两性霉素B降解产物研究及其HPLC方法与微生物测定法含量测定结果的量值统一
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摘要
抗生素是指由细菌、霉菌或其它微生物在生命过程中产生的具有抗病原体或其它活性的一类物质,具有在低微浓度下选择性地抑制或杀灭他种微生物或肿瘤细胞的能力。自一九四一年青霉素应用于临床以来,经各方微生物学家及生化学家的努力,迄今为止,已发现的抗生素种类已达几千种之多。依据美国疾病管制局的档案资料显示,目前临床使用的抗生素就有一百五十多种,药效涉及到抗肿瘤、抗真菌、抗病毒、抗革兰氏阴性细菌等等。按其结构可以分为：青霉素类,头孢菌素类,磷霉素类,万古霉素类,利福霉素类,多粘菌素类,氨基糖类,四环素类,大环内酯类等。多烯大环内酯类抗生素作为抗生素的重要组成部分,在临床使用中占据举足轻重的地位,临床常用的多烯类抗生素有制霉菌素、曲古霉素、两性霉素B等,其中两性霉素B主要用于深部真菌感染。
多烯抗生素溶解性能差,会发生聚合现象。由于结构复杂、性质不稳定,所以对其研究相对缓慢。通过对两性霉素B的临床应用现状考察,发现其仍然在深部真菌感染的治疗中起着不可替代的作用,所以各国科学家对其含量测定、毒副作用、体内药物动力学及临床应用等进行不懈的研究探索。多烯类抗生素的作用机制均类似,都是通过与真菌细胞膜的甾醇结合,改变膜的通透性,导致膜内容物外流从而使真菌细胞凋亡。国内外报道的有关含量测定方法的研究异彩纷呈,除了药典规定的微生物检定法,还有新兴的HPLC法,分光光度法等。对其杂质控制方面研究甚少,是进一步努力研究的方向。
两性霉素B是由结节性链霉菌(Streptomyces nodosus)产生的一种七烯类广谱抗真菌抗生素,分子式C47H73NO17,其抗真菌活性是在1956年被发现的,1960年两性霉素B在临床上被用于治疗多种深部真菌如白色念珠菌、新型隐球菌、曲霉菌等引起的心脏或全身感染。对某些严重的深部真菌病如新生隐球菌脑膜炎、侵袭性曲霉病,特别是对免疫缺陷或严重粒细胞缺乏的患者的治疗以及某些地方性真菌病如球孢子菌病、组织胞浆菌病、皮炎芽生菌病等仍需应用两性霉素B,因此迄今仍是许多危重深部真菌感染治疗的首选药物,尚无适当的替代药物,故被喻为“黄金标准”。鉴于两性霉素B在临床应用上的重要地位,本论文对两性霉素B的降解产物进行了较为系统的研究探索,得到尽可能多的杂质信息。在不同条件下加速破坏两性霉素B得到不同的降解产物,并通过适当的分离手段将其分离纯化,测定UV,NMR,MS数据,进行结构鉴定。对于其中一个降解产物两性霉素B(5)我们进行了较为系统全面的研究,使用微生物效价法测定活性,并通过斑马鱼动物模型测定其毒性。本论文的另一个重要研究内容是通过制备液相色谱纯化一批两性霉素B纯品,针对两性霉素B建立起一个高效简便且性能优于EP的HPLC方法并对其进行方法学验证,将HPLC纯度测定结果与微生物效价测定结果进行量值统一,使用简便的HPLC方法取代繁琐复杂的微生物检定法测定两性霉素B的效价,从而使两性霉素B的质量控制更加简便,快速,可靠。
The antibiotics are the class of substances which are produced in the living process of the bacteria, mold, or other microorganisms, they can selectively restrain or kill the microorganisms or the tumor cells at a low density. From 1941 when the penicillin was applied in the clinic till now, there are thousands of kinds of antibiotics found by the effort of microbiologist and biochemist. As the data based on the Center for Disease Control and Prevention (CDC) showed, there are more than one hundred and fifty kinds of antibiotics used in the clinic currently, including antineoplastic agents, antifungal agents, antiviral agents, anti gram-negative bacillus agents and so on. Antibiotics can be classified into the following categories by the structure:penicillins, cephalosporins, fosfomycins, vancomycins, rifomycins, polymyxins, amino sugars, tetracyclines, macrolides etc. Polyene macrolides play a very important role in the clinic as an essential part of the antibiotics. Amphotericin B, which belongs to the polyene macrolides is a conventional agent used for the cure of systemic fungal infections. The solubility of polyene macrolides is very poor, and the aggregation may occur. Due to the complicated structure, unstable chemical property, the research about polyene macrolides is relatively slow. After the review of the clinical use of amphotericin B, it was found that it is still an irreplaceable agent for curing the systemic fungal infections, so the scientists are keeping on the exploration and research on the detention, side effect, the pharmacokinetics and clinical application of amphotericn B. The polyene antifungal antibiotics, an antibiotic family amphotericin B belonging to, selectively associate ergosterol that located in fungal cell membrane to form transmembrane channels. As a result, cellular components such as potassium and magnesium ions, glucose, and amino acids, pass through the channels. There are various methods for the determination of amphotericin B, except for the Microbiological Potency assay, there are HPLC method and spectrophotometry method etc. However, in respect of its impurities control research, the further effort is needed. Amphotericin B was first isolated from Streptomyces nodosus in 1956 and has been used in the clinic since 1960 to treat a great variety of systemic fungal infections caused by Candida albicans, Cryptoccus neoformans and Aspergillus. Its molecular formula is C47H73NO17. For some serious deeply infected fungus diseases such as cereospinal menigitis of Cryptococcus neoformans, invasive aspergillosis, particularly the treatment of immunodeficiency or seriousagranulocytosis, and some local fungus, amphotericin B is still needed and irreplaceable. Amphotericin B is an important antifungal antibiotic referred to as a "golden standard" due to its high and broad spectrum of activity. In view of the importance of amphotericin B in clinical application, in this paper, the degradation products of amphotericin B have been researched systematically to explore for as much as possible information about the impurities. Different degradations of amphotericin B were obtained under different conditions to speed up the degradation reactions, then they were purified through the proper means, and the UV, NMR, MS data were determined to identify the structures for them. For one of the degradation products named amphotericin B (5), the systemic research has been done, including the primary activity test by Microbiological Potency assay, and the primary toxicity test on zebrafish animal model. The other major research content in this paper is purifying some pure amphotericin B sample by preparative HPLC, establishing a fully new HPLC method and doing the systematic methodology validation. A conclusion that the proposed HPLC method can replace the microbiological assay for potency determination of amphotericin B can be drawn after the method validation and the unification between potency and purity. In this way, the routine quality control for amphotericin B can become faster, simpler and more reliable.
多烯抗生素溶解性能差,会发生聚合现象。由于结构复杂、性质不稳定,所以对其研究相对缓慢。通过对两性霉素B的临床应用现状考察,发现其仍然在深部真菌感染的治疗中起着不可替代的作用,所以各国科学家对其含量测定、毒副作用、体内药物动力学及临床应用等进行不懈的研究探索。多烯类抗生素的作用机制均类似,都是通过与真菌细胞膜的甾醇结合,改变膜的通透性,导致膜内容物外流从而使真菌细胞凋亡。国内外报道的有关含量测定方法的研究异彩纷呈,除了药典规定的微生物检定法,还有新兴的HPLC法,分光光度法等。对其杂质控制方面研究甚少,是进一步努力研究的方向。
两性霉素B是由结节性链霉菌(Streptomyces nodosus)产生的一种七烯类广谱抗真菌抗生素,分子式C47H73NO17,其抗真菌活性是在1956年被发现的,1960年两性霉素B在临床上被用于治疗多种深部真菌如白色念珠菌、新型隐球菌、曲霉菌等引起的心脏或全身感染。对某些严重的深部真菌病如新生隐球菌脑膜炎、侵袭性曲霉病,特别是对免疫缺陷或严重粒细胞缺乏的患者的治疗以及某些地方性真菌病如球孢子菌病、组织胞浆菌病、皮炎芽生菌病等仍需应用两性霉素B,因此迄今仍是许多危重深部真菌感染治疗的首选药物,尚无适当的替代药物,故被喻为“黄金标准”。鉴于两性霉素B在临床应用上的重要地位,本论文对两性霉素B的降解产物进行了较为系统的研究探索,得到尽可能多的杂质信息。在不同条件下加速破坏两性霉素B得到不同的降解产物,并通过适当的分离手段将其分离纯化,测定UV,NMR,MS数据,进行结构鉴定。对于其中一个降解产物两性霉素B(5)我们进行了较为系统全面的研究,使用微生物效价法测定活性,并通过斑马鱼动物模型测定其毒性。本论文的另一个重要研究内容是通过制备液相色谱纯化一批两性霉素B纯品,针对两性霉素B建立起一个高效简便且性能优于EP的HPLC方法并对其进行方法学验证,将HPLC纯度测定结果与微生物效价测定结果进行量值统一,使用简便的HPLC方法取代繁琐复杂的微生物检定法测定两性霉素B的效价,从而使两性霉素B的质量控制更加简便,快速,可靠。
The antibiotics are the class of substances which are produced in the living process of the bacteria, mold, or other microorganisms, they can selectively restrain or kill the microorganisms or the tumor cells at a low density. From 1941 when the penicillin was applied in the clinic till now, there are thousands of kinds of antibiotics found by the effort of microbiologist and biochemist. As the data based on the Center for Disease Control and Prevention (CDC) showed, there are more than one hundred and fifty kinds of antibiotics used in the clinic currently, including antineoplastic agents, antifungal agents, antiviral agents, anti gram-negative bacillus agents and so on. Antibiotics can be classified into the following categories by the structure:penicillins, cephalosporins, fosfomycins, vancomycins, rifomycins, polymyxins, amino sugars, tetracyclines, macrolides etc. Polyene macrolides play a very important role in the clinic as an essential part of the antibiotics. Amphotericin B, which belongs to the polyene macrolides is a conventional agent used for the cure of systemic fungal infections. The solubility of polyene macrolides is very poor, and the aggregation may occur. Due to the complicated structure, unstable chemical property, the research about polyene macrolides is relatively slow. After the review of the clinical use of amphotericin B, it was found that it is still an irreplaceable agent for curing the systemic fungal infections, so the scientists are keeping on the exploration and research on the detention, side effect, the pharmacokinetics and clinical application of amphotericn B. The polyene antifungal antibiotics, an antibiotic family amphotericin B belonging to, selectively associate ergosterol that located in fungal cell membrane to form transmembrane channels. As a result, cellular components such as potassium and magnesium ions, glucose, and amino acids, pass through the channels. There are various methods for the determination of amphotericin B, except for the Microbiological Potency assay, there are HPLC method and spectrophotometry method etc. However, in respect of its impurities control research, the further effort is needed. Amphotericin B was first isolated from Streptomyces nodosus in 1956 and has been used in the clinic since 1960 to treat a great variety of systemic fungal infections caused by Candida albicans, Cryptoccus neoformans and Aspergillus. Its molecular formula is C47H73NO17. For some serious deeply infected fungus diseases such as cereospinal menigitis of Cryptococcus neoformans, invasive aspergillosis, particularly the treatment of immunodeficiency or seriousagranulocytosis, and some local fungus, amphotericin B is still needed and irreplaceable. Amphotericin B is an important antifungal antibiotic referred to as a "golden standard" due to its high and broad spectrum of activity. In view of the importance of amphotericin B in clinical application, in this paper, the degradation products of amphotericin B have been researched systematically to explore for as much as possible information about the impurities. Different degradations of amphotericin B were obtained under different conditions to speed up the degradation reactions, then they were purified through the proper means, and the UV, NMR, MS data were determined to identify the structures for them. For one of the degradation products named amphotericin B (5), the systemic research has been done, including the primary activity test by Microbiological Potency assay, and the primary toxicity test on zebrafish animal model. The other major research content in this paper is purifying some pure amphotericin B sample by preparative HPLC, establishing a fully new HPLC method and doing the systematic methodology validation. A conclusion that the proposed HPLC method can replace the microbiological assay for potency determination of amphotericin B can be drawn after the method validation and the unification between potency and purity. In this way, the routine quality control for amphotericin B can become faster, simpler and more reliable.
引文
[1]Gallis HA.Drew RH. Pickard WW. Amphotericin B:30 year of clinical experience. Rer Infect Dis,1990:12:308-29.
[2]Jin Li, Hui-Qin Zhu, Jing-Yun Li, Shao-Hong Jin, Chang-Qin Hu. Isolation, Structure Elucidation and Activity of an Unknown Impurity of Amphotericin B. J. Antibiot.2007,60(4):272-276.
[3]Tran-Dinh S, Herve M, Lebourguais 0, et al. Effects of amphotericin B on the glucose metabolism in S accharomyces cerevisiae cells. Studies by 13C,1H-NMR and biochemical methods. Eur J Biochem,1991,197 (1):271-279.
[4]P Minodier, S Robert, G Noel. First-line liposomal amphotericin B for Pediatric visceral leishmaniasis in southern France[J]. Arch Pediatr,2005,12(7):1102-1108.
[5]Adedayo Adedoyin,Christine E Suenson,Lois E Bolcsak,et al,A phomacokinetic Study of Amphotericin B Lipid Complex Infet al (Abelect) in Patients with Definite or Probable Systemic Fungal Infet alions[J].Antimicrob Agents Chemother,2000,44:2900-2902.
[6]Rodrigo Martino, Bertrand Dupont, Helen Huckle, et al. No signs of New Toxicity When Amphotericin B Lipid Complex (ABLC, ABELCET) and Caspofungin Are Used in Combination in the Treatment of FUO[J], Blood (Ash Annual Meeting Abseracts).2004,104(11):5077.
[7]国家药典委员会.中华人民共和国药典[S].北京：化学工业出版社,2005.
[8]The United States Pharmacopoeia Commission Inc The United States Pharmacopeia [S].Rochvile, MD:the United States Pharmacopeia Convention, Inc,2005:NF23.
[9]The British Pharmacopoeia Commission. The British Pharmacopoeia[S]. London:the Stationary Office,2003.
[10]Council of Europe. European Pharmacopoeia.6th ed. Strasbourg:Council of Europe; 2007.
[11]Espada R, Josa JM. HPLC assay for determination of amphotericin B in biological samples. Biomed Chromatogr.2008.4;22(4):402-7.
[12]严喜亚,李增民.高效液相色谱法测定两性霉素B含量和相关物质.中国抗生素杂志.2006.9.31(9)：551-4.
[1]J.Koyler-Brajtburg, G.Medoff et al. Classification of Polyene Antibiotics According to Chemical Structure and Biological Effects. Antimicrobial Agents and Chemotherapy.1979; 15:716-722.
[2]Hamilton-Miller, J. M. T.1973. Chemistry and biology of the polyene macrolide antibiotics. Bacteriol. Rev.37:166-196.
[3]Hammond, S. M.1977. Biological activity of polyene antibiotics. Prog. Med. Chem. 14:105-283.
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[2]Jin Li, Hui-Qin Zhu, Jing-Yun Li, Shao-Hong Jin, Chang-Qin Hu. Isolation, Structure Elucidation and Activity of an Unknown Impurity of Amphotericin B. J. Antibiot.2007,60(4):272-276.
[3]Tran-Dinh S, Herve M, Lebourguais 0, et al. Effects of amphotericin B on the glucose metabolism in S accharomyces cerevisiae cells. Studies by 13C,1H-NMR and biochemical methods. Eur J Biochem,1991,197 (1):271-279.
[4]P Minodier, S Robert, G Noel. First-line liposomal amphotericin B for Pediatric visceral leishmaniasis in southern France[J]. Arch Pediatr,2005,12(7):1102-1108.
[5]Adedayo Adedoyin,Christine E Suenson,Lois E Bolcsak,et al,A phomacokinetic Study of Amphotericin B Lipid Complex Infet al (Abelect) in Patients with Definite or Probable Systemic Fungal Infet alions[J].Antimicrob Agents Chemother,2000,44:2900-2902.
[6]Rodrigo Martino, Bertrand Dupont, Helen Huckle, et al. No signs of New Toxicity When Amphotericin B Lipid Complex (ABLC, ABELCET) and Caspofungin Are Used in Combination in the Treatment of FUO[J], Blood (Ash Annual Meeting Abseracts).2004,104(11):5077.
[7]国家药典委员会.中华人民共和国药典[S].北京：化学工业出版社,2005.
[8]The United States Pharmacopoeia Commission Inc The United States Pharmacopeia [S].Rochvile, MD:the United States Pharmacopeia Convention, Inc,2005:NF23.
[9]The British Pharmacopoeia Commission. The British Pharmacopoeia[S]. London:the Stationary Office,2003.
[10]Council of Europe. European Pharmacopoeia.6th ed. Strasbourg:Council of Europe; 2007.
[11]Espada R, Josa JM. HPLC assay for determination of amphotericin B in biological samples. Biomed Chromatogr.2008.4;22(4):402-7.
[12]严喜亚,李增民.高效液相色谱法测定两性霉素B含量和相关物质.中国抗生素杂志.2006.9.31(9)：551-4.
[1]J.Koyler-Brajtburg, G.Medoff et al. Classification of Polyene Antibiotics According to Chemical Structure and Biological Effects. Antimicrobial Agents and Chemotherapy.1979; 15:716-722.
[2]Hamilton-Miller, J. M. T.1973. Chemistry and biology of the polyene macrolide antibiotics. Bacteriol. Rev.37:166-196.
[3]Hammond, S. M.1977. Biological activity of polyene antibiotics. Prog. Med. Chem. 14:105-283.
[4]戴自英,刘裕昆,汪复.实用抗菌药物学.第2版[M].上海：上海科学技术出版社,1998.246-254.
[5]Harry Brik. Natamycin[J].Analytical Profiles of Drug Substances.1981,10:514-557.
[6]中华人民共和国卫生部药典委员会.中华人民共和国药典(二部).2005年版[S].北京：化学工业出版社,2005：240.
[7]Bolard J, Seigneuret M, Boudet G.1980. Interaction between phospholipid bilayer membranes and the polyene antibiotic amphotericin B:Lipid stateand cholesterol content dependence. Biochim Byophys Acta 599:280-293.
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