WO2009046618A1 - Deshydroxy vancomycin, the preparation, pharmaceutical composition and the use - Google Patents

Deshydroxy vancomycin, the preparation, pharmaceutical composition and the use Download PDF

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Publication number
WO2009046618A1
WO2009046618A1 PCT/CN2008/001524 CN2008001524W WO2009046618A1 WO 2009046618 A1 WO2009046618 A1 WO 2009046618A1 CN 2008001524 W CN2008001524 W CN 2008001524W WO 2009046618 A1 WO2009046618 A1 WO 2009046618A1
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Prior art keywords
vancomycin
gel
fermentation
purification
preparation
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PCT/CN2008/001524
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English (en)
French (fr)
Inventor
Bingxiang Xu
Haisong Xie
Huan YU
Wei Mao
Weidong Ye
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Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory
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Application filed by Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory filed Critical Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory
Priority to US12/680,650 priority Critical patent/US8338372B2/en
Priority to EP20080800525 priority patent/EP2208732B1/en
Publication of WO2009046618A1 publication Critical patent/WO2009046618A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
    • 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
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin

Definitions

  • the present invention relates to novel vancomycin derivatives.
  • the vancomycin derivative is fermented by a strain of the genus Amycolatopsis orientalis (Amycolatopsis Orientalis), and the microorganism strain is deposited as CGMCCNO.ll 83, and dehydroxyvancomycin is obtained by preparative chromatography. Book
  • Vancomycin is a bactericidal glycopeptide antibiotic developed in the 1950s and has a strong bactericidal action against Gram-positive cocci.
  • Staphylococcus epidermidis has resulted in the infection of methicillin-resistant Staphylococcus aureus, which is particularly important for the clinical effect of vancomycin. Therefore, vancomycin is currently the drug of choice for the treatment of serious infectious diseases caused by methicillin-resistant Staphylococcus aureus. Due to the unique mechanism of action of vancomycin, its peptide antibiotics are easily decomposed in the body, making it widely used clinically.
  • norvancomycin like vancomycin, can effectively inhibit the colonization and reproduction of Clostridium difficile in the intestine, thus eliminating its Pathogenicity. It is a special drug for the treatment of Clostridium difficile pseudomembranous colitis, has strong antibacterial effect, and is an effective drug for treating anaerobic infection and some Gram-positive bacteria. It is not easy to produce drug resistance during use, has no cross-resistance with other antibiotics, and has good pharmacokinetic properties. But it can't always replace imported vancomycin.
  • the object of the present invention is to obtain a novel vancomycin derivative produced by the vancomycin fermentation process.
  • the invention provides a dehydroxyvancomycin of formula I.
  • Formula I Dehydroxyvancomycin molecular structure Wherein 1 in the dehydroxy vancomycin molecule of Formula I is 11. Therefore, the difference between vancomycin and dehydroxyvancomycin is that R is H in the dehydroxy vancomycin molecule, and R is OH in the vancomycin molecule.
  • the invention also provides a preparation method of dehydroxy vancomycin, comprising the following steps:
  • step (3) purifying and purifying the above-mentioned fine filtrate to obtain dehydroxy vancomycin; wherein, in step (2), performing column chromatography separation and purification in a gel chromatography column containing a salt-water mobile phase;
  • step (3) chromatographic separation and purification are carried out in a macroporous adsorption resin chromatography column containing a buffer-methanol mobile phase.
  • the pH of the vancomycin concentrate was adjusted to 2.8.
  • Gel column chromatography the mobile phase is an aqueous solution of 0.1M NH4HCO 3.
  • the gels were a dextran gel, a polyacrylamide gel, an agarose gel, and a polystyrene gel.
  • the column chromatography solution is subjected to nanofiltration, concentration and desalting using a 0.22 um microporous membrane.
  • the macroporous adsorption resin is a Rohm and Haas macroporous adsorption resin.
  • a method for preparing dehydroxy vancomycin includes the following:
  • Shake flask seed medium loading 750ml shake flask volume is 100ml; culture temperature: 28 ° C; culture time: 48hr; well-grown shake flask seed solution pH 7.7 or so, appearance is milky yellow, centrifuged at 4000rpm for 10 min, bacteria
  • the amount of silk is about 22%; the microscopic hyphae are thick and strong, and no bacteria can be used as seeds to feed the next-stage seed tank culture or small-scale fermentation tank fermentation.
  • the fermentation test was carried out at three different temperatures of 25 °C, 28 °C, and 30 °C under shake flask conditions. The results showed that the fermentation level was the highest at 28 °C, and the by-products in the fermentation broth were higher than the other two temperature conditions. To be less, it was therefore determined that the vancomycin fermentation temperature was 28 °C.
  • the fermentation conditions of the fermentation medium under shaking conditions were 6.5, 6.8 and 7.0.
  • the results showed that the fermentation pH was the highest at the initial pH of the fermentation medium of 6.8, and the color of the fermentation broth was higher than that.
  • the two conditions were shallow and the by-products were also small, so the initial pH of the fermentation medium for vancomycin was determined to be 6.8.
  • Fermentation cycle The fermentation period of the shake flask is 6 days, and the fermentation cycle of the fermenter is 5-6 days.
  • Shake flask volume The fermentation level is the highest when the 750ml shake flask volume is 100ml.
  • vancomycin Since the pH of the fermentation broth is about 7.7 at the end of the fermentation of vancomycin, vancomycin is present in the form of a free base in the fermentation broth.
  • vancomycin is adsorbed onto the macroporous adsorption resin (D1300) by exchange. This exchange process requires that the antibiotic be present as a free acid. Therefore, it is necessary to acidify the fermentation broth with dilute hydrochloric acid and adjust the pH to about 3.2. In this acidification process, flocculant and help can be added at the same time.
  • a filter or the like makes the fermentation liquid easy to filter and obtains a fermentation filtrate of a better quality.
  • D1300 is used as the adsorption resin.
  • the pH of the fermentation filtrate should be adjusted back to about 4.5 with 4N NaOH.
  • the desorption was carried out using an 80% acidic aqueous ethanol solution having a pH of 2.0.
  • the desorption solution was analyzed by HPLC.
  • the area ratio of vancomycin was generally around 75%, and the average unit should be above 10,00 ( ⁇ g/ml).
  • the desorbed liquid obtained by the above process is desalted by nanofiltration concentration, decolorized by activated carbon by a needle, and an appropriate amount of yellow blood salt and zinc sulfate are added to remove iron ions.
  • the collected mixed liquid is collected by desalting and concentration, and then added with 4N hydrochloric acid to adjust the pH to about 2.8.
  • the activated carbon is decolorized, and the yellow salt and zinc sulfate are used to remove the iron ions to obtain a desalting and decolorizing concentrated liquid, which satisfies the requirements of subsequent column purification.
  • the chromatographic medium used in the column layer was finally determined to be Sephadex C-25, and the in-depth and meticulous research on column conditions, eluent and elution methods was carried out to determine the best.
  • the purification scheme adopts 0.1M NH4HC0 3 aqueous solution for gradient elution, and the eluent is collected in stages, and the qualified fractions are combined and subjected to nanofiltration to remove desalted to obtain the obtained chromatographic refining solution.
  • the concentrated solution is passed through a Sephadex C-25 gel chromatography column, and further separated and purified according to the process requirements.
  • the above chromatographic purification liquids are combined and desalted by nanofiltration to obtain a chromatographic refining solution.
  • the qualified refined liquid is decolorized by activated carbon and filtered by a 0.22 um microporous membrane to remove the heat source, the vancomycin hydrochloride filtrate can be obtained by freeze-drying. 3.
  • High purity dehydroxy vancomycin is obtained by chromatographic separation and purification.
  • the present invention also provides the use of dehydroxyvancomycin for the preparation of a medicament for treating a susceptible bacterial infection.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of dehydroxy vancomycin, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further comprises vancomycin and/or vancomycin hydrochloride.
  • the inventors used preparative chromatography to separate and purify the vancomycin hydrochloride sample to obtain high-purity dehydroxyvancomycin for subsequent scientific research work.
  • Figure 1 is an AU spectrum of vancomycin hydrochloride as shown in the present invention. Detailed ways
  • Example 1 Isolation and purification of vancomycin hydrochloride containing vancomycin hydrochloride
  • the fermentation broth was acidified with dilute hydrochloric acid and the pH was adjusted to about 3.2.
  • yellow blood salt locculant or filter aid
  • D1300 is used as the adsorption resin.
  • the pH of the fermentation filtrate should be adjusted back to about 4.5 with 4N NaOH.
  • the desorption was carried out using an 80% acidic aqueous ethanol solution having a pH of 2.0.
  • the desorption solution was analyzed by HPLC.
  • the area ratio of vancomycin was generally around 75%, and the unit average should be above ⁇ , ⁇ .
  • the desorbed liquid obtained by the above process is desalted by nanofiltration, and then decolorized by activated carbon, and added An appropriate amount of yellow blood salt and zinc sulfate are added to remove iron ions.
  • the collected mixed solution is desalted and concentrated by nanofiltration, and then added with 4N hydrochloric acid to adjust the pH to about 2.8, decolorized by adding activated carbon, yellow blood salt and zinc sulfate to remove iron ions to obtain a desalting and decolorizing concentrated liquid.
  • Example 1 contains vancomycin hydrochloride aseptic raw material powder containing dehydroxy vancomycin; experimental apparatus:
  • UV6000 variable wavelength ultraviolet spectroscopic detector (provided by Beijing Innovation Tongheng Technology Co., Ltd.);
  • Rohm and Haas macroporous adsorption resin (XAD1600 resin) 250ml glass chromatography column; several auxiliary glass instruments.
  • Injection volume 150mg
  • Buffer Dissolve 4 ml of triethylamine in 1996 ml of water, adjust the pH to 3.2 with H 3 P04 to prepare a buffer;
  • the obtained AU spectrum is shown in Fig. 1.
  • the peak No. 5 is the main peak of vancomycin hydrochloride
  • the peak No. 6 is our target impurity peak.
  • a 250 ml XAD1600 resin column was placed, and the collected liquid was adsorbed on the column. After the column was washed with a large amount of water, it was eluted with 50% aqueous methanol, and 60 ml of the eluate was collected.
  • the 60 ml eluate was concentrated to about 5 ml with a rotary evaporator, and sent to a lyophilized machine room to freeze-dry to obtain 35 mg of the target.

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Description

去羟基万古霉素及其制备方法、 和其药物组合物及其用途 技术领域
本发明涉及新的万古霉素衍生物。具体地说， 利用东方拟无枝酸 菌（Amycolatopsis Orientalis说),微生物菌种保藏号为 CGMCCNO.ll 83 的菌株发酵得到该万古霉素衍生物，通过制备色谱分离纯化得到去羟 基万古霉素。 书
背景技术 万古霉素是五十年代开发的一种有杀菌作用的糖肽类抗生素，对 革兰阳性球菌有强大的杀菌作用。特别是最近由于表皮葡萄球菌感染 有所增加， 出现了耐甲氧西林金葡萄球菌的感染， 使临床对万古霉素 的作用尤为重要。 因此， 万古霉素是目前临床上用于治疗由甲氧西林 耐药金葡菌引起的严重感染疾病的首选药物。由于万古霉素的作用机 制独特， 其多肽类抗生素在体内容易分解， 使其在临床上得到广泛应 用。
此外， 国内己有去甲万古霉素同类药物， 实验表明， 去甲万古霉 素同万古霉素一样，可以有效地抑制难辨梭状芽孢杆菌在肠道中的定 植和繁殖， 从而消除了它的致病性。它是治疗难辨梭状芽孢杆菌伪膜 性肠炎的特效药物， 抗菌作用强， 是治疗厌氧菌感染及一些革兰式阳 性菌的有效药物。使用中不易产生耐药性，与其他抗生素无交叉耐药， 具有良好的药代动力学性质。 但它始终不能取代进口万古霉素。
Figure imgf000004_0001
盐酸万古霉素分子结构 近年来， 迫切需要研发出治疗人类疾病的新抗生素。为了研发新 药， 通常对已知的抗生素物质进行分子结构上的结构修饰来取得突 破， 但是这样的结构修饰往往会导致新物质失去所需的活性。在万古 霉素的发酵产物有多个衍生物生成，发酵菌株的不同也会导致衍生物 的差异。在已报道的文献中，我们发现美国 lilly公司申请的专利中就 列出了多种万古霉素的衍生物（专利号 EP0159180), 分别为 M43A, M43B, M43C, M43D。 其结构如下：
Figure imgf000005_0001
M43A
Figure imgf000005_0002
Q£
Figure imgf000006_0001
Figure imgf000006_0002
|7ZSlOO/800ZN3/X3d 8Ϊ99藝 OOZ OAV 本申请的发明人在盐酸万古霉素的生产中从万古霉素的发酵产 物中发现了一种新的万古霉素衍生物去羟基万古霉素。通过对其分子 结构研究，我们确定该物质是一种不同于上述四种万古霉素衍生物的 新物质。具体地说， 去羟基万古霉素与万古霉素的结构极为相似， 鉴 于万古霉素良好的抗菌能力，可以推测一旦去羟基万古霉素被认识清 楚， 极有可能成为一种新的抗生素。
发明内容
本发明的目的是为了得到万古霉素发酵过程产生的一种新的万 古霉素衍生物。 具体地说， 本发明提供了一种通式 I的去羟基万古霉 素。
Figure imgf000007_0001
通式 I： 去羟基万古霉素分子结构 其中， 通式 I的去羟基万古霉素分子中 1 为11。 因此， 万古霉素 与去羟基万古霉素的不同点在于去羟基万古霉素分子中 R为 H，而万 古霉素分子中 R为 OH。
本发明还提供了去羟基万古霉素的制备方法， 包括以下步骤：
( 1 ) 利用东方拟无枝酸菌 （Amycolatopsis Orientalis), 微生物 菌种保藏号为 CGMCCNO.1183的菌株制备含有去羟基万古霉素的万 古霉素浓缩液；
(2)通过柱层析分离纯化上述浓縮液得到含有去羟基万古霉素的 盐酸万古霉素精滤液；
(3 ) 再经过色谱分离纯化上述精滤液制得去羟基万古霉素； 其中， 在步骤（2) 中， 在含有盐-水流动相的凝胶层析柱中进行 柱层析分离纯化；
在步骤（3 ) 中， 在含有缓冲液 -甲醇流动相的大孔吸附树脂色谱 柱中进行色谱分离纯化。
其中， 将所述万古霉素浓缩液 pH调整至 2.8。 在凝胶层析柱中， 所述流动相为 0.1M NH4HCO3水溶液。所述凝胶为葡聚糖凝胶、聚丙 烯酰胺凝胶、 琼脂糖凝胶和聚苯乙烯凝胶。
在柱层析分离纯化中， 还包括用 0.22um微孔滤膜对柱层析液进 行纳滤、 浓縮、 脱盐。
此外， 在色谱分离纯化中， 所述流动相为缓冲液： 甲醇 =92: 8。 所述缓冲液包括三乙胺和磷酸， 且 pH=3.2。 所述大孔吸附树脂为罗 门哈斯大孔吸附树脂。
鉴于去羟基万古霉素是万古霉素的副产物，在进行制备色谱分离 纯化之前， 二者的工艺过程是一致的， 均采用东方拟无枝酸菌菌株制 备去羟基万古霉素与万古霉素。因此，在进行制备色谱分离纯化之前， 采用东方拟无枝酸菌 （Amycolatopsis Orientalis), 微生物菌种保藏号 为 CG MCCNO.1183菌株制备去羟基万古霉素，该方法参见本申请人 于 2004年 10月 15日提交的中国第 200410067197.7号专利申请。 根 据本发明， 去羟基万古霉素的制备方法包括如下：
1、 利用东方拟无枝酸菌菌株制备含有去羟基万古霉素的万古霉素
( 1 ) 培养基配方的选择及确定
通过对培养基配方中的碳源、 氮源及无机盐等的种类及浓度 的研究， 确定了发酵水平较高且稳定的斜面、 种子及发酵培养基 配方。
(2) 斜面培养基
经培养基配方的筛选比较， 确定万古霉素产生菌的斜面培养 基采用高氏一号斜面培养基， 具体配方如下：
可溶性淀粉 2.0%;
NaCl 0.05%；
KN03 0.1%；
MgSO4 0.05%;
FeS04 0.001%；
KH2PO4 0.05%；
琼脂 2.2%
消后 pH 7.2-7.4
斜面接种后， 于 28°C培养 7天。 生长好的斜面丰满， 表面呈 灰白色， 菌落呈馒头状， 表面有浅灰色环纹， 孢子乳白色， 直径 在 3-5mm之间。
(3) 种子培养基
经培养基配方的筛选比较， 确定万古霉素产生菌的种子培养 基具体配方如下：
可溶性淀粉 4.0%;
葡萄糖 1.5%;
甘油 2.0%;
黄豆饼粉 (热搾） 2.0%;
KN03 0.6%；
KH2P04 0.02%；
MgCl2 0.02%；
泡敌 0.06%
消前 pH 6.8
摇瓶种子培养基装量： 750ml摇瓶装量为 100ml; 培养温度： 28°C ; 培养时间： 48hr; 生长良好的摇瓶种子液 pH7.7左右， 外观 呈乳黄色， 4000rpm离心 10 min, 菌丝量在 22%左右； 镜检菌丝 粗壮， 无杂菌即可作为种子接入下一级种子罐培养或小试发酵罐 发酵。
(4) 发酵培养基
经培养基配方的筛选比较， 确定万古霉素产生菌的发酵培养 基具体配方如下：
甘油 6.0%;
黄豆饼粉 (热搾) 2.0；
KH2P04 0.02%;
MgCl2 0.02%;
KNO3 0.6%；
CaCl2 0.3%;
消前 pH6.8 (5) 发酵条件的选择及确定
通过试验， 确定了万古霉素摇瓶及 10L玻璃罐发酵的发酵条 件。 包括发酵温度、 PH、 摇床转速、 摇瓶装量、 玻璃罐通气量、 搅拌转速等。
(6)发酵温度
通过在摇瓶条件下， 25 °C、 28°C、 30°C三个不同的温度发酵 试验， 结果证明在 28°C时的发酵水平最高， 发酵液中的副产物较 其余两个温度条件为少， 因此确定万古霉素发酵温度为 28°C。
(7) 发酵最适 pH
通过在摇瓶条件下发酵培养基的初始 pH为 6.5、 6.8及 7.0三 种不同的 pH发酵条件试验， 结果证明在发酵培养基的初始 pH为 6.8时的发酵水平最高， 发酵液的颜色较另外两种条件为浅， 副产 物也较少， 因此确定万古霉素的发酵培养基的初始 pH为 6.8。
(8) 其它发酵条件
发酵周期： 摇瓶发酵周期为 6天， 发酵罐发酵周期为 5-6天。 摇瓶装量： 750ml摇瓶装量为 100ml时发酵水平最高。
10L玻璃罐发酵参数：通气量 l :l(vol:vol)，搅拌转速 550rpm。
2、 含有去羟基万古霉素的盐酸万古霉素的分离纯化
( 1 )发酵液的预处理
由于在万古霉素的发酵终点时发酵液的 pH在 7.7左右， 因此在 发酵液中万古霉素是以游离碱的形式存在。 而在分离纯化的第一步， 即将万古霉素通过交换吸附到大孔吸附树脂 (D1300)上。 此交换过程 要求抗生素以游离酸的形式存在。 因此， 需要用稀盐酸将发酵液酸化 并使其 pH调至 3.2左右。 在此酸化过程中， 同时可加入絮凝剂及助 滤剂等使发酵液易于过滤且获得质量较好的发酵滤液。
(2) 吸附、 脱盐及脱色
为了进一步将发酵滤液中的万古霉素与其他的水溶性杂质相分 离， 需要将发酵滤液通过装有大孔羧酸型吸附树脂的树脂柱， 水洗除 去其他杂质后， 再采用乙醇水溶液将抗生素解吸下来。
采用 D1300 作为吸附树脂， 在将发酵滤液通过树脂柱进行吸附 前， 应首先用 4N NaOH将发酵滤液 pH回调至 4.5左右。 解吸采用 pH=2.0的 80%酸性乙醇水溶液。 解吸液经 HPLC分析检测， 万古霉 素积分面积比一般在 75%左右， 单位平均应在 10，00(^g/ml以上。
上述过程得到的解吸液经纳滤浓縮脱盐， 再经针用活性炭脱色， 并加入适量的黄血盐及硫酸锌除去铁离子。收集的上述混合收集液纳 滤脱盐浓缩， 再加入 4N盐酸调节 pH至 2.8左右， 加入活性炭脱色， 黄血盐及硫酸锌除去铁离子后得到脱盐脱色浓缩液，满足后续柱层纯 化的要求。
(3)柱层纯化
通过对大量分离介质的选择试验，最终确定柱层采用的层析介质 为 Sephadex C-25, 并进行了柱层条件、 洗脱剂、 洗脱方式等方面的 深入细致的研究，确定了最佳的纯化方案，该纯化工艺方案采用 0.1M NH4HC03水溶液进行梯度洗脱， 分段收集洗脱液， 合格部分合并经 过纳滤浓缩脱盐即为收得的层析精制液。
将上述浓缩液通过 Sephadex C-25凝胶层析柱， 依照工艺要求进 行进一步的分离纯化， 以上的层析纯化液合并， 通过纳滤浓缩脱盐， 即可得到层析精制液。 合格的精制液经活性炭脱色、 0.22um微孔滤 膜过滤除热源后，该盐酸万古霉素精滤液通过冷冻干燥可获得含有去 3、 再经过色谱分离纯化制得高纯度的去羟基万古霉素。
此外， 根据本发明的另一方面， 本发明还提供了去羟基万古霉素 在制备用于治疗易感细菌感染药物的用途。
而且， 本发明进一步提供了一种药物组合物， 其包含药学有效量 的去羟基万古霉素， 以及药学上可接受的载体或赋形剂。 其中， 所述 药物组合物还包括万古霉素和 /或盐酸万古霉素。
本发明人采用制备色谱对盐酸万古霉素样品进行分离纯化获得 高纯度去羟基万古霉素的， 以用于后续科研工作。 附图说明
图 1为本发明去羟基盐酸万古霉素的 AU光谱图。 具体实施方式
实施例 1: 含有去羟基盐酸万古霉素的盐酸万古霉素的分离纯化
(1)发酵液的预处理
用稀盐酸将发酵液酸化并使其 pH调至 3.2左右。 在此酸化过程 中， 同时可加入黄血盐（絮凝剂或助滤剂）等使发酵液易于过滤且获 得质量较好的发酵滤液。
(2)吸附、 脱盐及脱色
采用 D1300 作为吸附树脂， 在将发酵滤液通过树脂柱进行吸附 前， 应首先用 4N NaOH将发酵滤液 pH回调至 4.5左右。 解吸采用 pH=2.0的 80%酸性乙醇水溶液。 解吸液经 HPLC分析检测， 万古霉 素积分面积比一般在 75%左右， 单位平均应在 ΙΟ,ΟΟΟμ^ιηΙ以上。
上述过程得到的解吸液经纳滤浓缩脱盐， 再用活性炭脱色， 并加 入适量的黄血盐及硫酸锌除去铁离子。收集的上述混合收集液纳滤脱 盐浓缩， 再加入 4N盐酸调节 pH至 2.8左右， 加入活性炭脱色， 黄 血盐及硫酸锌除去铁离子后得到脱盐脱色浓缩液。
(3)柱层纯化
将上述浓缩液通过 Sephadex C-25 凝胶层析柱， 采用 0.1M N¾HC03水溶液进行梯度洗脱， 分段收集洗脱液， 合并层析纯化液， 经过 0.22um微孔滤膜纳滤浓缩脱盐制得的层析精制液。 该精制液经 活性炭脱色过滤除热源后，该精滤液通过冷冻干燥可获得含有去羟基 盐酸万古霉素的盐酸万古霉素无菌原料粉。 实施例 2: 再经过色谱分离纯化制得高纯度的去羟基万古霉素 实验原料：
实施例 1中含有去羟基万古霉素的盐酸万古霉素无菌原料粉； 实验仪器：
P6000高压制备泵；
UV6000可变波长紫外分光检测器 （由北京创新通恒科技有限公 司提供）；
C18 MONOMERIC 5 X25cm制备色谱柱 （GRACE);
旋转蒸发仪；
罗门哈斯大孔吸附树脂 （XAD1600树脂） 250ml玻璃层析柱； 辅助用玻璃仪器若干。
实验步骤：
1、 建立色谱分离条件
兼顾分离度和效率优化后的色谱分离条件为：
色谱柱： C18 MONOMERIC 5 X25cm制备色谱柱 （GRACE); 检测波长： 280nm;
进样量： 150mg;
流速： 60ml/min;
缓冲液：溶解 4ml三乙胺于 1996ml 水中，用 H3P04调 pH至 3.2 配成缓冲液；
流动相为缓冲液： 甲醇 =92: 8
载体： XAD1600树脂
得到的 AU光谱图， 参见图 1， 其中， 5号峰为盐酸万古霉素主 峰， 6号峰为我们的目标杂质峰。
2、 收集目标杂质馏分及除去三乙胺盐
收集了五十针馏分约 6L 收集液。
装 250ml XAD1600树脂层析柱， 将收集液上柱吸附。 上柱后用 大量的水洗充分后，用 50%的甲醇水溶液洗脱，收集到 60ml洗脱液。
3、 浓缩冻干
将 60ml洗脱液用旋转蒸发仪浓缩至 5ml左右， 送冻干机房冻干 得到 35mg目标物。
4、 结构鉴定
待测样品的 iHNMR图谱给出的质子信号峰， 总体与万古霉素的 相似。其中， 3个甲基单峰 δθ.85 (3Η, J=6.0Hz)， δ0.89 (3Η， J=6.5Hz) 和 δΐ.06 (3Η, J=6.5Hz), 以及 1个甲基单峰 82.26是待测样品结构 中存在与万古霉素相同的 N-methylleucine(A)和 vancosaminyl(Van)结 构单元的诊断信号。在芳香区, 3个 or /zo- eto-偶合的 ABX体系: δ7.16 ( 1H， brs, J meto<1.0Hz,H-E4)> 6.71 ( 1H, d， <8.0Hz，H-E7)、 6.76 ( 1H , brd «/=8Hz， J mefa<1.0Hz,H-E8 ) 7.41 ( 1H， brs， J " <1.0Hz,H-B5)、 7.13 ( 1H, d, Jorito=8.0Hz,H-B8) 和 7.16 ( 1H， d,
Figure imgf000016_0001
57.84 ( 1H， brs, «/meto<1.0Hz，H-F5)、 7.31 ( 1H, d, J^。=8.0Hz,H-F8)、 和 7.45 ( 1H, brd J=8Hz, Jmeto<1.0Hz,H-F9), 以及 2个 weto-偶合的 AB自旋体系： δ5.47( lH, brs,Jmeia<1.0Hz,H-D4) 和 5.20( 1H， brs, Jortho <1.0Hz，H-D8)、 6.38 C 1H, brs, Jmeto<1.0Hz,H-G6) 和 （1H， brs, Jmeto<1.0Hz,H-G8)o
本发明通过上面的实施例进行举例说明， 但是， 应当理解， 本发 明并不限于这里所描述的特殊实例和实施方案。在这里包含这些特殊 实例和实施方案的目的在于帮助本领域中的技术人员实践本发明。任 何本领域中的技术人员很容易在不脱离本发明精神和范围的情况下 进行进一步的改进和完善，因此本发明只受到本发明权利要求的内容 和范围的限制，其意图涵盖所有包括在由所附权利要求所限定的本发 明精神和范围内的备选方案和等同方案。

Claims

要 求
1、 一种如下通式 I的去羟基万古霉素:
Figure imgf000017_0001
其中， 式中 1 为11。
2、 一种如权利要求 1所述的去羟基万古霉素的制备方法， 所述 方法包括如下步骤：
( 1 ) 利用东方拟无枝酸菌菌株制备含有去羟基万古霉素的万古 霉素浓缩液；
(2)通过柱层析分离纯化上述浓缩液得到含有去羟基万古霉素的 盐酸万古霉素精滤液；
(3 )再经过色谱分离纯化上述无菌原料粉制得去羟基万古霉素; 其中， 在步骤 （2) 中， 在含有盐-水流动相的凝胶层析柱中进行 柱层析分离纯化；
在步骤（3) 中， 在含有缓冲液 -甲醇流动相的大孔吸附树脂色谱 柱中进行色谱分离纯化。
3、 如权利要求 2所述的制备方法， 将所述万古霉素浓縮液 pH 调整至 2.8。
4、 如权利要求 2所述的制备方法， 在凝胶层析柱中， 所述流动 相为 O.lM
Figure imgf000018_0001
5、 如权利要求 2所述的制备方法， 所述凝胶为葡聚糖凝胶、 聚 丙烯酰胺凝胶、 琼脂糖凝胶和聚苯乙烯凝胶。
6、 如权利要求 2所述的制备方法， 在柱层析分离纯化中， 还包 括用 0.22um微孔滤膜对柱层析液进行纳滤、 浓缩、 脱盐。
7、 如权利要求 2所述的制备方法， 在色谱分离纯化中， 所述流 动相为缓冲液： 甲醇 =92 : 8。
8、 如权利要求 2或 7所述的制备方法， 所述缓冲液包括三乙胺 和磷酸， 且 pH=3.2。
9、 如权利要求 2所述的制备方法， 所述大孔吸附树脂为罗门哈 斯大孔吸附树脂。
10、一种药物组合物， 其包含药学有效量的如权利要求 1所述的 去羟基万古霉素， 以及药学上可接受的载体或赋形剂。
11、 如权利要求 10所述的药物组合物， 还包括万古霉素和 /或盐 酸万古霉素。
12、一种如权利要求 10或 11所述的包含去羟基万古霉素的药物 组合物在制备用于治疗易感细菌感染药物的用途。
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US8748495B2 (en) 2009-03-09 2014-06-10 Zhejiang Medicine Co., Ltd. Xinchang Pharmaceutical Factory Method of preparing oil suspensions of carotenoid with low viscosity and high fluidity and use thereof

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US8338372B2 (en) 2012-12-25

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