域
本发明属于医药领域，涉及效价不低于600μg/mg的多黏菌素E2甲磺酸钠、其制备方法以及在制备治疗革兰氏阴性菌感染的药物中的用途。
背景技术
多黏菌素E(colistin)是一种由多种组分组成的多肽类抗生素，主要由E1和E2组成(或称为A或B)。在20世纪50年代，多黏菌素E就应用于临床，主要用于革兰氏阴性菌引起的感染，特别是由多重耐药铜绿假单孢菌、鲍曼不动杆菌、肺炎克雷伯菌等引起感染的治疗。临床使用的多黏菌素E类产品有两种，一种是口服或局部使用的硫酸多黏菌素E(也称硫酸黏菌素)，另一种是供注射用的多黏菌素E甲磺酸钠(Colistimethate Sodium)。这两种药物均是多组分药物，在硫酸多黏菌素E中，含有E1、E2、E3等至少5种组分(参见欧洲药典7.0)，同样在多黏菌素E甲磺酸钠中也至少含有对应的多黏菌素E1甲磺酸钠、多黏菌素E2甲磺酸钠等5种组份，然而，不同的组分可能会带来药效学、药代动力学和毒理学的差异，为了保证多黏菌素E产品的批间质量稳定性，需制备单一组分的多黏菌素E组合物。
中国专利申请CN102190710A公开了一种多黏菌素E2组合物，其中多黏菌素E2的结构如式I所示，实施例4公开了多黏菌素E2甲磺酸钠组合物的制备，所述组合物的效价为560μg/mg，尽管其效价高于市售产品，但经测定，所述组合物中修饰产物种类较多，较难保证产品的批间质量稳定性。
Figure PCTCN2017115467-appb-000001
中国专利申请CN104918951A公开了一种含有至少3个氨基被甲磺酸钠基双修饰的组合物，其中实施例2公开了10修饰的多黏菌素E2甲磺酸钠的制备，其相比于CMS对照物一般具有较低的活性。
因此，仍需提供高效价且产品批间质量稳定的多黏菌素E2甲磺酸钠。
发明内容
本发明一方面提供一种多黏菌素E2甲磺酸钠，其效价不低于600μg/mg。
在一些优选实施方案中，其效价不低于620μg/mg。
在一些更优选的实施方案中，其效价不低于640μg/mg。
在一些特别优选的实施方案中，其效价不低于660μg/mg。
在一些实施方案中，本发明的多黏菌素E2甲磺酸钠包含修饰产物，其中修饰指，多黏菌素E2结构中，Dbu的γ氨基被甲磺酸钠基(-CH2SO3Na)或甲醇基(-CH2OH)取代。优选的，Dbu的γ氨基被甲磺酸钠基(-CH2SO3Na)取代。
在本发明的一些优选实施方案中，本发明的多黏菌素E2甲磺酸钠包含4修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基(-CH2SO3Na)取代，其中，在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％。进一步优选的，4修饰产物占30％-43％。
在本发明的一些优选实施方案中，本发明的多黏菌素E2甲磺酸钠包含6修饰产物，即多黏菌素E2结 构中，Dbu的γ氨基被6个甲磺酸钠基(-CH2SO3Na)取代，其中，在HPLC图谱中以面积归一化法计，6修饰产物占35％-65％。进一步优选的，6修饰产物占45％-55％。
在本发明的一些优选实施方案中，本发明的多黏菌素E2甲磺酸钠包含8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被8个甲磺酸钠基(-CH2SO3Na)取代，其中，在HPLC图谱中以面积归一化法计，8修饰产物占5％-22％。进一步优选的，8修饰产物占7％-17％。
在本发明的一些更优选的实施方案中，本发明的多黏菌素E2甲磺酸钠包含4修饰产物、6修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基(-CH2SO3Na)或6个甲磺酸钠基(-CH2SO3Na)取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，6修饰产物占35％-65％。进一步优选的，4修饰产物占30％-43％，6修饰产物占45％-55％。
在本发明的一些更优选的实施方案中，本发明的多黏菌素E2甲磺酸钠包含4修饰产物、8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基(-CH2SO3Na)或8个甲磺酸钠基(-CH2SO3Na)取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，8修饰产物占5％-22％。进一步优选的，4修饰产物占30％-43％，8修饰产物占7％-17％。
在本发明的一些更优选的实施方案中，本发明的多黏菌素E2甲磺酸钠包含4修饰产物、6修饰产物、8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基(-CH2SO3Na)、6个甲磺酸钠基(-CH2SO3Na)或8个甲磺酸钠基(-CH2SO3Na)取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，6修饰产物占35％-65％，8修饰产物占5％-22％。进一步优选的，4修饰产物占30％-43％，6修饰产物占45％-55％，8修饰产物占7％-17％。
本发明再一方面提供一种制备效价不低于600μg/mg的多黏菌素E2甲磺酸钠的方法，包括：
(1)以硫酸多黏菌素E为原料，通过层析纯化得到纯度≥96％的硫酸多黏菌素E2；
(2)将步骤(1)得到的硫酸多黏菌素E2与甲醛溶液反应，其中硫酸多黏菌素E2与甲醛的摩尔比为1:10-20；
(3)向步骤(2)的反应产物中加入亚硫酸氢钠溶液，继续反应，其中硫酸多黏菌素E2与亚硫酸氢钠的摩尔比为1:10-20；
(4)通过超滤的方式纯化步骤(3)的反应液，超滤膜的截留分子量在1000-4000之间，向得到的超滤截留液中加入不超过超滤截留液5倍体积的水，再进行超滤，再向得到的超滤截留液中加入不超过超滤截留液5倍体积的水，如此反复多次，当电导率降至2.0mS/cm-3.0mS/cm时停止超滤；
(5)将步骤(4)所得的超滤截留液进行冷冻干燥，得到多黏菌素E2甲磺酸钠。
在一些实施方案中，多黏菌素E2甲磺酸钠的效价不低于620μg/mg；优选不低于640μg/mg；更优选不低于660μg/mg。
在一些实施方案中，步骤(1)中，使用反相层析进行纯化，层析填料选自反相硅胶基质(例如C18填料)或反相聚合物层析介质(例如以聚苯乙烯/二乙烯苯或聚丙烯酸酯为骨架的反相聚合物层析介质，优选为陶氏化学公司出品的Amberchrom XT30或者苏州纳微生物科技有限公司生产的NM-100)；层析的流动相选自乙醇的硫酸稀溶液或乙醇的盐酸稀溶液，pH控制在2.0-3.0；洗脱方式选自等度分段洗脱或梯度洗脱，优选为等度分段洗脱。
在一些实施方案中，步骤(2)中，硫酸多黏菌素E2与甲醛的摩尔比优选为1:12-18。
在一些实施方案中，步骤(2)中，控制反应温度在20-25℃。
在一些实施方案中，步骤(2)中，控制pH在6.5-7.5。
在一些实施方案中，步骤(2)中，控制反应时间在1.5-2.5小时，优选为2小时。
在一些实施方案中，步骤(3)中，硫酸多黏菌素E2与亚硫酸氢钠的摩尔比优选为1:12-18。
在一些实施方案中，步骤(3)中，控制pH在6.8-7.2。
在一些实施方案中，步骤(3)中，控制反应时间在15-20小时，优选为18小时。
在一些实施方案中，步骤(4)中的超滤膜的截留分子量优选为1000-3000。
在一些实施方案中，步骤(4)中，优选向超滤截留液中加入超滤截留液3-5倍体积的水，更优选向超 滤截留液中加入超滤截留液3.5-4.5倍体积的水，特别优选向超滤截留液中加入超滤截留液4倍体积的水。
在一些实施方案中，步骤(4)中，控制超滤时的温度在20-25℃。
在一些实施方案中，步骤(4)中，优选电导率降至2.2mS/cm-2.8mS/cm时停止超滤，更优选电导率降至2.4mS/cm-2.6mS/cm时停止超滤，特别优选电导率降至2.5mS/cm时停止超滤。
本发明再一方面提供多黏菌素E2甲磺酸钠在制备治疗革兰氏阴性菌感染的药物中的用途。优选的，所述革兰氏阴性菌选自大肠杆菌、铜绿假单孢菌、鲍曼不动杆菌、肺炎克雷伯菌或NDM-1细菌。
例如，本发明的多黏菌素E2甲磺酸钠可用于治疗敏感革兰阴性杆菌引起的急性或慢性感染，特别是针对碳青霉烯类抗生素耐药的肺炎克雷伯菌等肠杆菌科细菌(carbapenem resistant Enterobacteriaceae，CRE)和铜绿假单胞菌、鲍曼不动杆菌等引起的肺部感染、尿路感染、消化道感染或血液感染等，包括但不限于败血症、肺炎、膀胱炎、肾炎等。
本发明的制备方法，通过制备高纯度的硫酸多黏菌素E2，以及在超滤过程中保证超滤截留液中多黏菌素E2甲磺酸钠高浓度(即超滤截留液中加入不超过超滤截留液5倍体积的水进行超滤)，出乎意料地得到了高效价(不低于600μg/mg)且修饰产物稳定的的多黏菌素E2甲磺酸钠，在保证产品高效的同时也保证了产品的批间质量稳定，有利于保证临床疗效的一致性和用药安全性。
附图说明
图1：实施例1制备的硫酸多黏菌素E2的HPLC图谱。
图2：实施例3制备的多黏菌素E2甲磺酸钠HPLC图谱。
图3：实施例4制备的多黏菌素E2甲磺酸钠HPLC图谱。
图4：实施例5制备的多黏菌素E2甲磺酸钠HPLC图谱。
图5：实施例6制备的多黏菌素E2甲磺酸钠HPLC图谱。
具体实施方式
下面用具体实施例来进一步说明本发明的内容，但并不以任何方式意味着对本发明进行限制。
实施例1多黏菌素E2甲磺酸钠的制备
(1)纯化硫酸多黏菌素E2
称取工业级硫酸多黏菌素E(购自绿康生化股份有限公司)适量，加水溶解，制成每1ml含120mg的溶液，上色谱柱分离，色谱条件如下：
层析填料：聚苯乙烯/二乙烯苯为骨架的反相微球，型号为XT-30，产自美国陶氏(DOW)化学有限公司
柱规格：50×250mm，
柱体积(CV)：491ml
流速：20.0ml/min
上样量：300ml
洗脱条件：先用含3％乙醇的硫酸稀溶液(用0.05mol/L硫酸调溶液的pH值为2.4)洗脱5CV，再用含8％乙醇的硫酸稀溶液(用0.05mol/L硫酸调溶液的pH值为2.4)洗脱30CV，最后用含60％乙醇的硫酸稀溶液(用0.05mol/L硫酸调溶液的pH值为2.4)洗脱5CV。
收集：50ml/管。
收集样品的分析及处理：用分析型HPLC分析(分析条件参照硫酸多黏菌素E的欧洲药典7.0)，将纯度大于96％的收集液合并，用2mol/L氢氧化钠溶液调溶液的pH值为5.0后并于55℃下减压蒸馏浓缩后得到浓缩液140ml，其中含有5.6g硫酸多黏菌素E2，其HPLC图谱参见图1。
多黏菌素E2甲磺酸钠的制备
将步骤(1)得到的140ml硫酸多黏菌素E2浓缩液于25℃水浴10min后加入甲醛溶液4.4ml，用2.5mol/L NaOH调节pH使其维持在6.5-7.5之间，搅拌2h后加入40％NaHSO3溶液16ml，用2.5mol/L NaOH调节pH至7.0，水浴控制温度于25℃下反应18h。将反应液用截留分子量为3000Da的超滤膜(MILLIPORE公 司)进行脱盐处理，待超滤截留液的总体积剩余约50ml时补加去离子水到200ml，在超滤过程中控制料液温度在20-25℃之间，截留液的pH维持在6.5-7.5之间，环境温度控制在20-25℃，再按照同样的方式补加去离子水到200ml进行多次超滤，截留液的电导率降至2.5mS/cm左右时停止超滤。将脱盐后的多黏菌素E2甲磺酸钠溶液进行冷冻干燥，得6.3g多黏菌素E2甲磺酸钠。
实施例2多黏菌素E2甲磺酸钠修饰度分析
通过LC-MS对实施例1制备的多黏菌素E2甲磺酸钠进行修饰度分析。
(1)LC-MS检测方法
HPLC条件如下：
仪器：Shimadzu UFLC XR(LC-20AD XR泵；SIL-20AC XR自动进样器；CTO-20AC柱温箱；SPD-M20A检测器)
色谱柱：Waters Atlantis T3(150mm×4.6mm，3μm)620#
流动相：A相：10mM甲酸铵-乙腈(95:5)
B相：10mM甲酸铵-乙腈(50:50)
线性梯度洗脱，洗脱程序如下表：
Figure PCTCN2017115467-appb-000002
流速：1.0ml/min
柱温：30℃
检测波长：210nm
质谱条件如下：
仪器：AB SCIEX Triple TOF 4600
离子源：DuoSprayTMIon Source(ESI)
参数：
离子化方式：负离子
气帘气CUR(Curtain Gas)：35psi
雾化气GS1(Gas 1)：60psi
辅助加热气GS2(Gas 2)：60psi
喷雾电压ISVF(IonSpray Voltage Floating)：-4500V
雾化温度TEM(Temperature)：550℃
碰撞能量CE(Collision Energy)：35V
碰撞能量范围CES(Collision Energy Spread)：15V
去簇电压DP(Declustering Potential)：80V
扫描范围(Mass Range)：m/z 100-2000
(2)LC-MS分析结果
在质谱数据解析公式中：1155为多黏菌素E2的分子量，82为H2SO3的分子量，12为C的分子量。
表1多黏菌素E2甲磺酸钠LC-MS分析结果及数据解析
Figure PCTCN2017115467-appb-000003
Figure PCTCN2017115467-appb-000004
即，1.8min、2.5min、3.0min、3.4min、4.7min得到的多黏菌素E2甲磺酸钠为8修饰产物；
8.8min、9.1min、11.0min、11.4min、11.9min、13.0min、13.7min、15.1min得到的多黏菌素E2甲磺酸钠为6修饰产物；18.2min、19.6min、23.7min、24.7min、25.7min、26.5min、28.9min、30.0min得到的多黏菌素E2甲磺酸钠为4修饰产物。
经面积归一化法测定，8修饰产物占8.7％、6修饰产物占46.0％，4修饰产物占42.7％。
实施例3多黏菌素E2甲磺酸钠的制备
参照实施例1步骤(1)的方法纯化硫酸多黏菌素E2，得到浓缩液140ml，其中含有5.6g硫酸多黏菌素E2。
将步骤(1)得到的140ml硫酸多黏菌素E2浓缩液于25℃水浴10min后加入甲醛溶液4.4ml，用2.5mol/L NaOH调节pH使其维持在至6.5-7.5之间，搅拌2h后加入40％NaHSO3溶液16ml，用2.5mol/L NaOH调节pH至7.0，水浴控制温度于25℃下反应18h。将反应液用截留分子量为3000Da的超滤膜(MILLIPORE公司)进行脱盐处理，待超滤截留液的总体积剩余约50ml时补加去离子水到220ml，在超滤过程中控制料液温度在20-25℃之间，截留液的pH维持在至6.5-7.5之间，环境温度控制在20-25℃，再按照同样的方式 补加去离子水到220ml进行多次超滤，截留液的电导率降至2.5mS/cm左右时停止超滤。将脱盐后的多黏菌素E2甲磺酸钠溶液进行冷冻干燥，得6.3g多黏菌素E2甲磺酸钠。
采用下述HPLC对实施例3制备的多黏菌素E2甲磺酸钠进行修饰度分析。
HPLC条件如下：
色谱柱：XSELECT CSH C18(4.6×250mm；5μm)
流动相：A相：甲酸铵溶液(称取甲酸铵3.15g，加水溶解并定容至1L。)
B相：乙腈
线性梯度洗脱，洗脱程序如下表：
Figure PCTCN2017115467-appb-000005
流速：0.8ml/min
柱温：30℃
检测波长：230nm
所得HPLC图谱参见图2，保留时间在10-20min得到色谱峰为多黏菌素E2甲磺酸钠的8修饰产物；22-50min得到色谱峰为多黏菌素E2甲磺酸钠的6修饰产物；51-65min得到色谱峰为多黏菌素E2甲磺酸钠的4修饰产物。
经面积归一化法测定，8修饰产物占7.9％、6修饰产物占50.8％，4修饰产物占41.3％。
实施例4多黏菌素E2甲磺酸钠的制备
参照实施例1步骤(1)的方法纯化硫酸多黏菌素E2，得到浓缩液140ml，其中含有5.6g硫酸多黏菌素E2。
将步骤(1)得到的140ml硫酸多黏菌素E2浓缩液于25℃水浴10min后加入甲醛溶液4.4ml，用2.5mol/L NaOH调节pH使其维持在至6.5-7.5之间，搅拌2h后加入40％NaHSO3溶液16ml，用2.5mol/L NaOH调节pH至7.0，水浴控制温度于25℃下反应18h。将反应液用截留分子量为3000Da的超滤膜(MILLIPORE公司)进行脱盐处理，待超滤截留液的总体积剩余约50ml时补加去离子水到180ml，在超滤过程中控制料液温度在20-25℃之间，截留液的pH维持在至6.5-7.5之间，环境温度控制在20-25℃，再按照同样的方式补加去离子水到180ml进行多次超滤，截留液的电导率降至2.5mS/cm左右时停止超滤。将脱盐后的多黏菌素E2甲磺酸钠溶液进行冷冻干燥，得6.3g多黏菌素E2甲磺酸钠。
采用HPLC对实施例4制备的多黏菌素E2甲磺酸钠进行修饰度分析。
HPLC条件同实施例3。
所得HPLC图谱参见图3，保留时间在10-20min得到色谱峰为多黏菌素E2甲磺酸钠的8修饰产物；22-50min得到色谱峰为多黏菌素E2甲磺酸钠的6修饰产物；51-65min得到色谱峰为多黏菌素E2甲磺酸钠的4修饰产物。
经面积归一化法测定，8修饰产物占15.2％、6修饰产物占54.6％，4修饰产物占30.2％。
实施例5多黏菌素E2甲磺酸钠的制备
参照实施例1步骤(1)的方法纯化硫酸多黏菌素E2，得到浓缩液140ml，其中含有5.6g硫酸多黏菌素E2。
将步骤(1)得到的140ml硫酸多黏菌素E2浓缩液于25℃水浴10min后加入甲醛溶液4.4ml，用2.5mol/L NaOH调节pH使其维持在6.5-7.5之间，搅拌2h后加入40％NaHSO3溶液16ml，用2.5mol/L NaOH调节 pH至7.0，水浴控制温度于25℃下反应18h。将反应液用截留分子量为1000Da的超滤膜(MILLIPORE公司)进行脱盐处理，待超滤截留液的总体积剩余约50ml时补加去离子水到240ml，在超滤过程中控制料液温度在20-25℃之间，截留液的pH维持在至6.5-7.5之间，环境温度控制在20-25℃，再按照同样的方式补加去离子水到240ml进行多次超滤，截留液的电导率降至2.5mS/cm左右时停止超滤。将脱盐后的多黏菌素E2甲磺酸钠溶液进行冷冻干燥，得6.3g多黏菌素E2甲磺酸钠。
采用HPLC对实施例5制备的多黏菌素E2甲磺酸钠进行修饰度分析。
HPLC条件同实施例3。
所得HPLC图谱参见图4，保留时间在10-20min得到色谱峰为多黏菌素E2甲磺酸钠的8修饰产物；22-50min得到色谱峰为多黏菌素E2甲磺酸钠的6修饰产物；51-65min得到色谱峰为多黏菌素E2甲磺酸钠的4修饰产物。
经面积归一化法测定，8修饰产物占13.3％、6修饰产物占54.9％，4修饰产物占31.7％。
实施例6多黏菌素E2甲磺酸钠的制备
参照实施例1步骤(1)的方法纯化硫酸多黏菌素E2，得到浓缩液140ml，其中含有5.6g硫酸多黏菌素E2。
将步骤(1)得到的140ml硫酸多黏菌素E2浓缩液于25℃水浴10min后加入甲醛溶液4.4ml，用2.5mol/L NaOH调节pH使其维持在6.5-7.5之间，搅拌2h后加入40％NaHSO3溶液16ml，用2.5mol/L NaOH调节pH至7.0，水浴控制温度于25℃下反应18h。将反应液用截留分子量为1000Da的超滤膜(MILLIPORE公司)进行脱盐处理，待超滤截留液的总体积剩余约50ml时补加去离子水到190ml，在超滤过程中控制料液温度在20-25℃之间，截留液的pH维持在6.5-7.5之间，环境温度控制在20-25℃，再按照同样的方式补加去离子水到190ml进行多次超滤，截留液的电导率降至2.5mS/cm左右时停止超滤。将脱盐后的多黏菌素E2甲磺酸钠溶液进行冷冻干燥，得6.3g多黏菌素E2甲磺酸钠。
采用HPLC对实施例6制备的多黏菌素E2甲磺酸钠进行修饰度分析。
HPLC条件同实施例3。
所得HPLC图谱参见图5，保留时间在10-20min得到色谱峰为多黏菌素E2甲磺酸钠的8修饰产物；22-50min得到色谱峰为多黏菌素E2甲磺酸钠的6修饰产物；51-65min得到色谱峰为多黏菌素E2甲磺酸钠的4修饰产物。
经面积归一化法测定，8修饰产物占8.3％、6修饰产物占50.8％，4修饰产物占40.9％。
实施例7抗菌活性测定
采用美国药典公布的多黏菌素E甲磺酸钠效价测定方法，以多黏菌素E甲磺酸钠的美国药典标准品(批号：H3J047，效价为420μg/mg)作为对照，分别对实施例1、3-6制备的多黏菌素E2甲磺酸钠和参比制剂(美国JHP制药公司，批号：762042)进行活性测定，具体结果参见下表。
多黏菌素E甲磺酸钠效价测定结果
样品名称 效价(μg/mg)
实施例1制备的多黏菌素E2甲磺酸钠 685
实施例3制备的多黏菌素E2甲磺酸钠 710
实施例4制备的多黏菌素E2甲磺酸钠 697
实施例5制备的多黏菌素E2甲磺酸钠 689
实施例6制备的多黏菌素E2甲磺酸钠 708
参比制剂 428
另以大肠杆菌和铜绿假单孢菌为指示菌，比较这些多黏菌素E甲磺酸钠的抗菌活性差异，具体结果参 见下表。
多黏菌素E甲磺酸钠MIC(μg/mL)测定结果
样品名称 大肠杆菌(ATCC8739) 铜绿假单孢菌(ATCC9027)
实施例1制备的多黏菌素E2甲磺酸钠 2 2
实施例3制备的多黏菌素E2甲磺酸钠 2 2
实施例4制备的多黏菌素E2甲磺酸钠 2 2
实施例5制备的多黏菌素E2甲磺酸钠 2 2
实施例6制备的多黏菌素E2甲磺酸钠 2 2
参比制剂 4 4
实施例8毒性测定
取SD大鼠20只，分为多黏菌素E2甲磺酸钠组(5雌5雄)、参比制剂组(5雌5雄)，设不同的剂量，静脉推注，每天1次，连续7天，比较动物的死亡率。试验结果参见下表。
多黏菌素E甲磺酸钠对大鼠急性毒性(死亡率)比较结果
Figure PCTCN2017115467-appb-000006
参比制剂和多黏菌素E2甲磺酸钠给药后的毒性症状为：瘫软无力，耳朵、四肢、嘴巴红肿。死亡前全身瘫软无力，呼吸微弱。死亡后，全身水肿。解剖：未见明显的脏器异常。毒性反应的严重性与给药浓度相关，浓度越高越严重。
给药后参比制剂组动物较同剂量下多黏菌素E2甲磺酸钠组动物的反应严重。相同给药剂量下多黏菌素E2甲磺酸钠组动物死亡率较参比制剂组低，而且参比制剂有两个剂量组(80、40mg/kg)，在给药2次或3次的情况下动物就出现了死亡，同剂量下的多黏菌素E2甲磺酸钠在给药4次后仍未出现死亡或少量死亡。
总之，相同给药条件下多黏菌素E2甲磺酸钠的毒性作用显著弱于参比制剂。

Claims (10)

  1. 一种多黏菌素E2甲磺酸钠，其效价不低于600μg/mg，优选不低于620μg/mg，更优选不低于640μg/mg，特别优选不低于660μg/mg。
  2. 权利要求1的多黏菌素E2甲磺酸钠，其包含修饰产物，其中修饰指，多黏菌素E2结构中，Dbu的γ氨基被甲磺酸钠基或甲醇基取代，优选的，Dbu的γ氨基被甲磺酸钠基取代。
  3. 权利要求2的多黏菌素E2甲磺酸钠，其包含4修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基取代，其中，在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，进一步优选的，4修饰产物占30％-43％。
  4. 权利要求2的多黏菌素E2甲磺酸钠，其包含6修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被6个甲磺酸钠基取代，其中，在HPLC图谱中以面积归一化法计，6修饰产物占35％-65％，进一步优选的，6修饰产物占45％-55％。
  5. 权利要求2的多黏菌素E2甲磺酸钠，其包含8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被8个甲磺酸钠基取代，其中，在HPLC图谱中以面积归一化法计，8修饰产物占5％-22％，进一步优选的，8修饰产物占7％-17％。
  6. 权利要求2的多黏菌素E2甲磺酸钠，其包含4修饰产物、6修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基或6个甲磺酸钠基取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，6修饰产物占35％-65％，进一步优选的，4修饰产物占30％-43％，6修饰产物占45％-55％。
  7. 权利要求2的多黏菌素E2甲磺酸钠，其包含4修饰产物、8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基或8个甲磺酸钠基取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，8修饰产物占5％-22％，进一步优选的，4修饰产物占30％-43％，8修饰产物占7％-17％。
  8. 权利要求2的多黏菌素E2甲磺酸钠，其包含4修饰产物、6修饰产物、8修饰产物，即多黏菌素E2结构中，Dbu的γ氨基被4个甲磺酸钠基、6个甲磺酸钠基或8个甲磺酸钠基取代，其中在HPLC图谱中以面积归一化法计，4修饰产物占25％-50％，6修饰产物占35％-65％，8修饰产物占5％-22％，进一步优选的，4修饰产物占30％-43％，6修饰产物占45％-55％，8修饰产物占7％-17％。
  9. 权利要求1-8中任一项的多黏菌素E2甲磺酸钠的制备方法，包括：
    (1)以硫酸多黏菌素E为原料，通过层析纯化得到纯度≥96％的硫酸多黏菌素E2；
    (2)将步骤(1)得到的硫酸多黏菌素E2与甲醛溶液反应，其中硫酸多黏菌素E2与甲醛的摩尔比为1:10-20；
    (3)向步骤(2)的反应产物中加入亚硫酸氢钠溶液，继续反应，其中硫酸多黏菌素E2与亚硫酸氢钠的摩尔比为1:10-20；
    (4)通过超滤的方式纯化步骤(3)的反应液，超滤膜的截留分子量在1000-4000之间，向得到的超滤截留液中加入不超过超滤截留液5倍体积的水，再进行超滤，再向得到的超滤截留液中加入不超过超滤截留液5倍体积的水，如此反复多次，当电导率降至2.0mS/cm-3.0mS/cm时停止超滤；
    (5)将步骤(4)所得的超滤截留液进行冷冻干燥，得到多黏菌素E2甲磺酸钠。
  10. 权利要求1-8中任一项的多黏菌素E2甲磺酸钠在制备治疗革兰氏阴性菌感染的药物中的用途，优选的，所述革兰氏阴性菌选自大肠杆菌、铜绿假单孢菌、鲍曼不动杆菌、肺炎克雷伯菌或NDM-1细菌。
PCT/CN2017/115467 2016-12-09 2017-12-11 高效价的多黏菌素e2甲磺酸钠 WO2018103758A1 (zh)

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