专利名称：用于治疗细菌感染的多价噬菌体制备方法
技术领域：
本发明涉及噬菌体的复价株系、获得这些株系的方法及其在治疗细菌感染中的应用，特别是在那些有药物抗性的细菌株系感染中的应用。
背景技术：
噬菌体(phages)是一种多样性的病毒，其生活史专一地与细菌细胞相关。噬菌体特征在于溶原性的或裂解性的生活史。作为抗菌剂，裂解性噬菌体尤其有用，该噬菌体当感染对它们敏感的细菌细胞后可在该细胞中复制，从而导致该细胞的完全破坏(通过裂解作用)及新噬菌体的释放，新的噬菌体可攻击和破坏其他的细菌细胞。该过程可发生于体外或体内。
噬菌体的一个基本特征是其裂解活性的众所周知的高特异性。该特性可应用于如各种细菌的物种确定(噬菌体分类)(参见如专利说明GB 2285684、US 5824468和SU 543260以及国际专利通知WO 0100786和WO 0109370)。噬菌体的其他已知的应用包括其作为分子生物学工具的应用，如在特定的蛋白质的表达和选择中的应用(如专利描述US6027930)和在灭菌和清除培养基中的应用(如专利描述EP 0414304、EP 0290295和GB 2253859以及国际专利通知WO 9808944和WO9003122)。修饰的噬菌体用于疫苗的生产(如WO 9505454)。也使用了某些噬菌体来源的蛋白质(如EP 0510907、US 5470573、WO9607329)。分离噬菌体和获得噬菌体制备物的方法是众所周知的且经常进行完善(如GB 829266、CS 192212、RU 2109055)。
第二次世界大战后在Georgia的Tbilisi微生物学和病毒学研究所(Institute of Microbiology and Virology of Tbilisi)已大规模地应用了噬菌体治疗。那里在细菌感染的治疗和预防中应用了各种噬菌体制备物的储备。可用的数据显示噬菌体治疗的巨大有效性。相似的研究在波兰已进行了超过25年。在弗罗茨瓦夫的波兰科学院免疫学和试验治疗研究所的噬菌体实验室(Bacteriophage Laboratoryof the Institute of Immunology and Experimental Therapy of thePolish Academy of Sciences in Wroclaw)，将噬菌体治疗用于治疗由抗药形式的细菌和那些不对抗生素易感的细菌所导致的感染(参见Stefan lopek等人，Archivum Immunologiae et TherapiaeExperimentalis，1981，31，293；Stefan 等人，ArchivumImmunologiae et Therapiae Experimentalis，1983，31，267；Stefan 等人，Archivum Immunologiae et Therapiae Experimentalis，1984，32，317；Stefan 等人，Archivum Immunologiae etTherapiae Experimentalis，1985，33，219；Stefan 等人，Archivum Immunologiae et Therapiae Experimentalis，1985，33，241Stefan 等人，Archivum Immunologiae et TherapiaeExperimentalis，1987，35，569；Beata Weber-Dabrowska等人，Archivum Immunologiae et Therapiae Experimentalis，2000，48，31-37；Beata Weber-Dabrowska等人，Archivum Immunologiae etTherapiae Experimentalis，2000，48，547-551)。过去14年中在那里进行的噬菌体治疗显示噬菌体治疗的高效性，该治疗包括1473个具有不同组织和器官化脓性感染的患者。
在1289个病例中记录到疾病症状的完全消除和康复。必须强调的是在至少一打左右的病例中，噬菌体治疗呈现为消除致命性感染的唯一可能。进行的治疗涉及单独的患者。它由以下内容组成a)从获自患者的材料中分离的细菌株系的生长和鉴定，b)确定该株系对特定噬菌体的易感性及选择对该株系显示最高裂解活性的噬菌体，c)制备具有大量噬菌体颗粒的噬菌体溶菌产物，d)生产用于治疗的无菌噬菌体制备物。
该程序相当昂贵、费力且费时。有时，从获得研究材料起到获得用于治疗的完成的无菌噬菌体制备物要经过7-10天。这种延迟在某些疾病状况中太长。大规模的噬菌体治疗不能用目前采用的程序进行。伴随粘稠物阻塞症的感染呈现为一个特别的问题。这是一种遗传的全身疾病，该疾病由粘液分泌腺的功能异常构成，这使得患者倾向于患慢性支气管和肺病。分泌的粘液浓且粘，从而使其难由天然途径(咳嗽)清除。粘液在支气管中的累积和滞留以及在其清除中的损害造成了细菌感染的有利环境，从而导致慢性支气管炎和肺炎。导致伴随粘稠物阻塞症感染的最危险的病原体是金黄色葡萄球菌(Staphylococcus aureus)和假单胞菌属(Pseudomonas)的杆菌。这些微生物持久地复发感染导致呼吸系统中严重的病理学改变和早死。很少患者可幸存超过25年。用可获得的抗生素对这些感染的治疗造成了全世界范围的治疗问题，尤其是在刚过去的几年中。因为绝大多数细菌株系显示对所有抗生素包括最后手段的抗生素万古霉素的抗性，所以对这些感染的抗生素治疗已变得越来越低效了。因此，迫切需要向治疗难治的和高度危险的细菌感染的医学实践中引入可替代的治疗方法。
发明目的过去数年中细菌株系对所有抗生素的抗性已有广泛的传播，该抗生素包括最后手段的抗生素万古霉素。结果，用抗生素治疗由这些药物抗性形式的细菌引起的感染是无效的。该状况已造成了巨大的治疗问题。因而，迫切需要向治疗难治的细菌感染的医学实践中引入其他的治疗方法。
考虑到上述目的，本发明致力获得复价噬菌体株系，该噬菌体株系可用于生产在治疗细菌感染中具有保证的效力的制备物，而无需对每一个病例单独选择噬菌体。在此，本发明的目的是提供允许获得高纯度的噬菌体制备物的途径，该制备物尤其不含内毒素。
本发明的一个特别的目的是获得多价的噬菌体制备物，该制备物可有效应用于治疗与粘稠物阻塞症相关的细菌感染，而无需每次单独选择噬菌体。
发明概述本发明提供获得复价噬菌体株系的方法，通过该方法a)积聚足够数目(n)的在给定区域随机出现的确定属的细菌病原体的不同株系，在此分离的细菌株系优选为药物抗性的，b)积聚足够数目的对该给定属的至少一个细菌株系特异的不同噬菌体株系，c)确定所积聚的噬菌体株系对每种所积聚的细菌株系的裂解活性，然后计算p值，该值为由给定的噬菌体裂解的菌株的数目与所有积聚的细菌株系的数目的比例。
d)对于结果所得的p值，检验n是否满足条件n≥pqd2z1-α2]]>其中q＝1-pd是不大于0.1的常数，最适地不大于10％pz1-α是依赖于置信因子1-α的正态分布的随机变量，该置信因子不小于0.95，e)选择满足在d)中限定的标准且具有不小于2/n的p值(优选地不小于0.5)的噬菌体株系。
每一个分离的噬菌体的裂解活性范围是关于在给定区域存在的病原体细菌株系的群体而确定的。为此，必须积聚足够大的随机选择的药物抗性细菌株系的集合。参考确立的群体大小为n个株系的细菌大样本(在描述的例子中，对于在波兰分离的假单胞菌属和葡萄球菌属的药物抗性株系，n分别为845和880)，计算了成功频率或由给定的噬菌体裂解的菌株株系的数目与所研究的株系的总数目的比例。人们认为该频率是成功概率p。失败概率则是q＝1-p。
关键的是选择的样本必须具有的大小n，从而可认为该样本是给定属株系的普通群体的代表(如假单胞菌属或葡萄球菌属)。为此，确定了下面使样本具有代表性的标准P(|v-p|＜d)＝P(-d＜v-p＜d)＝1-α样本频率v和成功概率p之间的差的绝对值小于预先指定的值d的概率为1-α，这相应于置信水平1-α。1-α的值通常设定为0.95或0.98。
样本数目n应该满足条件n≥pqd2z1-α2]]>其中z1-α是正态分布的随机变量。对于1-α＝0.95，z1-α＝1.96，而对于1-α＝0.98，z1-α＝2.33。
基于该标准和从所表征的噬菌体获得的频率p的值，通过给d和置信范围1-α指定合理的值，可能证实用于确定给定噬菌体的裂解活性范围的量级n及确定在指定的d和1-α的值时，对该n获得的裂解活性对于给定属的细菌株系的普通群体是否可认为是正确的。
噬菌体株系优选在步骤b)中从源自环境的样品中分离，通过使该样品经过孔径为0.2-0.4μm的膜滤器、向滤液中添加培养基并使其与确定属的细菌的液体培养基混合、使其在约37℃的温度温育约1小时、取出一部分悬浮液并将其涂于有固体培养基的平板上、于约37℃温育2～24小时、分离单一秃斑周围的培养基样品、将其转移到确定属的细菌的液体培养基中并进行温育直到培养物清澈，然后通过使溶菌产物经过孔径为0.2-0.4μm的膜滤器而获得噬菌体制备物，在此优选重复接种固体培养基并将秃斑再次分离5次。
将在步骤c)中的细菌株系优选接种到固体培养基上，在该培养基上沉积了一部分在步骤b)中获得的噬菌体制备物，于约37℃温育约4小时，其后将温度设定于约4℃进行2～24小时，借此噬菌体株系的裂解活性通过至少单个秃斑的出现而证实。
可对裂解物实施进一步的纯化，特别关于内毒素，在此使含有噬菌体的混合物与含有纤维素或其部分酯化的衍生物接触，然后用可除去杂质尤其是可除去内毒素的溶液冲洗，其后将纯化的噬菌体洗涤出来。内毒素可有效地用水、非解离性物质的溶液或可能缓冲的浓度不大于0.1M的盐溶液进行洗脱。噬菌体级分也可有效地用非解离性物质的溶液或任何缓冲液或可能缓冲的浓度大于0.05M的盐溶液进行洗脱。同样地，内毒素和噬菌体的洗脱是在-25℃～+100℃之间的温度中进行的。优选地，内毒素和噬菌体洗脱可用含有机溶剂的水性盐溶液进行。该有机溶剂最好选自二甲基亚砜、二甲基甲酰胺、异丙醇和丙酮，且可使用以有机或无机酸部分酯化的纤维素作为基质。用乙酸、硝酸、硫酸或磷酸部分酯化的纤维素作基质，特别是其中0.01～5％的葡萄糖分子被酯化的纤维素可用作基质，优选其中0.25～1％的葡萄糖分子，更优选0.5～1％的葡萄糖分子被酯化。
病原性细菌株系优选为假单胞菌属或葡萄球菌属。
本发明的进一步的方面是治疗或预防由细菌病原体导致的感染的药物，该药物含有活性试剂和可能的药物学可接纳的载体，从而该活性试剂包含对该属的细菌特异的复价噬菌体株系且由本发明的方法获得。
根据本发明，该药物可具有如下特征，作为对葡萄球菌属细菌特异的复价噬菌体株系，它含有至少一种株系的选自下述的噬菌体S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCMF/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，优选为噬菌体S1、S2和S4或S5。根据本发明的一个方面生产的该药物用于治疗或预防患粘稠物阻塞症的人的感染，以及作为对葡萄球菌属细菌特异的复价噬菌体株系，它含有至少一种选自F/00002、F/00004和F/00007的噬菌体株系。
根据本发明，该药物可具有如下特征，作为对假单胞菌属细菌特异的复价噬菌体株系，它含有至少一种株系的选自下述的噬菌体P1(PCM F/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCMF/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，最适为噬菌体P7、P20和P6。根据本发明的某些方面生产的该药物用于治疗或预防患粘稠物阻塞症的人的感染，以及作为对假单胞菌属细菌特异的复价噬菌体株系，它含有至少一个选自F/00010、F/00013和F/00018的噬菌体株系。
本发明进一步的目的是应用根据本发明的方法获得的复价噬菌体株系以开发治疗或预防由细菌病原体导致的细菌感染的药物。
对于生产治疗或预防由葡萄球菌属细菌导致的感染的药物，有利的是应用至少一种选自S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/0006)、S6(PCMF/00006)和S7(PCM F/00007)的噬菌体株系，最适为噬菌体S1、S2和S4或S5。对于制造治疗或预防患粘稠物阻塞症的人的由葡萄球菌属细菌导致的感染的药物，有利的是使用至少一种选自F/00002、F/00004和F/00007的噬菌体株系。
同样地，对于制造治疗或预防由假单胞菌属细菌导致的感染的药物，有利的是使用至少一种选自P1(PCM F/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCMF/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)的噬菌体株系，最适为噬菌体P7、P20和P6。对于制造治疗或预防患粘稠物阻塞症的人的由假单胞菌属细菌导致的感染的药物，有利的是使用至少一种选自F/00010、F/00013和F/00018的噬菌体株系。
本发明进一步的目的是对葡萄球菌属细菌特异的复价噬菌体株系，该噬菌体株系选自S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCMF/00006)和S7(PCM F/00007)，最适选自S1、S2、S4和S5。
本发明一个目的也是对假单胞菌属细菌特异的复价噬菌体株系，该噬菌体株系选自P1(PCM F/00008)、P2(PCM F/00009)、P3(PCMF/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，最适选自P7、P20和P6。
在研究中使用了我们自己的噬菌体集合，该噬菌体集合的活性是针对人的细菌感染中为最频繁的病原学因子的细菌物种。所述的实施例涉及如下多价噬菌体，该噬菌体对葡萄球菌株系(金黄色葡萄球菌)包括其甲氧基苯青霉素抗性的株系起裂解作用，及对蓝脓(blue pus)杆菌(铜绿假单胞菌(Pseudomonas aeruginosa))起裂解作用，该杆菌特别出现于那些患粘稠物阻塞症的人中。这些物种当前是导致高死亡率的严重感染的原因。
本发明的优点向治疗中引入含有用本发明方法获得的多价噬菌体的多价噬菌体制备物是在与细菌感染的斗争中的一个主要发展，该细菌感染对于抗生素治疗不易感。某些经济利益也与该新技术相关。它使得在原料成本中有显著的减少并缩短了从收集用于鉴定的材料到获得治疗性噬菌体制备物之间的时间。因此，噬菌体制备物将可供患者即时使用，而不需患者等待单一的噬菌体制备物的生产。
包含用本发明的方法获得的几个多价噬菌体的裂解性噬菌体制备物的广泛范围从其在治疗细菌感染中应用的看来是非常重要的优点。众所周知，在一个细菌群体中，对噬菌体抗性的形式可以以约1×10-7的频率出现。存在2个噬菌体时，该突变的概率为约1×10-14，且3个噬菌体时仅为1×10-21。利用这种安排，对噬菌体抗性的细菌株系的问题在实际上不存在。
包含多价噬菌体的示例性的药物的极大的应用性需要特别的强调。它们通常可在医学实践中应用并允许有效操作有危险的细菌感染。同样可能的是在外用的药物的制备中应用根据本发明获得的噬菌体，从而将其应用于如对皮肤病感染的治疗和预防中。也已发现经口给予根据本发明获得的噬菌体溶菌产物可增加对将来可能的细菌感染的抗性。
在某些情况中，噬菌体治疗可与抗生素治疗结合使用。已显示在细菌感染治疗中使用的多价噬菌体制备物是高效的。在脓毒病的治疗(88％的治愈率)和具有粘稠物阻塞症的患者的感染的治疗中观察到了特别的效力。
附图简述
图1显示对噬菌体S1-S7获得的统计学分析的结果。
图2显示对噬菌体P1-P20获得的统计学分析的结果。
图3显示所选噬菌体株系对于从粘稠物阻塞症患者中分离的属于葡萄球菌属的细菌株系的效率的统计学分析结果。
图4显示所选噬菌体株系对于从粘稠物阻塞症患者中分离的属于假单胞菌属的细菌株系的效率的统计学分析结果。
为对本发明实质有更好的理解，将其在下文通过实施例进行阐明。
实施例1.噬菌体分离葡萄球菌属和假单胞菌属的噬菌体从城市污水中分离。使该污水经过孔大小为0.2-0.4μm的膜滤器，该膜滤器捕获了细菌但允许噬菌体通过。从结果所得的滤液中，加入浓缩的液体培养基，并且将不同的滤液稀释物与新鲜的细菌液体培养物混合(金黄色葡萄球菌或铜绿假单胞菌)。使样品于37℃温育1小时，其后用移液管从每一个样品中吸取0.2ml的悬浮液并用玻璃棒涂于含有琼脂培养基的平板上，然后将平板于37℃温育过夜。如果寻找的噬菌体存在于用含有新鲜细菌培养物的适当的滤液稀释物接种的平板上，那么则观察到细菌的均一(云状的)生长和分离的透明小区域，即所谓的秃斑，该斑点通常含有几百万个噬菌体颗粒。将单一的秃斑连同其周围的琼脂一起用铂环切下，然后转移到新鲜的细菌液体培养基上并进行温育直到培养物清澈。在使其经过孔大小为0.2-0.4μm的膜滤器后，滤液仅含有噬菌体，该膜滤器阻止了残存于裂解物中的任何细菌。获得的噬菌体的不同稀释物对琼脂培养基上各种细菌的接种和光秃斑点的再次分离进行了5次，这使得能够获得噬菌体的纯系。
实施例2.确定金黄色葡萄球菌株系对与之特异的噬菌体微生物的易感性。
易感性用由Wahl描述的培养基进行确定。将具有该培养基的平板在37℃的温度干燥30分钟、用通过振荡而混合新鲜的4-小时的金黄色葡萄球菌液体培养物覆盖，将过量的悬浮液用移液管去除并再次于37℃干燥30分钟。在一个分为6个部分的平板上，要研究的株系的易感性对6种不同但特定的噬菌体进行测定。将1滴1/10的噬菌体稀释物置于每一个部分的表面。将平板在温箱中于37℃温育约4小时，其后将其置于冰箱中直到第二天。如果菌株对该确定的噬菌体具有高的易感性，那么在噬菌体引入到用细菌生长物均匀覆盖的培养基中的位置即可见到透明的区域，这是细菌细胞的完全破坏(裂解作用)的结果。株系的较低的易感性由出现单一的光秃斑点来证实。
实施例3.确定假单胞菌属株系对与之特异的噬菌体微生物的易感性。
确定敏感性的方法与在实施例2中描述的相似。尽管在该情况中，原培养基是有磷酸盐缓冲液补充的琼脂，且沉积了噬菌体的平板的温育时间为5小时。
实施例4.选择对于葡萄球菌属和假单胞菌属具有最广的活性谱的噬菌体——获得复价噬菌体株系。
噬菌体根据在实施例1中描述的方法分离。确定了从全波兰分离的各种葡萄球菌属菌种的845个药物抗性临床株系对来自特异于葡萄球菌属的噬菌体集合的的噬菌体的易感性，以及不同假单胞菌属菌种的880个药物抗性临床株系对来自特异于假单胞菌属的噬菌体集合的噬菌体的易感性。
对两组株系对各噬菌体的易感性的分析允许选择具有出乎意料的高活性和广谱裂解作用的噬菌体。这些噬菌体在表1和2中给出。
表1.保藏于波兰弗罗茨瓦夫的波兰微生物保藏所(PolishCollection of Microorganisms)(PCM)的对葡萄球菌属有高裂解活性的噬菌体。
表2.保藏于波兰弗罗茨瓦夫的波兰微生物保藏所(PCM)的对假单胞菌属有高裂解活性的噬菌体。
对噬菌体S1-S7和P1-P20的结果进行了统计学分析以确定对于具体的噬菌体所获得的p值对于各个属的细菌的普通群体是否可认为是正确的。该结果在图1和2中给出。对大多数噬菌体计算的n值比研究样品的细菌株系的数目小，这提示裂解谱的范围至少对于在波兰出现的临床株系是正确的。考虑到这些结果，可认为在表1和表2中列出的噬菌体是关于本发明的多价噬菌体。
出乎意料的是，在对葡萄球菌属的噬菌体的情况中，7种活性最大的噬菌体中的3种即S1、S2和S4或S5显示对95％的所研究的葡萄球菌属株系的裂解活性。同样地，对假单胞菌属选择的21种噬菌体中的3种即P7、P20和P6显示对87％的所研究的葡萄球菌属株系的裂解活性。
人们也可将遭受粘稠物阻塞症的患者认为是“出现细菌感染的给定区域”，如在本发明的本质中所定义的。在对该组患者中出现的病原体的研究中，使用了137个细菌株系，其中的84个鉴定为假单胞菌属的而53个鉴定为金黄色葡萄球菌。所有的株系均自全波兰的粘稠物阻塞症患者的材料中分离。分析所研究的假单胞菌属和金黄色葡萄球菌株系对噬菌体的易感性使得能够选择具有最高裂解活性和最宽的作用范围的噬菌体。出乎意料的是，在假单胞菌属的噬菌体的情况中，21种活性噬菌体中的3种，即F/00010、F/00013(原文有误，译者)和F/00018显示对75％的假单胞菌属株系的裂解活性(关于成功概率参见图3，其中噬菌体3＝F/00010、噬菌体6＝F/00013且噬菌体11＝F/00018)。在对葡萄球菌属选择的噬菌体中，3种即F/00002、F/00004和F/00007显示对98％的葡萄球菌属株系的裂解活性(关于成功概率参见图4，其中噬菌体1＝F/00002、噬菌体3＝F/00004且噬菌体4＝F/00007)。
实施例5.使对葡萄球菌属的噬菌体和对革兰氏阴性杆菌的噬菌体复制，从而获得噬菌体溶菌产物。
将约5％的噬菌体溶菌产物添加到新鲜的4-小时的金黄色葡萄球菌培养物中(对数期)，该培养物培养于补充了葡萄糖的液体培养基中。通过振荡使试管或瓶子混合并于37℃温育3小时。在出现清澈的培养物(裂解)之后，使样品冷藏至下一日。使裂解物通过孔大小为0.2-0.4μm的膜滤器以去除任何残存的未裂解的细菌细胞。活的噬菌体颗粒的浓度通过Gratia的双层方法确定。将不添加噬菌体溶菌产物的温育的培养物用作对照。
复制对革兰氏阴性杆菌(假单胞菌属)的噬菌体。将蛋白胨水用于复制假单胞菌属的噬菌体。将5％的噬菌体溶菌产物添加到新鲜的4-小时的铜绿假单胞菌培养物中，通过振荡使试管或瓶子混合并于37℃温育5小时。在培养物清澈之后，使样品在冰箱中存放至下一日，其后使其通过孔大小如上的膜滤器进行过滤。溶菌液中活的噬菌体颗粒的数目用Gratia的方法确定。
无菌的液体噬菌体溶菌产物显示持久的裂解活性。当冷藏时，它们可保持其活力达几年，一些情况下超过10年。
实施例6.多价噬菌体制剂的生产及应用。
由实施例5中描述的方法获得的源自实施例4中所选的具有广谱裂解活性的噬菌体的无菌液体溶菌物或其浓缩的或进一步纯化的形式(参加下面的实施例)用于多价噬菌体药物的制备中。
针对葡萄球菌属的示例性多价噬菌体制剂是含有不同量的噬菌体S1、S2和S4或S5的裂解物或这些裂解物的衍生物的混合物。对于粘稠物阻塞症患者的葡萄球菌属引起的感染，应该使用来自噬菌体F/00002、F/00004和F/00007的制剂。
针对假单胞菌属的示例性多价噬菌体制剂是含有不同量的噬菌体P7、P20和P6的裂解物或这些裂解物的衍生物的混合物。对于粘稠物阻塞症患者的假单胞菌属引起的感染，应该使用来自噬菌体F/00010、F/00013和F/00018的制剂。
该药物在饭前30分钟和预先中和胃液(如用Gel Aluminiphosphorici、纯化的苏打或VIchy水)之后以相当于10ml的裂解物的量每日3次经口服给予。当直接向伤口应用该制剂时，应该在24小时内进行3次。通过局部应用，伤口不应该用消毒剂清洁，这是因为这样将导致噬菌体的失活。如果需要，伤口可用无菌的培养基或0.9％的NaCl生理溶液进行清洁。
在某些情况中，噬菌体治疗可与抗生素治疗结合应用。在细菌感染的治疗中所用的多价噬菌体制剂已显示高的效力。在脓毒病的治疗(88％的治愈率)和粘稠物阻塞症患者的感染的治疗中观察到了特别的效力。
实施例7.噬菌体制备物的进一步纯化，从含有噬菌体的混合物中去除内毒素。
在几个医学应用中或者归因于给予的方法(如静脉内地)，需要高纯度的噬菌体制备物，特别不含细菌内毒素。根据本发明的方法，利用噬菌体对含有纤维素的基质，或最适地其酯化的衍生物的亲和力。在本实施例中使用一种可购得的纤维素的硫酸衍生物，该衍生物特征在于低水平的酯化(8μmole/ml凝胶)。将该基质用于从含有噬菌体的混合物中去除内毒素。在浸洗基质和去除内毒素之后，纯化的下一步是洗脱吸附的噬菌体。
将1ml含有纤维素的硫酸衍生物的基质加入到无菌层析柱中。通过用以70％乙醇浸泡的玻璃丝封住柱子的底部而防止基质从柱子中的流出。
制备了下面的洗脱缓冲液缓冲液I0.01M磷酸盐缓冲液，pH 7.6；缓冲液II含有1M氯化钠的0.01M磷酸盐缓冲液，pH 7.6。
进入洗脱液的组合物的盐于145℃烘焙1小时。
溶液用蒸馏的无热原的(apyrogenic)水制备。
层析前，用1ml基质填充层析柱，该层析柱用5ml缓冲液I冲洗然后用5ml缓冲液II冲洗。
将柱子用10ml缓冲液I冲洗以备用于实际的层析。内毒素的去除是通过用在Sepharose 4B上对浓缩的噬菌体溶菌产物Ps PCMF/00018进行分子筛程序获得的大分子级分实现。
将0.2ml的噬菌体混合物转移到含有1ml纤维素的硫酸衍生物基质的层析柱中。获取0.2ml体积的级分。第一个级分用3ml缓冲液I洗脱。在这些情况下，未结合的内毒素从柱子中流出。第二个级分用缓冲液II洗脱。级分II含有纯化的噬菌体。
级分中内毒素单位的结果加到含有纤维素的硫酸衍生物基质上的层析的材料含有2500单位的内毒素/ml。用缓冲液I洗脱的级分含有600-1000单位的内毒素/ml而用缓冲液II洗脱的级分含无内毒素的噬菌体(约1单位/ml)。
噬菌体制备物中内毒素的含量用美国Charleston的CharlesRiver Endosafe公司的凝胶方法确定。
以下布达佩斯条约对为专利程序进行的微生物保藏的国际认证各个菌株的保藏证明和存活证明BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNTTION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
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1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
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BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOP THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or dnte of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPFST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date ofthe new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has beenrequested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date ofthe new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DF POSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the infomation has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status ofinternational depositary authority isconverted into a deposit under the Budapest Treaty， such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALPECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority ws acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of th test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATFMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last pege)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international deposiary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date ofthe transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the foliowing page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PUR POSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositay authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DE POSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international deposita ry authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a dcposit under the Budapest Treaty，such date is thc date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acqulred；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNTTION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negativeForrr BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut of international depositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or atansfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PUR POSE OF PATENT PROCEDUREINTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSITissued pursuant to Rule 7.1 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified at the bottom of this page
1Where Rule 6.4(d)applies，such date is the date on which the statut ofinternationaldepositary authority was acquired；where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority isconverted into a deposit under the Budapest Treaty，such date is the date on which the microorganism was received by theinternational depositary authority.
Form BP/4(sole page)
BUDAPEST TREATY ON THE INTERNATIONALRECOGNITION OF THE DEPOSIT OF MICROORGANISMSFOR THE PURPOSES OF PATENT PROCEDUREINTERNATIONAL FORM
VIABILITY STATEMENTissued pursuant to Rule 10.2 by theINTERNATIONAL DEPOSITARY AUTHORITYidentified on the following page
1Indicate the date of the original deposit or，where a new deposit or a transfer has been made，the most recent relevant date(date of the new deposit or date of the transfer).
2In the cases referred to in Rule 10.2(a)(ii)and(iii)，refer to the most recent viability test.
3Mark with a cross the applicable box.
From BP/9(first page)
4Fill in if the information has been requested and if the results of the test were negative.
Form BP/9(second and last page)
权利要求
1.获得复价噬菌体株系的方法，该方法特征在于a)积聚足够数目(n)的在给定区域随机出现的特定物种的病原体细菌株系，在此分离的细菌株系最适为抗药性株，b)积聚足够数目的对至少一种给定种的细菌株系特异的噬菌体株系，c)确定所积聚的噬菌体株系对每种所积聚的细菌株系的裂解活性，然后估计p值，该值为由给定的噬菌体裂解的细菌株系的数目与所有积聚的细菌株系的数目的比例。d)从结果所得的p值，检验n是否满足条件n≥pqd2z1-α2]]>其中q＝1-pd是不大于0.1的常数，最适地不大于10％pz1-α是依赖于置信因子1-α的正态分布的随机变量，该置信因子不小于0.95e)选择满足d)中的标准且具有不小于2/n的p值，最适地不小于0.5的噬菌体株系。
2.权利要求1的方法，其中在步骤b)中噬菌体株系从源自环境的样品中分离，通过使该样品经过孔大小为0.2-0.4μm的膜滤器、向获得的滤液中添加培养基并使其与确定属的细菌液体培养物混合、在约37℃的温度温育约1小时、取出一部分悬浮液并将其应用于有固体培养基的平板、于约37℃温育2～24小时、分离单个秃斑周围的培养基样品、将其转移到该确定属的细菌的液体培养物中并进行温育直到培养物发亮，然后通过使溶菌产物经过孔大小为0.2-0.4μm的膜滤器而获得噬菌体产物，在此优选固体培养基的接种和秃斑的再次分离操作重复5次。
3.权利要求1或2的方法，其中在步骤c)中所研究的细菌株系接种到各个固体培养基上，在该培养基上转移有一部分在步骤b)中获得的噬菌体制备物，于约37℃的温度温育约4小时，其后将其于约4℃的温度放置约2～24小时，其后噬菌体株系的裂解活性通过至少单个秃斑的出现而证实。
4.权利要求1的方法，其中病原体细菌株系是葡萄球菌属或假单胞菌属的。
5.权利要求1或2的方法，该方法包含对溶菌产物的进一步纯化，尤其是除内毒素，在此使含有噬菌体的混合物与含有纤维素或其至少部分酯化的衍生物相接触，然后用去除杂质，尤其是内毒素，的溶液洗涤，其后将纯化的噬菌体洗脱。
6.权利要求5的方法，其中用水、非解离性物质的溶液或浓度不大于0.1M的盐溶液，尤其是缓冲的盐溶液进行内毒素的洗脱。
7.权利要求5的方法，其中用非解离性物质的溶液，或任何缓冲液，或浓度大于0.05M的盐溶液，尤其是缓冲的盐溶液进行噬菌体的洗脱。
8.权利要求5的方法，其中内毒素和噬菌体的洗脱在-25℃～100℃之间的温度中进行。
9.权利要求5的方法，其中内毒素和噬菌体的洗脱用含有机溶剂的水性盐溶液进行。
10.权利要求9的方法，其中有机溶剂选自二甲基亚砜、二甲基甲酰胺、异丙醇和丙酮。
11.权利要求5的方法，其中用以有机或无机酸部分酯化的纤维素作为基质。
12.权利要求5的方法，其中使用以乙酸、硝酸、硫酸或磷酸部分酯化的纤维素。
13.权利要求5的方法，其特征在于使用其中0.01％～5％的葡萄糖分子，尤其是0.25％～1％的葡萄糖分子被酯化的纤维素。
14.一种治疗或预防由细菌病原体引起的感染的药物，该药物含有活性剂和可能的药物学可接纳的载体，其特征在于该活性剂包含对细菌特异的复价噬菌体株系，其属于根据权利要求1-13的方法中获得的类型。
15.权利要求14的药物，其中对葡萄球菌属细菌特异的所述复价噬菌体株系为至少一种选自下述的噬菌体株系S1(PCM F/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，尤其是噬菌体S1、S2和S4或S5。
16.权利要求14的药物，其特征在于它用于治疗或预防粘稠物阻塞症患者中产生的感染，以及作为对葡萄球菌属细菌特异的复价噬菌体株系，它含有至少一种选自F/00002、F/00004和F/00007的噬菌体株系。
17.权利要求14的药物，其特征在于作为对假单胞菌属细菌特异的复价噬菌体株系，它含有至少一种选自下述的噬菌体株系P1(PCMF/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，尤其是噬菌体P7、P20和P6。
18.根据权利要求14的药物，其特征在于它用于治疗或预防粘稠物阻塞症患者中产生的感染，以及作为对假单胞菌属细菌特异的复价噬菌体株系，它含有至少一种选自F/00010、F/00013和F/00018的噬菌体株系。
19.由根据权利要求1-13的方法获得的复价噬菌体株系在生产用于治疗或预防由病原体细菌引起的感染的药物中的用途。
20.权利要求19的用途，其中为生产治疗或预防由葡萄球菌属细菌导致的感染的药物，应用至少一种选自S1(PCM F/00001)、S2(PCMF/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)的噬菌体株系，尤其是噬菌体S1、S2和S4或S5。
21.权利要求19的用途，其中为生产治疗或预防粘稠物阻塞症患者中由葡萄球菌属细菌导致的感染的药物，使用至少一种选自F/00002、F/00004和F/00007的噬菌体株系。
22.权利要求19的用途，其中为生产治疗或预防由假单胞菌属细菌引起的感染的药物，应用至少一种选自P1(PCM F/00008)、P2(PCMF/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)的噬菌体株系，尤其是噬菌体P7、P20和P6。
23.权利要求19的用途，其中为生产治疗或预防粘稠物阻塞症患者中由假单胞菌属细菌引起的感染的药物，使用至少一种选自F/00010、F/00013和F/00018的噬菌体株系。
24.对葡萄球菌属细菌特异的复价噬菌体株系选自S1(PCMF/00001)、S2(PCM F/00002)、S3(PCM F/00003)、S4(PCM F/00004)、S5(PCM F/00006)、S6(PCM F/00006)和S7(PCM F/00007)，尤其是选自S1、S2、S4和S5。
25.对假单胞菌属细菌特异的复价噬菌体株系选自P1(PCMF/00008)、P2(PCM F/00009)、P3(PCM F/00010)、P4(PCM F/00011)、P5(PCM F/00012)、P6(PCM F/00013)、P7(PCM F/00014)、P8(PCM F/00015)、P9(PCM F/00016)、P10(PCM F/00017)、P11(PCM F/00018)、P12(PCM F/00019)、P13(PCM F/00020)、P14(PCM F/00021)、P15(PCM F/00022)、P16(PCM F/00023)、P17(PCM F/00024)、P18(PCM F/00025)、P19(PCM F/00026)和P20(PCM F/00027)，尤其是选自P7、P20和P6。
全文摘要
提供了噬菌体的复价株系、获得这些株系的方法及其在治疗细菌感染中的应用，特别是针对那些抗药性菌株，尤其是发生于粘稠物阻塞症患者中的。
文档编号A61P11/00GK1541101SQ02815833
公开日2004年10月27日 申请日期2002年7月18日 优先权日2001年7月18日
发明者B·威必尔达布罗斯卡, B 威必尔达布罗斯卡, M·姆尔兹克, 瓤, A·格斯克, 箍, J·伯拉特恩斯克, 囟魉箍, M·鲁萨克－泽拉首斯卡, 泽拉首斯卡, D·斯皮尔, ざ 申请人:波兰科学院免疫和实验治疗研究所