CN1242052C - 诺卡氏菌的生物纯培养物 - Google Patents
诺卡氏菌的生物纯培养物 Download PDFInfo
- Publication number
- CN1242052C CN1242052C CNB031487955A CN03148795A CN1242052C CN 1242052 C CN1242052 C CN 1242052C CN B031487955 A CNB031487955 A CN B031487955A CN 03148795 A CN03148795 A CN 03148795A CN 1242052 C CN1242052 C CN 1242052C
- Authority
- CN
- China
- Prior art keywords
- nocathiacin
- culture
- antibiotic
- substratum
- nocardia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明公开了新颖的微生物ATCC-202099的生物纯培养物。
Description
本申请是申请日为1999年7月13日的题为“诺卡沙星抗菌素”的中国专利申请99808680.0的分案申请。
技术领域
本发明涉及新颖的噻唑肽类抗菌素化合物诺卡沙星I,II和III及其药用组合物和用抗菌素诺卡沙星I,II和III治疗细菌感染的方法。
背景技术
对于一些抗菌药物如内酰胺抗菌素、β-大环内酯、喹诺酮和万古霉素来说,耐药菌的出现已成为世界健康难题。(Cohen,M.L.Antimicrobial resistance:prognosis for public health.TrendMicrobiol.1994,2,422-425)。临床实践中最显著的问题是耐甲氧苯青霉素的金黄色葡萄球菌(MRSA)菌株的发生率增加。目前,治疗多种MRSA感染的最有效药物是万古霉素。同时,也出现了有关MRSA分离菌耐万古霉素的报道。其他一些最近出现的与临床相关的多药耐药菌是肠球菌。一种耐青霉素和其他抗菌素的公众易感染的重要的病原体—肺炎链球菌也成为世界健康难题。包括美国在内的一些国家已出现肺炎链球菌多药耐药菌。这种医源性的(nosocomial)和公众易感染的耐药病原体的出现和传播大大威胁着世界范围公众健康。所以迫切需要发明一种治疗多药耐药菌感染的新型药物。本发明即致力于这种研究。
新型的噻唑肽抗菌素如下所述(在此所指的是诺卡沙星(nocathiacin)I,II和III,或是诺卡沙星抗菌素)。它在毫微摩尔水平对革兰氏阳性菌有抑制活性。这里所述的新颖抗菌素从诺卡氏菌种ATCC-202099的液体培养基中分离制得。已知的噻唑肽类抗菌素有藓霉素和诺西肽,有报道glycothiohexide-α在体外对革兰氏阳性菌有抑制活性,但没报道体内活性。在此披露的抗菌素对全身性金黄色葡萄球菌感染的鼠模型显示了体内活性。
以前,J.E.Lee t等(U.S.临时申请60/093,021,1998年7月16日申请)和Sassaki,T.等,J.of Antibiotics 51,No.8,第715-721页(1998年8月25日出版)曾描述过诺卡沙星I。本发明诺卡沙星抗菌素与诺西肽(Prange T.等,J.Am Chem Soc.99,6418(1977);Benazet,F.等.Experientia 36,414(1980);Floss,H.G.等.J.Am Chem Soc.115,7557(1993);glycothiohexides(Steinberg,D.A.等,J.Ant ibiot.47,887(1994);M.D.Lee等,J.Antibiot.47,894(1994)M.D。Lee等,J.Antibiot.47,901(1994);US 5,451,581,1995年),及抗菌素S-54832A(U.S4,478,831,1984年)相关但又有明显差异。
发明内容
本发明涉及新颖的噻唑肽类抗菌素化合物诺卡沙星I,II和III。本发明的抗菌素可由诺卡氏菌种(Nocardiasp.)(WW-12651,ATCC-202099株)的发酵液体培养基中分离和纯化而获得。
本发明也涉及了药用组合物和用抗菌素诺卡沙星I,II和III治疗细菌感染的方法,及用于生产抗菌素的诺卡氏菌种菌株的生物学纯培养基。本发明包括诺卡沙星抗菌素的所有可药用衍生物,如可药用的盐和其酯类。
人们希望这种在体内外抑制革兰氏阳性菌的化合物能象其它抗菌素一样用于哺乳动物特别是人类,标题化合物在治疗细菌感染方面的利用正基于此。本发明化合物具有抗菌活性,尤其是抑制革兰氏阳性菌。
附图说明
图1所示为诺卡沙星I的紫外吸收图谱.
图2所示为诺卡沙星I的红外吸收图谱.
图3所示为诺卡沙星I在氘化二甲亚砜中测定的1H-NMR图谱(500MHz).
图4所示为诺卡沙星I在氘化二甲亚砜中测定的13C-NMR图谱(125MHz).
图5所示为诺卡沙星II的紫外吸收图谱.
图6所示为诺卡沙星II的红外吸收图谱.
图7所示为诺卡沙星II在氘化二甲亚砜中测定的1H-NMR图谱(500MHz).
图8所示为诺卡沙星II在氘化二甲亚砜中测定的13C-NMR图谱(125MHz).
图9所示为诺卡沙星III的紫外吸收图谱.
图10所示为诺卡沙星III的红外吸收图谱.
图11所示为诺卡沙星III在氘化二甲亚砜中测定的1H-NMR图谱(500MHz).
图12所示为诺卡沙星III在氘化二甲亚砜中测定的13C-NMR图谱(125MHz).
具体实施方式
该诺卡沙星I,II和III的抗生素化合物是在发酵过程中发现的。标题所示诺卡沙星化合物从Nocardia sp.(WW-12651,ATCC-202099)的发酵液体培养基中分离,经提取和色谱分离得以纯化获得到无定形固体。
微生物描述
用于产生诺卡沙星抗菌素的微生物是从新墨西哥州采集的土壤样品中分离的Nocardia sp.。培养物(菌株WW-12651)1998年3月4日贮藏在Rockville,MD的美国典型培养物储藏中心(ATCC),储藏编号为ATCC-202099。ATCC的储藏满足所有的布达佩斯条约要求。休眠的培养物也保存在布里斯托尔-迈尔斯施责宝公司的药物研究所培养物储藏中心,第五Research Parkway,Wallingford,CT 06492。这里描述的特殊微生物之外,应该理解一种突变体,例如包括X-射线在内的化学或物理诱变剂导致的突变,和那些通过分子生物学技术改变遗传组成的菌也可以培养产生诺卡沙星抗菌素。
根据国际链霉菌属项目(ISP)指南的建议,菌株WW-12651的显微研究在ISP的形态学培养基(ISP2,ISP3,ISP4,ISP5和ISP7)中进行,在28℃孵育下的第7、14和21天进行镜检观察。
在ISP4培养基上生长为奶油色的菌落,气生菌丝体是白色分节的。在光学显微镜下,生长的菌丝体中可见孢子链,而穗网状菌丝体中很少或未见孢子链。这种有机体的形态学分类为非链霉菌类型。在盐-淀粉琼脂中(ISP4)可见浅棕橙色reverse颜色。在任何的ISP培养基中都没有扩散色素的产生。在酪氨酸琼脂(ISP7)中没有形成类黑素,用改良的Arai和Mikami黑色素形成试验也未检测到。
细胞壁的氨基酸成分是丙氨酸,L-谷氨酸,天冬氨酸和间-二氨基庚二酸异构体,细胞壁的糖成分是半乳糖,阿拉伯糖和核糖。碳源利用研究显示当作为单碳源结合无机盐琼脂(ISP9)时,葡萄糖、甘露醇、蔗糖、木糖和果糖(较差)可被利用。阿拉伯糖,肌醇,棉子糖和鼠李糖在ISP9培养基作为唯一碳源时,不被利用(ISP9)。基于以上性质和霉菌酸的分析,可以确定这种微生物属于Nocardia属。
Nocardia sp.(菌株WW-12651)的发酵
诺卡沙星抗菌素的生产可以在下述条件的合适的营养培养基中通过培养Nocardia sp.菌株WW-12651完成,优选置于浸没有氧条件下,直至在发酵中检测出一定量的诺卡沙星,通过使用合适的溶剂从生长的菌丝中提取活性成分获得,浓缩包含所需成分的溶液,然后从培养基的其它代谢物中色谱分离浓缩的成分。
任何益于培养菌生长的温度如16℃和40℃都会影响Nocathiacin的生产,但优选在22-32℃进行。液体培养基有必要孵育一段时间以保证Nocathiacin的生产充分进行,用HPLC通常监测1-6天,生产中旋转振动器转速为50-300rpm,优选150-200rpm。
微生物的生长可以使用适当的培养基通过普通的工艺完成。简而言之,碳源包括葡萄糖、果糖、甘露糖、麦芽糖、半乳糖、甘露醇和甘油、其它糖和糖醇、淀粉和其他的碳水化合物,或碳水化合物的衍生物如葡聚糖、cerelose和复杂的营养物如燕麦粉、玉米粉、小米、谷类等。用于培养基中的碳源的精确数量一定程度取决于培养基的其它成分,同时也发现培养基中含0.5-10%的碳水化合物是令人满意的。这些碳源能单独或合并用于同一个培养基中。一些优选碳源将在下面阐述。
N源包括甘油、精氨酸、苏氨酸、蛋氨酸等氨基酸及铵盐和来源复杂的酵母提取物、玉米提取物、可溶性馏出物、大豆粉、棉籽粉、鱼粉等,不同N源能单独使用在0.05-5%重量范围的培养基中。
能合并用于培养基的营养无机盐是普通的盐类,它们能产生Na、K、Mg、Ca、P、S、Cl、碳酸等离子,也包括钴、锰、铁、钼、锌、镉等的痕量金属。
典型地说,Nocardia sp.(菌种WW-12651)生长在含100ml营养培养基的500ml烧瓶中:其中每升去离子水含20g淀粉;5g葡萄糖;3g N-Z case;2g酵母提取物;5g鱼肉提取物;5g碳酸钙。培养物在250r pm的旋转振荡器中32℃孵育3天。营养培养基与一定量的每升去离子水中含100g蔗糖和200g甘油的冷冻保护液混合。混合物的每4ml一份转移至无菌冷冻管中(5ml容量),在干冰-丙酮浴中冷冻,得到的冷冻的营养培养基在-80℃保存。
用于生产诺卡沙星抗菌素产品的种子培养物通过下面过程制得。将4ml含冷冻保护液的培养基移至含100ml无菌营养培养物(同前述相同成分)的500ml烧瓶中。种子培养基在250r pm的旋转振荡器中28℃-32℃孵育3天。这种种子培养基每4ml培养在含100ml下列培养基的烧瓶中:每升去离子水含HY Yest 421,10g,20g葡萄糖,10gNutrisoy,这是优选的培养物。该培养物在180rpm-250rpm的旋转振荡器中24℃-32℃孵育。最佳的诺卡沙星抗菌素通常在发酵4-5天后获得。
当用诺卡沙星I,II或III化合物用作治疗细菌感染的药学组分时,它们可以与同一种或几种可药用的载体结合,例如溶剂、稀释剂等。也可以以某种形式口服,如片剂、胶囊剂、可分散的散剂、颗粒剂或含有例如约0.05-5%混悬剂的混悬液、含有例如约10-50%糖浆的糖浆剂、含有例如约20-50%乙醇的酏剂等,或以含0.05-5%混悬剂(混悬于等渗介质中)的混悬液或无菌注射液形式非胃肠道给药。这里所说的药用制剂可以是约0.05-90%的活性成分与载体结合,但更常见的是重量比为约5%和60%。
活性组分的有效剂量,可以依据所采用的特殊化合物、给药方法和所治疗疾病的严重程度不同而不同,因此一般说来,当本发明化合物按照大约0.5-500mg/每千克动物体重的每天剂量服用,优选一日两至四次给予不同剂量,或用缓释形式可以获得满意的疗效。对于大多数大的哺乳动物,每日总剂量1-100mg,优选从2-80mg。适宜内服的剂型含与固体或液体的药用载体混合的0.5-500mg活性成分,这种给药方式可以选择提供最佳的治疗效果。例如,以几种不同的剂量每日给药或根据治疗形势的特殊需要而成比例地减小剂量。
这些活性组分可以口服给药及静脉、肌内或皮下途径给药。固体载体包括淀粉、乳糖、磷酸二钙、微晶纤维素、蔗糖和高岭土,液体载体包括无菌水、聚乙二醇、非离子表面活性剂和食用油如玉米、花生和芝麻油,这些辅料与活性成分的性质和所需给药的特殊形式相匹配。在药用化合物制剂中通常使用的辅料是有利的,例如矫味剂、着色剂、防腐剂和抗氧剂如VE、抗坏血酸、BHT和BHA。
这些活性化合物也可以非肠道或腹膜内给药。像游离碱和药理学可接受的盐。这样的活性成分的溶液或混悬液可以通过在水中与表面活性剂如羟丙基纤维素混合来制备。胶体溶液(Dispersions)可以在甘油、液态聚乙二醇和这几种混合油中制备。在一般的储存和使用条件下,这些制剂含有防腐剂以防止细菌繁殖。
用于注射的药用剂型包括无菌水溶液或胶体分散体。所有情况,这种剂型必须是无菌的和必须是一定程度上具有通针性的流体。它必须在生产和储存中是稳定的且有抗细菌、真菌污染的能力。载体可以是溶剂或分散介质,包括水、乙醇、多元醇(如甘油、丙二醇和液体聚乙二醇)及适当的混合物和植物油。
术语“可药用的盐”包括溶化物、水合物、酸加成的盐和四价盐。酸加成的盐可由含碱性氮的诺卡沙星I,II或III化合物制得,可药用的有机或无机酸包括(但不限于)盐酸、氢溴酸、硫酸、磷酸、硫酸甲烷、醋酸、枸橼酸、丙二酸、丁二酸、反丁烯二酸、马来酸、磺酸或酒石酸。四价盐从碱性诺卡沙星I,II或III化合物和烷基或芳烷基卤化物中获得,优选甲基或苄基溴。
人们已知的本发明诺卡沙星化合物可包括不同的立体异构体。
MREF分析方法的描述
Nocardia sp.(ATCC202099)培养物中制得的粗提物,在高效普筛分析检测中发现了诺卡沙星抗菌素化合物的生物活性。这种筛选以孵育在琼脂生长培养基中的粪便肠球菌A28152的生长抑制为基础。粪便肠球菌可以耐许多抗菌素如青霉素G、万古霉素、环丙沙星、替考拉宁、四环素、链霉素、庆大霉素、红霉素、氯林肯霉素和雷米封,但对氯霉素敏感,其次是亚胺培南。因此MREF的名字表示多药耐药性粪便肠球菌。
粪便肠球菌菌种A28152接种到Brain Heart Infusion Broth营养培养基中,于37℃搅拌生长至对数期。定期地取出0.2ml培养物并移至含96孔塑料平底盘中,在595nm下检测光密度。当光密度在0.2-0.4时,经1000×g离心10分钟可得到细菌。细胞团混悬于Mue11er-HintonII生长培养基中,混悬液接种到含1%Difco的琼脂的熔化Mueller-HintonII生长培养基中,温度为48℃,得到一个1×107细胞/毫升的接种细胞密度。25ml的细胞混悬液倾入无菌的长方形盘中,一个特殊的与盘相配的塑料盖子密合于盘子顶端,其间培养基仍为熔化状态。盖子上有标准规格8×12排列的塑料钉。培养基在室温下15分钟凝胶,除去带针的盖子,小的凹形印迹留在曾与熔化培养基接触过的胶化培养基上,这些印迹作为样品的负载区。
待筛选检测的样品在100%的二甲亚砜(DMSO)中溶解浓度为300μM。每6μl体积的样品分别加入盘上载样区中。加样完毕,培养板于37℃培养24小时。环绕样品区,可看到一个透明的环状区,这便是生长抑制环形区外,细菌不可遏制地生长,琼脂培养基由于细菌生长而变得混浊。而同样的条件下单纯用DMSO不能产生任何可检测到的生长抑制。氯霉素和DMSO作为阳性和阴性对照品孵育在每一盘中。
采用薄层色谱(TLC)生物图象(biogram)确认Nocardia sp.(ATCC202099)生产培养物得到的活性物质,其材料包括用于琼脂扩散分析的生物检测皿(购自Nunc-Nalge International)。此皿为243×243×18mm,可提供530cm2的微生物培养区。把TLC盘放到生物检测皿上,并将琼脂覆盖物缓慢倾入以覆盖TLC盘和生物检测皿底部。琼脂覆盖物包括用0.5%去纤维蛋白的羊血补充的200ml Mueller-HintonII琼脂基和浓度为1×106细胞/毫升的耐药粪便肠球菌菌株A28152。冷却后,TLC生物图像于室温下孵育约18小时。孵育后,由于这种肠球菌中α-溶血细胞活性的原因,血琼脂变为红-棕色。如果化合物能抑制这种的生长,可见由于抑制了溶血而产生的非常明显的红色区。此方法考虑到了所需生物活性与适宜色谱馏分之间的相应关系,因此促进了活性成分及其分离物活性的确认。
这种用于MREF检测的方法包括检测两种不同的对比(contrast)剂。第一种是2,3,5-三苯基-2H-四吡咯氯化物(终浓度0.003%)补充到200ml Todd Hewitt琼脂基中。第二种方法是将0.5%的羊血补充到200ml Mueller-Hinton琼脂基中。两个方法均可显示明显的生长抑制区域,但是,用血琼脂孵育时间越短,分辨率(resolution)越好。考察了细胞接种浓度范围,但1×106细胞/毫升的结果最佳。
分离及结构特性
采用多药耐药性粪便肠球菌(MREF)琼脂扩散分析测定Nocardias p.发酵生成的诺卡沙星抗菌素的纯化。用乙酸乙酯提取,再进行溶剂分配、Sephadex LH-20和/或硅胶层析。这些步骤产生一种能在MREF琼脂扩散分析中显示活性的诺卡沙星抗菌素混合物。最后,单一的诺卡沙星抗菌素的纯化通过正相或反相高效液相制备层析完成,光谱数据显示该组分属于噻唑肽类抗菌素。在2D NMR阳离子电雾化(electrospray)HRMS研究和MS/MS数据基础上排布了诺卡沙星I,II和III的结构(见下式)。
其中W是
和R是OH(诺卡沙星I)或R是H(诺卡沙星II);和W是H和R是OH(诺卡沙星III)。
一般方法
材料:
己烷,氯仿(ACS纯),和甲醇,乙腈(色谱纯)购自FisherScientific公司。这些溶剂不是再纯化和重蒸馏的。用于色谱层析实验的水是指在室内(in-house)去离子水通过了一种微孔4cartridge试剂级的水系统(10兆欧Milli-Q水)。Sephadex LH-20g购自Pharmacia LKB.Uppsala,Sweden。Dicalite(硅藻土)产自GrefcoMinerals,Torrance,CA。LiChroprep硅60,25-40μm购自EMSeparations,NJ,E.Merek的美国合作伙伴,德国。
薄层色谱分析(TLC)
已涂上薄层色谱的硅胶GHLF板(型号10×20cm,250微米)购自Analtech,Inc,Newark,DE。组分点样使用2微升的微量管(带一次性吸管),板在已用氯仿-甲醇(9:1V/V)平衡的槽中展开。层析所得的组分在UV长波光下和/或用浓硫酸喷雾加热后可显示。
HPLC分析
诺卡沙星抗菌素纯化是用HPLC分析检测的。APEX 5μODS柱,直径4.6mm×长度15cm。(产于Jones Chromatography公司,Lakewood公司)。Hewlett Packard 1100型液相色谱仪分析,254nm下紫外检测。使用乙腈和0.01M磷酸钾缓冲液PH 3.5的梯度系统,按照D.J.Hook等(I.Chromatogr.385,99(1987))的方法,洗脱液的泵流速为1.2ml/分钟。
HPLC制备
下列组件用于组建HPLC制备系统:Beckman仪器公司。(SomersetNJ),Beckman“黄金系统”126程序化溶剂系统装置;Beckman 166程序化检测装置;Beckman“黄金系统”翻译711U软件;IBM PS/255SX系统控制器;YMC公司(Wilmingtan,NC)HPLC制备柱(正相):PVA-硅胶5μm粒径,孔径120°,直径20mm×长度150mm,配以Diol 25μ粒径,孔径120°,直径10mm×长度10mm的drop-in保护柱);流动相氯仿-甲醇梯度,流速为10ml/分钟,290nmUV检测。另一柱(反相):YMC公司,ODS-AQ 5μ粒径,孔径120°,直径20mm×长度150mm,配以ODS-A25μ粒径,孔径120°,直径20mm×长度10mm的dropin保护柱);流动相0.1M醋酸-丙酮梯度,流速为10ml/分钟,360nmUV检测。
分析设备
用阳离子电喷雾型Finnigan SSQ 7000单四极质谱仪进行低分辨率质谱分析。MS/MS的测定,采用阳离子电喷雾型Finnigan SSQ 7000串联四极质谱仪进行,氩碰撞气或Finnigan LCQ离子扑获质谱分析仪。高分辨率MS数据测定采用Finnigan MAT 900磁扇形质谱仪,阳离子电喷雾型,ppg reference。Hewlett-Packard 8452A二极管矩阵分光光度仪用于UV图谱测定,Perkin Elmer 2000傅里叶变换光谱仪用于红外测定。1H-NMR和13C-NMR数据用Bruker DRX-500分别与500.13和125.76MHz,使用Nalorac微探针。化学位移是相对于溶剂的数据(DMSO-d6,δH 2.49;δc 39.6)。圆二性色谱数据采用JascoJ-720分光偏振仪。
实施例
下述实施例阐述了Nocathiacin抗菌素的制备及其生物学特性。那些合理的、本领域技术人员能预见的变化并不偏离本发明的范畴。
Nocathiacin I、II的发酵和纯化
发酵制备Nocathiacin I和II
从冷冻的Nocardia sp.ATCC202099的营养培养基中取出4ml,接种到100ml下列种子培养液的500ml烧瓶中:每升去离子水含20g淀粉、5g葡萄糖、3g N-Ccase、2g酵母提取物、5g鱼肉提取物和3g碳酸钙。培养物于转速为250rpm的旋转振动器中32℃下孵育3天。4ml生成的培养物,分别接种到200个含100ml下列
生产培养基的500ml烧瓶中:每L去离子水含20g葡萄糖、5g蛋白胨、10g红星淀粉和5g Allophosite。
生产培养场于转速为250rpm的旋转振动器中32℃下孵育5天,然后处理以回收Nocathiacin I和II。
实施例1:从摇瓶中发酵
粗提取物的制备
用大约8L乙酸乙酯剧烈振荡0.5小时提取Nocardia sp.ATCC-202099(20L.)发酵培养基(包括菌丝体的全培养基)。两相混合物预先用3L.(1Kg)预涂层混合,然后大的Coors Buchner漏斗(内径27cm,外径28.5cm,深9cm)真空下过滤。分离灰黄色乙酸乙酯提取物并用旋转蒸发仪在真空下蒸发干燥,得到大约7.2g残余物A。
液-液分配残余物A
用含10%水的甲醇溶解残余物A(7.2g)。转移溶液到一分离漏斗中,用同体积的己烷提取4次。除去己烷层。加入38ml水将甲醇相稀释为含35%水的甲醇液,用同体积的氯仿提取3次。氯仿预先用含35%水的甲醇液饱和。己烷、氯仿和甲醇提取液用旋转蒸发仪在真空下蒸发干燥。主要在氯仿中可检测到Nocathiacin抗菌素(1.3g残余物B)。
Sepadex LH-20色谱分离残余物B
残余物B(1.3g)溶于10ml 1∶1氯仿-甲醇加到3×100cm的Glenco柱中,此柱用100g_Sepadex LH-20的1∶1氯仿-甲醇悬液填充。弃去前75ml,以2-3ml/分钟的流速每8-10ml一次收集馏分。馏分固化在硅胶TLC profiles(氯仿-甲醇9∶1,长波紫外和/或硫酸高铈喷雾)。用此方法,可在第10-15个馏分检测到黄色荧光性Nocathiacin抗菌素混合物。合并馏分并蒸发干燥(284mg残余物C)。
硅胶液相色谱分离残余物C
将Nocathiacin抗菌素的富集馏分(残余物C)预吸附到2g默克LiChroprep硅胶60上,然后加到填有这种吸附剂的2.5×115cm釉化过滤漏斗中。于house真空下先用1∶1己烷-氯仿(100ml)、再用氯仿并且逐渐增加氯仿比例(例如2.5,5,7.5,10,12.5,15,和25%甲醇/氯仿溶液,每份100ml)洗脱。馏分固化在硅胶TLC profiles(氯仿-甲醇9∶1,长波紫外和/或硫酸高铈喷雾)。用此方法,可在5-12.5%甲醇/氯仿馏分中检测到Nocathiacin I抗菌素。可在15-15%甲醇/氯仿馏分中检测到Nocathiacin II抗菌素。合并馏分并蒸发干燥(残余物D,Nocathiacin I,230mg),(残余物E,NocathiacinII,49mg)
分离Nocathiacin I和H
用Ba ckman黄金系统的制备HPLC系统(YMC ODS-AQ C18柱)进一步纯化残余物D和E。典型的进样量为25-50mg/50-100μl二甲亚砜溶液。先用氯仿再经pre-programmed concave梯度至8∶2的氯仿:甲醇洗脱30分钟。流速为10ml/分钟。紫外290nm检测。用这种方法获得Nocathiacin
I(保留时间17分钟的峰,总收率56mg)和Nocathiacin
II(保留时间19.5分钟的峰,总收率6.7mg)。
纯化方案
下表描述了Nocathiacin抗菌素的纯化方案:
Nocathiacin I的物理-化学特性II III
形态: 灰黄色无定形固体
分子式: C61H60N14O18S5
分子量: 1436
质谱: HR-ESIMS [M+H]+m/z 1437.285
ESI-MS/MS fragmentation ions:m/z 1266,1248,
1221,1204,1186,1154,788
红外光谱: 主要波长(cm-1)3392,3108,2932,1740,
1721,1694,1670,1640,1533,1478,1420,1384,1320,
1250,1207,1128,1091,1037,1014,751.
紫外光谱: λmax(MeOH)222,290,364nm(1ogε4.89,4.52,
4.17)
圆二色谱: CDλnm(Δε)(MeOH)212(+34.1),239(-50.5),
267(+20.8),307(-8.7),364(+5.5)
HPLC保留时间 25.6分钟,(C18;0.01M的pH3.5磷酸钾缓冲液
(Rt)
-乙腈梯度洗脱(J Chromatogr.385,99,(1987))
1H-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ2.49):
δ10.05(1H,s),9.07(1H,s),8.65(1H,s),8.57(1H,
d.J=8.0Hz),8.51(1H,s),8.50(1H,s),8.24(1H,s),
8.07(1H,s br),7.89(1H,d,J=11.2Hz),7.86(2H,s),
7.70(1H,d,J=8.4Hz),7.61(1H,sbr),7.34(2H,m),
7.16(1H,d,J=6.9Hz),6.40(1H,s),5.98(1H,d,
J=12.0Hz),5.77(1H,s),5.73(1H,dd,J=10.9,4.4
Hz),5.70(1H,d,J=8.7Hz),5.22(1H,m),5.02(1H,
d,J=11.4Hz),4.94(1H,d,J=3.7Hz),4.75(1H,d,
J=10.2Hz),4.51(1H,d,J=11.0Hz),4.29(1H,d,
J=9.6Hz),4.19(1H,m),4.13(1H,d,J=10.1Hz),
4.00(1H,d,J=95Hz),3.88(3H,s), 3.78(1H,d br,
J=5.9Hz),2.52(6H,s),2.49(1H,m),2.11(1H,s br),
1.99(3H,s),1.95(1H,m),1.81(1H,d,J=13.9Hz),
1.43(3H,s),1.21(1H,m),1.16(3H,s,br),0.59(3H,
d,J=6.3Hz).
13C-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ39.6):
δ171.8,168.1,167.7,167.4,167.0,165.2,164.0,
163.1,161.5,161.1 160.5,160.3,159.1,158.6,154.6,
151.9,149.7,149.6,148.8,145.8,142.5,135.3,135.0,
134.5,129.9,128.1,127.0,126.3,125.5,124.0,123.1,
119.8,119.4,112.7,111.2,109.7,103.7,95.1,79.3,
70.6,68.4,67.5,66.1,65.1,64.5,63.3,56.1,55.5,50.2,
49.8,44.4,40.0,30.5,18.0,13.1.
Nocathiacin II的物理-化学特性
形态: 灰黄色无定形固体
分子式: C61H60N14O17S5
分子量: 1420
质谱: HR-ESIMS[M+H]+m/z 1421.297
ESI-MS/MS fragmentation ions:m/z 1250,1206,
1188,1170,1156,1138,788
红外光谱: 主要波长(cm-1)3387,1725,1656,1534,
1478,1423,13118,1249,1192,1087,1014,886,793,
751.
紫外光谱: λmax(MeOH)220,295,364nm(logε5.01,4.67,
4.36)
圆二色谱: CDλnm(Δε)(MeOH)211(+41.5),235(-56.1),
258(+21.0),277(+13.4),307(-10.0),364(+5.6)
HPLC保留时间 21.1分钟,(C18;0.01M的pH3.5磷酸钾缓冲液-
(Rt) 乙腈梯度洗脱(J Chromatogr.385,99,(1987))
1H-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ2.49);
δ9.10(1H,s),8.59(1H,d,J=8.5Hz),8.51(1H,s),
8.45(1H,s),8.22(1H,s),8.15(1H,s),8.05(1H,s
br),7.69(1H,s br),7.66(1H,s),7.51(1H,d,J=8.3
Hz),7.47(1H,s),7.30(1H,s),7.25(1H,dd,J=8.0,
7.2Hz),7.12(1H,d,J=7.1Hz),7.02(1H,d,J=8.4
Hz),6.49(1H,s),6.03(1H,d,J=12.1Hz),5.78(1H,
m),5.70(1H,d,J=8.6Hz),5.66(1H,s),5.30(1H,d,
J=7.7Hz),5.00(1H,d,J=13.0Hz),4.98(1H,d,
J=10.5Hz),4.94(1H,d,J=4.8Hz),4.83(1H,d,
J=11.2Hz),4.72(1H,m),4.37(1H,d,J=9.6Hz),
4.23(1H,m),4.11(1H,d,J=9.9Hz),4.03(1H,d,
J=7.7Hz),3.89(3H,s),3.75(1H,m),2.49(6H,s),
2.14(1H,m),2.01(3H,s),1.94(1H,m),1.79(1H,
d,J=14.2Hz),1.40(3H,s),1.23(1H,m),1.04(3H,
s,br),0.54(3H,d,J=6.5Hz).
13C-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ39.6):
δ172.0,168.2,167.1,167.0,165.5,165.3,163.2,
162.9,161.6,161.2,160.6,160.1,158.7,156.3,149.9,
149.0,148.2,145.9,138.8,136.9,134.3,128.8,127.9,
127.2,126.6,125.6,125.2,124.7,124.1,123.9,122.8,
117.9,116.3,115.6,109.9,101.9,95.1,79.0,70.7,
68.4,67.6,66.3,66.0,64.0,62.5,56.1,55.1,51.4,50.6,
44.4,40.5,30.6,18.1,18.0,13.0.
Nocathiacin抗菌素的生物学评价
实施例2:Nocathiacin I和II的抗菌活性
为证明其抗菌活性,采用常规培养基稀释法(用营养培养基(Difcos)连续稀释)测定了本发明Nocathiacin抗菌素对多种细菌的最小抑菌浓度(MIC)。结果见表1,这表明Nocathiacin抗菌素可用于治疗细菌感染。
表1
菌名 菌株号 MIC(ug/ml) MIC(ug/ml)
Nocathiacin I Nocathiacin II
肺炎链球菌 A9585 0.001 0.015
肺炎链球菌/青霉素 A27881 0.001 0.015
intermediate
肺炎链球菌/耐青霉素 A28272 0.001 0.015
酿脓链球菌 A9604 0.001 0.125
粪便肠球菌 A20688 0.03 0.5
粪便肠球菌 A27519 0.03 0.5
粪便肠球菌+50%小牛血清 A20688 0.25 0.5
粪便肠球菌 A24885 0.015 0.5
粪便肠球菌/耐硫链丝菌 SC15829 0.025 0.5
肽(10ug/ml)
鸟肠球菌 A27456 0.015 0.5
金黄色葡萄球菌 A9537 0.001 0.06
金黄色葡萄球菌/β-青霉 A15090 0.007 0.5
素酶阳性
金黄色葡萄球菌+50%小牛 A15090 0.015 0.5
血清
金黄色葡萄球菌 A24407 0.007 0.5
/QC/ATCC#29213
金黄色葡萄球菌/耐异甲 A27218 0.003 0.5
氧苯青霉素
金黄色葡萄球菌+50%小牛 A27218 0.007 0.5
血清
金黄色葡萄球菌/耐同甲 A27223 0.003 0.125
氧苯青霉素
金黄色葡萄球菌+50%小牛 A27223 0.001 0.125
血清
细球菌luteus A9852 0.003 0.125
枯草杆菌 A9506A 0.001 0.125
表皮葡萄球菌 A24548 0.003 0.125
溶血葡萄球菌 A27298 0.03 0.25
大肠杆菌 A15119 >128 >128
大肠杆菌 A22292 >128 >128
大肠杆菌/AcrA∷Kan A28901 >128 >128
肠炎沙门菌 A9531 >128 >128
粘膜炎莫拉氏菌/β-青霉 A22344 0.06 0.125
素酶阳性
粘膜炎莫拉氏菌/β-青霉 A25409 0.06 0.125
素酶阳性
流感嗜血杆菌/β-青霉素 A20191 >128
酶阴性
流感嗜血杆菌/β-青霉素 A20183 >128
酶阴性
流感嗜血杆菌/β-青霉素 A21515 >128
酶阳性
沙门菌/WT A27207 >128 >128
沙门菌/RE A27208 >128 >128
实施例3:Nocathiacin I在全身性金黄色葡萄球菌感染模型中的体内抗菌活性
用雌性IcR小鼠全身性感染模型来评价Nocathiacin I的体内抗菌活性。动物用6.5×106CFU的金黄色葡萄球菌感染,该菌混悬于7%的粘蛋白中事先过夜培养。Nocathiacin I溶解于由10%二甲亚砜、5%吐温80和85%水组成的待试制剂中。以100mg/kg的总剂量(于感染后1和4小时,2×50mg/kg)皮下给予该溶液0。9只小鼠在实验期间全部存活并且无中毒症状。测得Nocathiacin I的PD50小于6.25mg/kg。
发酵制备Nocathiacin III
可在任何益于培养菌生长的温度如16℃和40℃进行Nocathiacin III的生产,但优选在22-26℃进行。液体培养基有必要孵育一段时间以保证Nocathiacin III的生产充分进行,用HPLC通常监测1-5天,生产中旋转振动器转速为50-300rpm,优选150-200rpm。
产物Nocathiacin III可从培养基中回收,方法与其他已知生物活性物质的回收相同。经采用常规溶剂如乙酸乙酯提取培养基,用常规树脂(如阴或阳离子交换树脂、非离子吸附树脂)和常规吸附剂(如活性炭、硅胶、纤维素和氧化铝)处理,结晶、重结晶,和/或反相制备HPLC纯化,得到Nocathiacin III。
下述实施例阐述了发酵法制备Nocathiacin III。那些合理的、本领域技术人员能预见的变化并不偏离本发明的范畴。
发酵
实施例4:
从冷冻的Nocardia sp.ATCC202099的营养培养基中取出4ml,接种到100ml下列种子培养基的500ml烧瓶中:每ml去离子水含20g日本可溶性淀粉、5g葡萄糖、3g N-C case、2g酵母提取物、5g鱼肉提取物和3g碳酸钙。培养物于转速为250rpm的旋转振动器中28℃下孵育3天。4ml生成的培养物,分别接种到10个含100ml下列生产培养基的500ml烧瓶中:每ml去离子水含10g HY酵母412、20g葡萄糖和10g Nutrisoy。生产培养物于转速为250rpm的旋转振动器中28℃下孵育2天,然后回收Nocathiacin III。
实施例5:
从冷冻的Nocardia sp.ATCC202099的营养培养基中取出4ml,接种到100ml下列种子液的500ml烧瓶中:每ml去离子水含20g日本可溶性淀粉、5g葡萄糖、3g N-C case、2g酵母提取物、5g鱼肉提取物和3g碳酸钙。培养物于转速为250rpm的旋转振动器中28℃下孵育3天。取4ml生成的培养物,分别接种到5个含100ml新鲜种子培养基的500ml烧瓶中,培养物于转速为250rpm的旋转振动器中28℃下孵育3天。将5个烧瓶中的培养基混合,然后取4ml合并培养物分别接种到100个含100ml下列生产培养基的500ml烧瓶中:每ml去离子水含10g HY酵母412、20g葡萄糖和10g Nutri soy。生产培养物于转速为180rpm的旋转振动器中24℃下孵育4天,然后回收Nocathiacin III。
实施例6:Nocathiacin III的分离
粗提取物的制备
用大约1L乙酸乙酯剧烈振荡提取Nocardia sp.ATCC202099发酵培养基(包括菌丝体)。离心分离两相混合物。水相再用乙酸乙酯提取(0.5L)。合并灰黄色乙酸乙酯提取物并用旋转蒸发仪在真空下蒸发干燥,得到大约198mg残余物A。
液-液分配残余物A
用10ml 10%水/甲醇溶解残余物A(198mg)。转移溶液到一分离漏斗中,用同体积的己烷提取3次。除去己烷层。加入3.8ml水将甲醇相稀释为含35%水的甲醇液,用同体积的氯仿提取3次。氯仿预先用含35%水的甲醇液饱和。己烷、氯仿和甲醇提取液用旋转蒸发仪在真空下蒸发干燥。主要在氯仿中可检测到Nocathiacin抗菌素,(106mg残余物B)。
分离Nocathiacin III
用Backman黄金系统的制备HPLC系统(YMC ODS-AQ C18柱)进一步纯化残余物B。典型的进样量为50mg/250μl二甲亚砜溶液。用0.1M乙酸铵-乙腈(55∶45v/v)线性梯度洗脱至乙腈洗脱。流速为10ml/分钟。紫外360nm检测。用这种方法获得Nocathiacin III(保留时间9分钟的峰,总收率12mg)。
Nocathiacin III的物理-化学特性
形态: 淡黄色无定形固体
分子式: C52H43N13O16S5
分子量: 1265
质谱: HR-ESIMS[M+H]+m/z 1266.162
ESI-MS/MS fragmentation ions:m/z 1248,1222,
1204,1186,788
红外光谱: 主要波长(cm-1)3382,1720,1667,1643sh,
1534,1510,1477,1420,1250,1207,1126,1015,750.
紫外光谱: λmax(MeOH)nm 224,290,364(logε4.85,4.52,
4.11).
圆二色谱: CDλnm(Δε)(MeOH)212(+38.7),238(-47.6),
266(+21.2),305(-11.4),362(+5.1).
HPLC保留时间 19.3分钟,(C18;0.01M的pH3.5磷酸钾缓冲液-
(Rt) 乙腈梯度洗脱(J Chromatogr.385,99,(1987))
1H-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ2.49):
δ10.05(1H,s),8.93(1H,s),8.59(1H,s),8.51(1H,
s),8.46(1H,s),8.37(1H,d,J=8.9 Hz),8.20(1H,s),
8.05(1H,s),7.91(1H,d,J=10.9Hz),7.77(1H,s br),
7.74(1H,s br),7.69(1H,d,J=8.4Hz),7.60(1H,s),
7.38(1H,d,J=7.5Hz),7.34(1H,dd,J=8.2,7.2Hz),
7.18(1H,d,J=7.0Hz),6.37(1H,s),6.07(1H,d,
J=7.1Hz),5.92(1H,d,J=12.2Hz),5.89(1H,d,
J=10.3Hz),5.74(1H,dd,J=10.8,4.8Hz),5.72(1H,
s),5.24(1H,m),5.02(1H,d,J=12.5Hz),4.71(1H,
d,J=10.4Hz),4.54(1H,d,J=11.2Hz),4.50(1H,m),
4.20(1H,m),4.13(1H,d,J=10.4Hz),4.02(1H,dd,
J=9.4,7.2Hz),3.87(3H,s),3.73(1H,d,J=9.6Hz),
1.97(3H,s),1.15(3H,s br).
13C-NMR 化学位移(相对于溶剂DMSO-d6,信号为δ39.6):
δ174.5,172.2,168.1,167.8,166.6,165.3,164.4,
163.0,161.2,160.4,159.3,158.8,154.7,152.9,149.6,
148.8,146.0,141.6,135.7,135.0,134.5,129.9,128.6,
127.1,126.4,126.3,125.6,125.3,124.2,123.1,119.7,
119.5,112.5,111.3,109.8,103.7,81.4,67.9,67.3,
65.3,64.5,63.5,56.2,55.7,49.7,49.5,18.0,13.2.
实施例7:Nocathiacin的抗菌活性
为证明其抗菌活性,采用常规培养基稀释法(用营养培养基(Difcos)连续稀释)测定了NocathiacinIII抗菌素对多种细菌的最小抑菌浓度(MIC)。结果见表2,这表明NocathiacinIII抗菌素可用于治疗细菌感染。
表2
菌名 菌株号 MIC(ug/m1)
Nocathiacin III
肺炎链球菌 A9585 ≤0.002
肺炎链球菌/青霉素intermediate A27881 ≤0.002
肺炎链球菌/耐青霉素 A28272 ≤0.002
粪便肠球菌 A20688 0.03
粪便肠球菌 A27519 0.03
粪便肠球菌+50%小牛血清 A20688 0.25
粪便肠球菌 A24885 0.03
鸟肠球菌 A27456 0.03
金黄色葡萄球菌/β-青霉素酶阳 A15090 0.007
性
金黄色葡萄球菌+50%小牛血清 A15090 0.03
金黄色葡萄球菌/QC/ATCC#29213 A24407 0.007
金黄色葡萄球菌/耐同甲氧苯青霉 A27223 0.007
素
金黄色葡萄球菌+50%小牛血清 A27223 0.007
表皮葡萄球菌 A24548 0.007
葡萄球菌haemolyticus A27298 0.007
粘膜炎莫拉氏菌/β-青霉素酶阳 A22344 0.06
性
粘膜炎莫拉氏菌/β-青霉素酶阳 A25409 0.06
性
流感嗜血杆菌/β-青霉素酶阴性 A20191 >64
流感嗜血杆菌/β-青霉素酶阴性 A20183 >64
流感嗜血杆菌/β-青霉素酶阳性 A21515 >64
Claims (1)
1.保藏号为ATCC-202099的微生物诺卡氏菌种的一种生物纯的培养物。
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US6287827B1 (en) | 1999-05-05 | 2001-09-11 | Bristol-Myers Squibb Company | Halo- or hydroxy-substituted nocathiacin antibiotics |
US20020055465A1 (en) * | 2000-08-14 | 2002-05-09 | Wenying Li | Nocathiacin antibiotics prepared by biotransformation or chemical methods |
US20020065219A1 (en) * | 2000-08-15 | 2002-05-30 | Naidu B. Narasimhulu | Water soluble thiazolyl peptide derivatives |
US6713448B2 (en) | 2001-04-25 | 2004-03-30 | Wyeth Holdings Corporation | Substantially pure glycopeptide antibiotics AC-98-1; AC-98-2; AC-98-3; AC-98-4 and AC-98-5 |
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WO2006086012A2 (en) * | 2004-09-22 | 2006-08-17 | Merck & Co., Inc. | Antibiotic compound |
WO2007127135A2 (en) * | 2006-04-24 | 2007-11-08 | Merck & Co., Inc. | Antibiotic compounds |
US20080132500A1 (en) * | 2006-10-20 | 2008-06-05 | Kun Liu | Antibiotic compounds |
US20080242597A1 (en) * | 2006-10-20 | 2008-10-02 | Kun Liu | Antibiotic compounds |
CN102028933A (zh) * | 2010-11-18 | 2011-04-27 | 中国药科大学 | 含脂质材料的诺卡沙星抗生素的药物组合物 |
CN102008436B (zh) * | 2010-11-18 | 2012-07-18 | 中国药科大学 | 含乳化剂的诺卡沙星抗生素药物组合物 |
CN102018953B (zh) * | 2010-11-18 | 2013-05-01 | 南京碧迪可医药科技有限公司 | 含诺卡沙星抗生素的药物组合物 |
CN102952760B (zh) * | 2011-08-25 | 2014-04-02 | 天津工业生物技术研究所 | 一株能将奎宁酮转化为(s)-3-奎宁醇的红串红球菌及转化方法 |
CN102952761B (zh) * | 2011-08-25 | 2014-01-15 | 天津工业生物技术研究所 | 一株能将奎宁酮转化为(r)-3-奎宁醇的诺卡氏菌及转化方法 |
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CH634853A5 (de) | 1978-06-08 | 1983-02-28 | Sandoz Ag | Schwefelhaltige metabolite, ihre herstellung und verwendung in arznei- und futtermitteln. |
US5451581A (en) | 1993-05-21 | 1995-09-19 | Lee; May D. | Antibiotic LL-14E605β and O-methyl-LL-14E605β |
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Cited By (1)
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CN101153274B (zh) * | 2006-09-29 | 2010-12-01 | 中国科学院上海生命科学研究院 | 提高利福霉素产量的方法 |
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TR200003587T2 (tr) | 2001-06-21 |
CA2337398A1 (en) | 2000-01-27 |
AU745333B2 (en) | 2002-03-21 |
NZ509306A (en) | 2002-07-26 |
AU5098199A (en) | 2000-02-07 |
KR20010071913A (ko) | 2001-07-31 |
NO20010250D0 (no) | 2001-01-15 |
CN1309566A (zh) | 2001-08-22 |
CN1133437C (zh) | 2004-01-07 |
EP1096939A1 (en) | 2001-05-09 |
IL139650A0 (en) | 2002-02-10 |
US6218398B1 (en) | 2001-04-17 |
HUP0102903A2 (hu) | 2002-05-29 |
JP2002520365A (ja) | 2002-07-09 |
WO2000003722A1 (en) | 2000-01-27 |
EP1096939A4 (en) | 2001-08-22 |
ID27775A (id) | 2001-04-26 |
NO20010250L (no) | 2001-03-07 |
CN1515674A (zh) | 2004-07-28 |
PL345657A1 (en) | 2002-01-02 |
BR9912042A (pt) | 2001-04-03 |
CZ2001182A3 (cs) | 2002-01-16 |
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