CN1313145C青蒿素相关性内过氧化物与携带铁的 蛋白质之间的共价缀合物及其使用方法|CN1313145C Covalent conjugates between artemisinin-related peroxides and iron-carrying proteins and their methods of use.
Entry posted by Roger in Artemisinin Patent｜青蒿素专利
53 views
CN1313145C青蒿素相关性内过氧化物与携带铁的 蛋白质之间的共价缀合物及其使用方法
CN1313145C Covalent conjugates between artemisinin-related peroxides and iron-carrying proteins and their methods of use.
青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物及其使用方法
本发明在一个方面中提供了青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。在某些实施方案中，该共价缀合物包括阿替林酯和全运铁蛋白。本发明在另一个方面中提供了给予本发明的共价缀合物以治疗癌症和由可结合携带铁的蛋白质的病原体所导致的感染的方法。
青蒿素相关性内过氧化物与携带铁的 蛋白质之间的共价缀合物及其使用方法
相关申请的交叉引用
本申请要求按照35U.S.C.119于2002年6月6日提交的美国临时申请号60/386,928的利益。
发明领域
本发明涉及青蒿素(artemisinin)相关性内过氧化物(endoperoxide)与携带铁的蛋白质之间的共价缀合物和这些缀合物在治疗癌症和由可结合携带铁的蛋白质的病原体导致的感染中的应用。
发明背景
青蒿素是从植物黄花蒿(Artemisia annua L)中分离的倍半萜内酯类，该植物的提取物已经用于治疗疟疾至少1600年。青蒿素分子含有内过氧化桥，它与铁原子反应形成自由基。青蒿素的抗疟作用是因其与寄生虫内的血红素发生反应形成自由基而使细胞死亡所导致的。癌细胞具有显著高于正常细胞的铁流入。因此，已经证实青蒿素和青蒿素类似物对建立的肿瘤和肿瘤细胞系具有细胞毒性(例如，参见Woerdenbag等(1993)《天然产物杂志》(J.Nat.Prod.)56(6)：849-56；Lai &Singh(1995)《癌症通讯》(Cancer Lett.)91：41-6；Efferth等(2001)《国际肿瘤学杂志》(Int.J.Oncol.)18：767-73；Li等(2001)《生物有机药物化学通讯》(Bioorg.Med.Chem.Lett.)11：5-8；Singh& Lai(2001)《生命科学》(Life Sci.)70：49-56；Efferth等(2002)《生物化学与药理学》(Biochem.Pharmacol.)64：617-23；Efferth等(2002)《血细胞、分子和疾病》(Blood Cells，Molecules & Diseases)28(2)：160-8；Sadava等(2002)《癌症通讯》(Cancer Lett.)179：151-6)。
已经描述了青蒿素的许多类似物和其它具有生物活性的含有内过氧化桥(endoperoxide bridge)的化合物(例如，参见美国专利US5,180,840；美国专利US5,216,175；美国专利US5,225,427；Cumming等(1998)《药物化学杂志》(J Med.Chem.)41(6)：952-64；Posner等(1999)《药物化学杂志》(J Med.Chem.)42：300-4；Li等(2001)《生物有机药物化学通讯》(Bioorg.Med.Chem.Lett.)11：5-8；Wu等(2001)《欧洲药物化学杂志》(Eur.J.Med.Chem.)36：469-79；Posner等(2003)《药物化学杂志》(J Med.Chem.)46：1060-5)。已经用于治疗疟疾的青蒿素类似物包括二氢青蒿素、蒿甲醚、青蒿琥酯、蒿乙醚、二氢青蒿素丙基碳酸酯和artelinic acid。
青蒿素是相对安全的药物，甚至在高剂量下也只有极少且低微的副作用。已经将6天70mg/kg/天的口服剂量用于治疗人疟疾。在用青蒿琥酯治疗癌症患者后，没有观察到明显的不良副作用(口服剂量50mg/天；肌内剂量60mg/天；为期9个月)(Singh &Verma(2002)《肿瘤学学报》(Arch.Oncol.)10(4)：279-80)。还已将青蒿素和青蒿素类似物用于治疗皮肤病，诸如银屑病、起泡性皮肤病、病毒疣、上皮软疣(mulluscum contagiosum)和痔(例如，参见美国专利US4,978,676；美国专利US5,219,880)。还将青蒿素和青蒿素类似物用于预防疟疾。
已经证实给予铁盐或携带铁的蛋白质全运铁蛋白(holotransferrin)增加了癌细胞和植入的肿瘤对青蒿素及其类似物的敏感性(Lai & Singh(1995)《癌症通讯》(Cancer Lett.)91：41-46；Moore等(1995)《癌症通讯》(Cancer Lett.)98：83-7；Singh & Lai(2001)《生命科学》(Life Sci.)70：49-56；Sadava等(2002)《癌症通讯》(Cancer Lett.)1179：151-6)。
还证实某些病原体通过结合携带铁的宿主蛋白质而获得铁。例如，细菌性脑膜炎的病原体脑膜炎奈瑟氏球菌表达携带铁的化合物的细胞表面受体，诸如运铁蛋白(transferrin)和乳铁蛋白(lactoferrin)(Evans & Oakhill(2002)《生物化学协会学报》(Biochem.Soc.Trans.)30(4)：705-7)。目前，尚无疫苗可用于脑膜炎奈瑟氏球菌B型菌株即西方世界中最流行的菌株。此外，已经证实人胃炎、胃和十二指肠溃疡和腺癌的病原体幽门螺杆菌(Helicobacter pylori)可通过结合人乳铁蛋白而获得铁(Husson等(1993)《感染性免疫》(Infect.Immun.)61(6)：2694-7)。
本领域中仍需要有效力增加的青蒿素组合物，以用于治疗癌症和由可结合携带铁的宿主蛋白的病原体导致的疾病。还需要用于治疗癌症和由可通过摄入携带铁的宿主蛋白而获得铁的病原体所导致的感染的方法。本发明满足了这些需求。
发明概述
本发明在一个方面中提供了新化合物和包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物的组合物。青蒿素相关性内过氧化物可以通过酰肼部分、肼部分或氨氧基部分与携带铁的化合物连接。在某些实施方案中，所述的青蒿素相关性内过氧化物具有如下结构：
其中n＝1-3，m＝0-3，Ar＝芳基，且Y＝-(C＝O)NH-、-NH-或-O-。有代表性的存在于本发明共价缀合物中的青蒿素相关性内过氧化物包括阿替林酯(artelinate)和二氢青蒿素。
有代表性的存在于本发明共价缀合物中的携带铁的蛋白质包括蛋白质中的运铁蛋白家族、与中性明胶酶相关的脂质运载蛋白(neutralgelatinaseassociated lipocalin，NGAL)、血红素蛋白和其它携带铁的蛋白质。因此，所述的共价缀合物可以包括：例如阿替林酯与全运铁蛋白、阿替林酯与全乳铁蛋白(hololactoferrin)或阿替林酯与血红蛋白的缀合物。本发明还提供了包含本发明共价缀合物和药物上可接受的载体的组合物。本发明的共价缀合物可用于治疗癌症和由可结合携带铁的蛋白质的病原体所导致的感染。
本发明在另一个方面中提供了对需要的受试者给予本发明的共价缀合物的方法。在本发明该方面中适合于给药的典型共价缀合物包括：例如阿替林酯与全运铁蛋白之间、阿替林酯与全乳铁蛋白之间和阿替林酯与血红蛋白之间的共价缀合物。
在某些实施方案中，本发明提供了治疗癌症的方法，通过对有此需要的人或动物受试者给予有效量的组合物来进行，该组合物包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。可以通过局部或全身性给予所述的共价缀合物，或可以将它们直接注入肿瘤。可以单独或与一种或多种其它治疗剂一起给予所述的共价缀合物。例如，可以与例如可通过增加缀合物中携带铁的蛋白质的细胞表面受体数来增加向细胞内的铁转运的活性剂一起给予所述的共价缀合物。
本发明还提供了治疗由可结合携带铁的蛋白质的病原体所导致的感染的方法，通过对有此需要的人或动物受试者给予有效量的组合物来进行，该组合物包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。在一个实施方案中，所述的病原体包括幽门螺杆菌，其中携带铁的蛋白质包括人乳铁蛋白，且其中所述青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。在其它典型的实施方案中，所述的病原体包括脑膜炎奈瑟氏球菌，其中所述携带铁的蛋白质包括人运铁蛋白，且其中所述青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。
在另外的实施方案中提供了治疗幽门螺杆菌感染的方法，通过对有此需要的人或动物受试者给予有效量的组合物来进行，该组合物包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。
优选实施方案的详细描述
本发明在一个方面中提供了包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物的组合物。本文所用的术语″共价缀合物″指的是其中青蒿素相关性内过氧化物与携带铁的蛋白质共价连接在一起的化合物。术语″青蒿素相关性内过氧化物″指的是含有内过氧桥(endoperoxide bridge)的化合物，它可与铁原子反应而形成自由基，导致细胞死亡。青蒿素相关性内过氧化物化合物还可以在有铜和锰存在的情况下形成自由基。有代表性的青蒿素相关性内过氧化物如本文所述，不过，显然其它内过氧化物也可用于该目的。
一般来说，青蒿素相关性内过氧化物选自由倍半萜内酯类及其醇类、碳酸酯类、酯类、醚类和磺酸酯类、阿替夫林、1，2，4-三烷类和1，2，4，5-四烷类组成的组。青蒿素相关性内过氧化物可具有如下结构：
其中R为 或 ，R1为氢、羟基、烷基或具有下列通式：
-O-R2， 或
其中R2为烷基或芳基且n为1-6；或者其药学上可接受的盐。本文所用的术语″烷基″指的是含有1-6个碳原子、优选1-4个碳原子的低级烷基。本发明的烷基可以是直链或支链基团。术语″芳基″指的是含有4-14个主链碳或杂原子的单环和多环芳族基团，包括碳环芳基和杂环芳基。碳环芳基是其中所有的环原子均为碳的芳基。杂环芳基含有1-4个杂原子作为环原子，剩余的环原子为碳。有代表性的芳基包括例如苯基和苄基。药物上可接受的盐包括碱金属或碱土金属盐，优选钠或钾盐。
例如，本发明的青蒿素相关性内过氧化物包括：青蒿素，其中R为
二氢青蒿素(R1＝-OH)；artesunicacid(R1＝-OCO(CH2)2CO2H)；以及青蒿琥酯；蒿甲醚(R1＝-OCH3)；和蒿乙醚(R1＝-OC2H5)。本发明其它有代表性的内过氧化物化合物包括artelinicacid、二氢青蒿素丙基碳酸酯、阿替夫林(Ro.42-1611)及其类似物(Biirgen等(1994)Sixth Int.Cong.Infect.Dis.Abst.427，p.152，Prague)、1，2，4-三烷类(Peters等(1993)Ann.Trop.Med.Parasit.87(1)：9-16)和1，2，4，5-四烷类(Vennerstrom等(1992)《药物化学杂志》(J Med Chem.)35(16)：3023-3027)。青蒿素的其它合适的结构类似物描述在例如美国专利US5,216,175和US5,180,840、Cumming等(1998)《药物化学杂志》(J Med Chem.)41(6)：952-64和PCT专利申请WO 97/01548、WO 99/33461和WO 00/42046中。
青蒿素相关性内过氧化物的来源可以为天然(例如分离自植物)、合成或半合成的。例如，可以通过在微生物宿主中表达相关合成途径中的酶来产生自由基发生剂(例如，参见Martin等(2003)《天然生物技术》(Nature Biotechnol.)，在线公开：2003年6月1日，doi：10.1038/nbt833)。
本文所用的术语″携带铁的蛋白质″指的是适合于将铁转运至细胞的蛋白质。本发明共价缀合物中携带铁的蛋白质可以为哺乳动物蛋白，例如人蛋白质。典型的携带铁的蛋白质包括蛋白质中的运铁蛋白家族、与中性明胶酶相关的脂质运载蛋白(neutral gelatinase-associatedlipocalin，NGAL)、血红素蛋白和其它结合铁的蛋白质。铁在细胞生长中起着关键的作用。蛋白质中的运铁蛋白家族包括运铁蛋白、乳铁蛋白、卵运铁蛋白(ovotransferrin)和黑运铁蛋白(melanotransferrin)(Aisen & Harris(1989)在《铁载体和铁蛋白》(Iron Carriers and IronProteins)中所述(ed.Loehr，VCH，New York)273-320页；Baker(1994)《高级无机化学》(Adv.Inorg.Chem.)41：389-463)。所有这些蛋白质均为结构相关性的单链糖蛋白，含有670-690个氨基酸。运铁蛋白将铁和血红素从循环中转运入细胞。运铁蛋白的载铁形式(全运铁蛋白)结合细胞表面上的运铁蛋白受体，并通过受体介导的胞吞作用被摄入细胞内，在此释放铁。乳铁蛋白在从人乳中吸收铁的过程中具有关键作用。乳铁蛋白的另一种功能是阻止铁接触传染原。不同于运铁蛋白，乳铁蛋白可以在低pH下稳定地结合铁并吸收入肠中。
NGAL是与运铁蛋白家族无关的携带铁的蛋白质，推测它可以将铁递送至分化着的上皮细胞处(Kaplan(2002)《细胞》(Cell)111：603-6)。血红素蛋白为诸如血红蛋白、肌红蛋白、血红素结合蛋白、细胞色素、过氧化氢酶和过氧化物酶这类携带血红素作为辅基的蛋白质。例如，血红素结合蛋白是60kDa的血清糖蛋白，它以极高的亲合力从血流中螯合血红素，并将其转运至肝(Baker等(2003)《美国国家科学院学报》(Proc.Natl.Acad Sci.US.A.)100(7)：3579-83)。
在本发明的共价缀合物中，青蒿素相关性内过氧化物与携带铁的蛋白质连接，连接的方式可以是能保留携带铁的蛋白质的活性和青蒿素相关性内过氧化物的活性二者的任何方式。例如，如实施例1中所述，可以通过下列步骤将青蒿素相关性内过氧化物与糖基化的携带铁的蛋白质连接：首先制备青蒿素相关性内过氧化物的酰肼衍生物，然后使其与携带铁的蛋白质上的一个或多个氧化多糖基团连接。为了制备青蒿素相关性内过氧化物的酰肼衍生物，可以首先通过下列步骤形成青蒿素相关性内过氧化物的酯：将1-羟基苯并三唑(HOBt)加入到青蒿素相关性内过氧化物中，随后添加乙基二甲基乙基碳二亚胺。然后使HOBt酯与肼反应生成酰肼衍生物。随后使该酰肼衍生物与携带铁的蛋白质共价连接，其中已用氧化剂(诸如高碘酸钠)氧化了多糖基团。任何含有-(C＝O)NH-基团的青蒿素相关性内过氧化物均可以用于制备可与运铁蛋白或其它携带铁的糖蛋白连接的酰肼衍生物(例如，参见Gumming等(1998)《药物化学杂志》(J Med.Chem.)41(6)：952-64)。
还可以通过首先制备青蒿素相关性内过氧化物的肼或氨氧基衍生物而将青蒿素相关性内过氧化物与糖基化的携带铁的蛋白质连接起来。为了制备青蒿素相关性内过氧化物的肼或氨氧基衍生物，首先通过下列步骤形成青蒿素相关性内过氧化物的卤化物：将卤代醇加入到青蒿素相关性内过氧化物中，随后添加三氟化硼合乙醚。然后可以使所述的卤化物或青蒿素相关性内过氧化物与肼或羟基胺反应而分别生成肼或氨氧基衍生物。卤代基团可以被对甲苯磺酰基或甲磺酰基取代而用于反应。然后可以使肼或氨氧基衍生物与携带铁的蛋白质共价连接，其中已用氧化剂(诸如高碘酸钠)氧化了多糖基团。任何含有-(C＝O)NH-、-NH-或-O-基团的青蒿素相关性内过氧化物均可以用于制备可与运铁蛋白或其它携带铁的糖蛋白连接的肼衍生物或氨氧基衍生物。
适合于按照这种方式连接的青蒿素相关性内过氧化物包括可以从中制备酰肼、肼或氨氧基衍生物而不破坏内过氧桥活性的化合物。一般来说，通过间隔基(诸如烃链)将内过氧桥与酰肼、肼或氨氧基隔开。还可以将醚类、酯类、酰胺类、硫化物和二硫化物用作间隔基。例如，可以用苯环将内过氧化桥与如阿替林酯上的酰肼、肼或氨氧基隔离开。因此，适用于本发明的青蒿素相关性内过氧化物包括、但不限于如下结构的内过氧化物：
其中n＝1-3，m＝0-3，Ar＝芳基，例如苯基、萘基、芘基、吡啶基或嘧啶基，且Y＝-(C＝O)NH-、-NH-或-O-。有代表性的存在于本发明共价缀合物中的青蒿素相关性内过氧化物包括阿替林酯和二氢青蒿素。
用于将青蒿素相关性内过氧化物与携带铁的糖蛋白相连接的方法包括硼酸酯介导的与携带铁的糖蛋白上的糖残基的连键(例如，参见美国专利US5,919,708)。因此，含有硼酸酯基的青蒿素相关性内过氧化物将与蛋白质表面上的糖残基的1，2-二醇部分反应。
就非糖基化的携带铁的蛋白质而言，可以如实施例2中所述通过使用氨基酸侧链使青蒿素相关性内过氧化物与该蛋白质连接。例如，青蒿琥酯和阿替林酯带有可以用N-羟基琥珀酰亚胺(例如，参见Lewis等(1994)《生物缀合物化学》(Bioconj.Chem.)5(6)：655-76)和水溶性碳化二亚胺试剂、诸如N-乙基(etheyl)-N′-二甲氨基乙基碳化二亚胺活化成活性酯的游离羧酸基。可以将这种酯与非糖基化的携带铁的蛋白质的水溶液混合而将青蒿素相关性内过氧化物与该蛋白质表面上的Lys残基连接。
可以通过本领域中的标准方法，例如通过使用凝胶过滤色谱法、离子交换和反相或疏水相互作用高压液相色谱法(HPLC)纯化所述的共价缀合物。可以通过使用本领域中的标准方法，例如离子喷雾质谱法，测定与一个分子的携带铁的蛋白质相结合的青蒿素相关性内过氧化物分子的数量。共价缀合物中青蒿素相关性内过氧化物与携带铁的蛋白质的比例取决于所用的携带铁的蛋白质和形成缀合物的方法。例如，使用实施例1中所述的方法获得每分子的全运铁蛋白含有1-10个分子的青蒿素相关性内过氧化物的共价缀合物。
本发明还提供了包括本发明共价缀合物的组合物。在某些实施方案中，如实施例1中所述，本发明的组合物包括阿替林酯与人全运铁蛋白之间的共价缀合物。在其它实施方案中，所述的组合物包括阿替林酯与人全乳铁蛋白之间的共价缀合物。
本发明的共价缀合物可用于治疗癌症。由于大部分癌细胞快速分裂，所以它们具有比正常细胞更高的铁摄取速率，且表达的细胞表面运铁蛋白受体的浓度也高于正常细胞。已经证实给予铁盐或全运铁蛋白增加了癌细胞对青蒿素及其类似物的敏感性(Moore等(1995)《癌症通讯》(Cancer Lett.)98：83-7；Singh & Lai(2001)《生命科学》(LifeSci.)70：49-56；Sadava等(2002)《癌症通讯》(Cancer Lett.)1179：151-6)。因此，通过将青蒿素相关性内过氧化物与携带铁的蛋白质、诸如全运铁蛋白共价连接，可增加青蒿素相关性内过氧化物对癌症的功效和选择性，因为内过氧化物和铁同时被转运至或转运入同一细胞。
本发明的共价缀合物还可用于治疗具有共价缀合物中所述携带铁的蛋白质的受体的致病生物体所导致的感染。为了建立成功的感染，病原体必须克服宿主施加的严格铁限制。为了克服这种限制，许多病原体从携带铁的宿主蛋白中获得铁(例如，参见Cornelissen，(2003)Front.Biosci.8：D836-47)。例如，细菌性脑膜炎的病原体脑膜炎奈瑟氏球菌表达携带铁的蛋白质-运铁蛋白和乳铁蛋白的细胞表面受体(Evans&Oakhill(2002)《生物化学协会学报》(Biochem.Soc.Trans.)30(4)：705-7)，人胃炎和消化性溃疡的病原体幽门螺杆菌表达人乳铁蛋白的受体(Husson等(1993)《感染性免疫》(Infect.Immun.)61(6)：2694-7)，而金黄色葡萄球菌表达血红蛋白受体(Mazmanian等(2003)《科学》(Science)299：906-9)。因此，本发明的共价缀合物可用于杀死其中具有共价缀合物中所述携带铁的蛋白质的受体的致病生物体。例如，青蒿素相关性内过氧化物与人运铁蛋白或人全乳铁蛋白之间的共价缀合物可以用于治疗脑膜炎奈瑟氏球菌导致的细菌性脑膜炎或幽门螺杆菌导致的胃炎和消化性溃疡病。
按照本发明的方法，当对癌细胞给药时，青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物具有优于分别给予青蒿素相关性内过氧化物和携带铁的蛋白质所达到的细胞毒性活性。此外，本发明的共价缀合物可有效杀伤带有携带铁的蛋白质的受体的病原体。
本发明在第二个方面中提供了对有此需要的受试者给予组合物的方法，该组合物包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。青蒿素相关性内过氧化物与携带铁的蛋白质的共价缀合物如上所述。例如，如实施例1中所述，某些实施方案提供了阿替林酯与全运铁蛋白的共价缀合物。
这些方法适合于任意动物受试者，诸如人体受试者。例如，需要有包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物的组合物的受试者可以是癌症患者。如上所述，快速增殖的细胞、诸如癌细胞一般具有较高的细胞表面运铁蛋白受体浓度。该方法提供了选择性递送内过氧化物部分和铁的机制，它可以对快速增殖的细胞、诸如癌细胞产生反应。因此，本发明提供了治疗癌症的方法，通过对有此需要的人或动物受试者给予有效量的化合物来进行，该化合物包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物。其它存在细胞过度增殖且可以用本发明的共价缀合物治疗的疾病包括、但不限于再狭窄、自身免疫病、关节炎、移植物排斥、炎症性肠病或医疗操作后诱发的增殖。在某些实施方案中，所述的方法包括给予化合物的步骤，该化合物包括阿替林酯与人全运铁蛋白之间的共价缀合物。
还可以给予包括青蒿素相关性内过氧化物与携带铁的蛋白质之间的共价缀合物的化合物和组合物，以便治疗由表达共价缀合物中所述携带铁的蛋白质的细胞表面受体的病原体所导致的感染。本文所用的术语″治疗病原体导致的感染″指的是抑制病原体生长和/或预防或改善与该感染相关的疾病症状。
具有携带铁的宿主蛋白的受体的典型病原体如上所述，包括表达人运铁蛋白的受体的脑膜炎奈瑟氏球菌和表达人乳铁蛋白的受体的幽门螺旋菌。目前已经证实金黄色葡萄球菌表达血红蛋白受体(Mazmanian等(2003)《科学》(Science)299：906-9)，单核细胞增生利斯特氏菌(Listeria monocytogenes)和炭疽芽孢杆菌(Bacillus anthracis)也表达类似的蛋白质(Cabanes等(2002)《微生物学趋势》(Trends.Microbiol.)10(5)：238-45)。表达携带铁的宿主蛋白的受体的典型病原体实例如表1中所示。一旦该携带铁的蛋白质与病原体表达的受体相结合，则铁或血红素一般从携带铁的蛋白质中释放出来，并被转运入细胞(例如，参见Gray-Owens &Schryyers(1996)《微生物学趋势》(Trends.Microbiol.)4(5)：185-91；Wandersman &Stojiljkovic(2000)《最新微生物学观点》(Curer.Op.Microbiol.)3：215-20)。
表1.人和动物病原体中携带铁的蛋白质的受体
| 病原体 | 宿主 | 疾病 | 受体 |
| 牛莫拉氏菌粘膜炎莫拉氏菌腔隙莫拉氏菌脑膜炎奈瑟氏球菌淋病奈瑟氏球菌放线共生放线杆菌(Actinobacillusactinomycetecomitans)马驹放线杆菌胸膜肺炎放线杆菌(Actinobacilluspleuropneumoniae)羔羊嗜血菌鸟巴斯德氏菌流感嗜血杆菌副鸡嗜血杆菌(Haemophilusparagallinarum)睡眠嗜血杆菌(Haemophilus somnus)副猪嗜血杆菌杜克氏嗜血杆菌 | 牛人人人人人马猪绵羊家禽人家禽牛猪人 | 角膜结膜炎中耳炎角膜结膜炎脑膜炎淋病青少年牙周炎败血症肺炎败血症窦炎脑膜炎，中耳炎传染性鼻炎血栓栓塞性脑膜脑炎格拉瑟病生殖器溃疡病 | 运铁蛋白，乳铁蛋白1运铁蛋白，乳铁蛋白1运铁蛋白，乳铁蛋白1运铁蛋白，乳铁蛋白1血红蛋白1，19运铁蛋白，乳铁蛋白，血红蛋白1，18运铁蛋白1运铁蛋白1运铁蛋白1运铁蛋白1运铁蛋白1运铁蛋白，血红蛋白1，20运铁蛋白1运铁蛋白1运铁蛋白1血红蛋白15 |
| 病原体 | 宿主 | 疾病 | 受体 |
| 溶血巴斯德氏菌多杀巴斯德氏菌金黄色葡萄球菌表皮葡萄球菌(Staphyloccusepidermidis)肺炎链球菌Leishmaniachagasi大肠埃希氏杆菌K88Tritrichonomasfoetus苍白密螺旋体肺炎枝原体百日咳博德特氏菌Trichonomasvaginalis杀鲑气单胞菌幽门螺杆菌小肠结肠炎耶尔森氏菌创伤弧菌牙龈卟啉单胞菌(Porphyromonasgingivalis) | 牛，绵羊，山羊牛人人人人猪牛人人人人鱼人人鳗鱼(eel)人 | 航运热，巴斯德菌病肺炎，败血症菌血症，肺炎，心内膜炎，脓毒性关节炎，骨髓炎，深脓肿，食物中毒心内膜炎，眼内炎，败血症，膀胱炎肺炎，脑膜炎，菌血症，中耳炎利什曼病肠病发生毛滴虫病梅毒肺炎百日咳阴道病疖病胃炎，胃和十二指肠溃疡，胃腺癌，淋巴瘤肠炎食物中毒，败血症，创伤感染牙周病 | 运铁蛋白1运铁蛋白1运铁蛋白血红蛋白2，13运铁蛋白2乳铁蛋白3运铁蛋白，乳铁蛋白4运铁蛋白5乳铁蛋白血红蛋白6乳铁蛋白7乳铁蛋白8乳铁蛋白9乳铁蛋白10运铁蛋白，乳铁蛋白11乳铁蛋白12血红蛋白，肌红蛋白，血红素结合蛋白，过氧化氢酶，清蛋白-血红素14血红蛋白16血红蛋白17 |
1 Gray-Owen & Schryvers(1996)Trends Microbiol.4(5)：185-91
2 Modun et al.(1998)Infect.Immun.66(8)：3591-6
3 Hammerschmidt et al.(1999)Infect.Immun.67(4)：1683-7
4 Britigan et al.(1998)Adv.Exp.Med.Biol.443：135-40
5 Grange et al.(1997)Adv.Exp.Med.Biol.412：357-61
6 Tachezy et al.(1996)Exp.Parasitol.83(2)：216-28
7 Alderete et al.(1988)Genitourin.Med.64(6)：359-63
8 Tryon & Baseman(1987)Microb.Pathog.3(6)：437-43
9 Redhead et al.(1987)J.Gen.Microbiol.133(4)：891-8
10 Peterson & Alderete(1984)J.Exp.Med.160(2)：398-410
11 Chart & Trust(1983)J.Bacteriol.156(2)：758-64
12 Dhaenens et al.(1997)Infect.Immun.65(2)：514-8
13 Mazmanian et al.(2003)Science 299：906-9
14 Bracken et al.(1999)J.Bacteriol.181(19)：6063-72
15 Al-Twafiq et al.(2000)J.Infect.Dis.181(3)：1049-54
16 Fouz et al.(1997)Microbiol.Lett.156(2)：187-91
17 Simpson et al.(2000)J.Bacteriol.182(10)：5737-48
18 Chen et al.(1996)Infect.Immun.64：5008-14
19 Stojiljkovic et al.(1996)J.Bacteriol.179(15)：4670-78
20 Frangipane et al.(1994)FEMS Microbiol.Lett.118：243-8
本发明该方面的方法提供了选择性递送内过氧化物部分和铁的机制，它可以通过使共价缀合物与携带铁的蛋白质的受体结合而对致病生物体的细胞膜产生直接的反应。按照本发明的方法，结合的共价缀合物中的内过氧化物部分与从携带铁的蛋白质中释放的铁或血红素反应，从而在病原体附近产生有害自由基。因此，本发明提供了治疗幽门螺杆菌导致的疾病的方法，通过对有此需要的人体受试者给予有效量的包括青蒿素相关性内过氧化物与人全乳铁蛋白之间的共价缀合物的组合物来进行。本发明还提供了治疗脑膜炎奈瑟氏球菌导致的疾病的方法，通过对有此需要的人体受试者给予有效量的包含青蒿素相关性内过氧化物与人全运铁蛋白之间的共价缀合物的组合物来进行。
可以通过给予有效量的包括本发明共价缀合物的组合物治疗的其它典型的感染包括局部细菌感染，诸如龈炎、皮肤和眼部感染。
所述共价缀合物的有效量一般可达最大耐受剂量，但浓度并非关键且可以广泛改变。当然，临床医师使用的精确量随化合物、给药途径、患者的身体情况和其它因素而改变。可以将每日剂量作为单剂量给药或可以将其分成多次剂量给药。
实际给予的本发明缀合物的用量应是治疗有效量，本文所用的该术语表示产生实质性的有益作用所需的用量。可以根据由体外或动物模型试验系统得到的剂量-反应曲线外推出有效剂量。一般还将动物模型用于确定理想的剂量范围和给药途径。然后将这类信息用于确定用于人或其它哺乳动物的有用剂量和给药途径。有效剂量的测定也属于本领域技术人员的能力范围。因此，实际给予的用量取决于治疗所施用的个体，并优选是优化用量，以便获得所需作用而没有显著副作用。
可以通过标准制药步骤在细胞培养物或实验动物中测定本发明共价缀合物的治疗功效和可能的毒性(例如ED50，即有效治疗50％群体的剂量；和LD50，即使50％群体致死的剂量)。治疗与毒性作用之间的剂量比为治疗指数，可以将其表示为LD50与ED50之比。表现出高治疗指数的共价缀合物特别适合于实施本发明的方法。可以将从细胞培养试验和动物研究中获得的数据用于配制人或其它哺乳动物所使用的剂量范围。这类缀合物的剂量优选属于包括几乎没有或无毒性的ED50的循环浓度范围内。剂量一般在该范围内改变，这取决于所用的剂型、受试者的敏感性和给药途径。因此，最佳用量可以根据给药方法的不同而改变，并且一般为以相同或相似形式给药的常用药剂的用量。
可以将本发明的共价缀合物单独或与一种或多种其它治疗活性剂一起给药。例如，在治疗癌症的过程中，可以将该缀合物与治疗剂联用，所述的治疗剂包括、但不限于：雄激素抑制剂，诸如氟他胺和luprolide；抗雌激素药，诸如他莫昔芬；抗代谢药和细胞毒性剂，诸如柔红霉素、氟尿嘧啶、氟尿苷、干扰素α、氨甲蝶呤、普卡霉素、巯基嘌呤、硫鸟嘌呤、多柔比星、卡莫司汀、洛莫司汀、阿糖胞苷、环磷酰胺、多柔比星、雌莫司汀、六甲蜜胺、羟基脲、异环磷酰胺、丙卡巴肼、丝裂菌素制剂、白消安、米托蒽醌、卡铂、顺铂、链佐星、博来霉素、放线菌素和伊达比星；激素，诸如甲羟孕酮、雌莫司汀、炔雌醇、雌二醇、亮丙立德、甲地孕酮、奥曲肽、己烯雌酚、氯烯雌醚、依托泊苷、鬼臼毒素和戈舍瑞林；氮芥衍生物，诸如美法仑、苯丁酸氮芥、methlorethamine和塞替派；类固醇，诸如倍他米松；和其它抗肿瘤药，诸如活牛分枝杆菌(Mycobacterium bovis)、dicarbazine、天冬酰胺酶、亚叶酸、米托坦、长春新碱、长春碱和泰索帝。在每种情况中的适宜用量随特定活性剂的不同而改变，本领域技术人员易于得知或易于通过常规实验测定。
还可以将本发明的共价缀合物与例如可增加缀合物中携带铁的蛋白质的细胞表面受体数量而增加铁向细胞内的转运的活性剂联合给药。例如，已经证实胰岛素、胰岛素样生长因子I和表皮生长因子会引起细胞表面上的运铁蛋白受体数量增加(例如，参见Davis等(1987)《生物化学杂志》(J Biol.Chem.)261(19)：8708-11；Davis等(1986)《生物化学杂志》(J Biol.Chem.)262(17)：13126-34)。因此，在某些实施方案中，将本发明含有运铁蛋白的共价缀合物与胰岛素、胰岛素样生长因子I或表皮生长因子联合给药。
可以通过任意有效途经、例如通过非肠道或口服来给予本发明的共价缀合物。给药方法包括局部(例如皮肤贴剂)、吸入、口含、动脉内、皮下、髓内、静脉内、鼻内、直肠内、眼内给药和其它常用方式。例如，可以将所述的共价缀合物直接注入肿瘤、注入肿瘤附近或注入对肿瘤供血的血管。
可以将本发明的共价缀合物配制成组合物，该组合物中另外含有合适的药物上可接受的载体，包括赋形剂和其它有利于对哺乳动物受试者给予该共价缀合物的其它化合物。有关配制和给药的更具体的技术可以在最新版《Remington氏药物科学》(″Remington′s PharmaceuticalSciences″)(Maack Publishing Co，Easton PA)中找到。
可以使用本领域中众所周知的药物上可接受的载体配制用于口服给药的组合物，其剂量适合于口服给药。这类载体能够将含有本发明的共价缀合物的组合物配制成适合于受试者摄取的片剂、丸剂、锭剂、胶囊、液体、凝胶、糖浆剂、浆液、混悬液等。可以将口服应用的组合物与固体赋形剂一起配制，任选研磨所得混合物，并且如果需要，在加入合适的其它化合物后将该颗粒混合物进行加工，以制成片剂或锭剂药芯。合适的赋形剂包括碳水化合物或蛋白质填料。它们包括、但不限于：糖类，包括乳糖、蔗糖、甘露糖醇或山梨醇；来自玉米、小麦、稻、马铃薯或其它植物的淀粉；纤维素，诸如甲基纤维素、羟丙基甲基纤维素或羧甲基纤维素钠；和树胶，诸如阿拉伯胶和黄耆胶；以及蛋白质，诸如明胶和胶原蛋白。如果需要，可以加入崩解剂或加溶剂，诸如交联聚乙烯吡咯烷酮、琼脂、藻酸或其盐，诸如藻酸钠。
给锭芯包合适的包衣层，诸如浓糖溶液，其还可以含有阿拉伯树胶、滑石、聚乙烯吡咯烷酮、卡波姆凝胶、聚乙二醇和/或二氧化钛、漆用溶液和合适的有机溶剂或溶剂混合物。可以向片剂或锭剂包衣层中加入染料或色素以用于产品识别或表征活性化合物用量(即剂量)。
例如，可以将用于口服给药的共价缀合物配制成由明胶制成的推入配合式胶囊以及由明胶和诸如甘油或山梨醇这类包衣材料组成的密封软胶囊。推入配合式胶囊可以含有共价缀合物，其中混有：填充剂或粘合剂，诸如乳糖或淀粉；润滑剂，诸如滑石或硬脂酸镁；和任选的稳定剂。在软胶囊中，可以将共价缀合物溶于或悬浮于合适的含有或不含稳定剂的液体中，诸如脂肪油、液体石蜡或液体聚乙二醇。
非肠道给药用组合物包括一种或本发明共价缀合物的水溶液。为了进行注射，可以用生理上相容的缓冲液、诸如Hank′s溶液、林格液或生理缓冲盐水将本发明的共价缀合物配制成水溶液。含水的注射混悬液可以含有增加该混悬液粘度的物质，诸如羧甲基纤维素钠、山梨醇或葡聚糖。另外，可以将共价缀合物的混悬液制备成适宜的油性注射混悬液。合适的亲脂性溶剂或载体包括：脂肪油，诸如芝麻油；或合成的脂肪酸酯类，诸如油酸乙酯或甘油三酯类；或脂质体。该混悬液还可以任选含有合适的稳定剂或增加共价缀合物溶解度的试剂以便制备高浓度溶液。
为了进行局部或鼻部给药，一般将适合于透入特定屏障的渗透剂用于制剂中。它们的实例为2-吡咯烷酮、N-甲基-2-吡咯烷酮、二甲基乙酰胺、二甲基甲酰胺、丙二醇、甲醇或异丙醇、二甲亚砜和月桂氮酮。还可以包括其它试剂以使制剂在美容上可接受。它们的实例为脂肪、蜡、油、染料、香料、防腐剂、稳定剂和表面活性剂。还可以包括诸如本领域中公知的那些角质分解剂。实例为水杨酸和硫。为了进行局部给药，组合物可以为用于全身递送所述化合物的经皮软膏或贴剂的形式，可以按照常规方式制备(例如，参见Barry，《皮肤病用制剂》(Dermatological Formulations)(Drugs and the PharmaceuticalSciences--Dekker)；Harrys Cosmeticology(Leonard Hill Books)。
为了进行直肠给药，可以以栓剂或滞留型灌肠剂的形式给予组合物。可以通过将共价缀合物与合适的无刺激性赋形剂混合来制备这类组合物，所述的无刺激性赋形剂在常温下为固体，而在直肠温度下为液体，由此在直肠中熔化而释放药物。合适的赋形剂包括、但不限于可可脂和聚乙二醇类。
这些不同类型的添加剂各自的用量对本领域技术人员而言是显而易见的，最佳用量与设计用于相同类型给药的其它已知制剂相同。例如，角质层渗透促进剂一般是约0.1％-约15％浓度。
可以按照与本领域中已知类似的方式制备含有本发明共价缀合物的组合物(例如，通过常规的混合、溶解、成粒、制锭、研磨、乳化、包囊、包埋或冻干法)。还可以通过常规方式(例如包衣)改变组合物成以提供适当释放特性，例如缓释或靶向释放。
可以盐的形式提供包含共价缀合物的组合物，并且可以与许多酸成盐，包括、但不限于盐酸、硫酸、乙酸、乳酸、酒石酸、苹果酸、琥珀酸等。盐倾向于比相应的游离碱形式更易溶于水性或其它质子溶剂。
在制备了制成含有共价缀合物和可接受的载体的这类组合物后，可以将它们放入合适的容器并标记使用。
下列实施例仅解释了目前已知用于实施本发明的最佳方式，但不应用于限制本发明。
实施例1
本实施例描述了本发明有代表性的组合物的制备方法，该组合物含有一种或多种与全运铁蛋白分子共价连接的青蒿素相关性内过氧化物分子。
Artelinic acid hydrazide的合成(ART-NH-NH2)：制备青蒿琥酯的酰肼衍生物的尝试并不成功，因环化反应而导致形成二氢青蒿素。如上所述由二氢青蒿素合成阿替林酯(Shrimali等(1998)《印度化学杂志》(Indian J Chem.)37B：1161-1163)。将artelinic acid(0.1g，0.24mmol)溶于无水乙腈(0.48mL)。向该溶液中加入1-羟基苯并三唑(HOBt)(0.038g，0.29mmol)，随后添加乙基二甲基乙基碳化二亚胺(0.055g，0.29mmol)。在室温下搅拌该反应混合物并通过薄层色谱法(TLC)监测，直到所有的酸均转化成HOBt酯。
将肼(0.46mL，0.48mmol)在乙腈(0.48mL)中的溶液冷却至0℃，并将上述反应混合物加入到该体系中，同时将温度维持在0-10℃。正如通过TLC测定的，在10分钟后反应完全。将该反应混合物倾入水(5mL)中、用乙酸乙酯(3×10mL)萃取并用盐水洗涤。分离有机相、用无水硫酸钠干燥并蒸发至干。通过使用甲醇/氯仿梯度的硅胶色谱法纯化粗产物而得到纯的产物(0.078g)，产率为76％。
阿替林酯-全运铁蛋白缀合物的合成：在室温下使用10mmol/L高碘酸钠将溶于1ml 0.1mol/L pH 5.5的乙酸钠的全运铁蛋白(2mg)在其聚糖水平氧化30分钟。将该溶液上样到安装了UV监测器的短Sephadex G-25柱。用0.1mol/L pH 5.5的乙酸钠洗脱该柱并收集蛋白质级分。向氧化的全运铁蛋白中加入过量的ART-NH-NH2溶液并将该反应体系在室温下保持过夜，同时轻轻振摇。然后将阿替林酯-全运铁蛋白缀合物溶液上样到Sephadex G-25柱上，以除去过量的ART-NH-NH2。用pH 7.5的0.1mol/L Tris-HCl缓冲液洗脱该柱，并收集蛋白质级分。将缀合物储存在4℃下。
通过疏水相互作用HPLC纯化阿替林酯-全运铁蛋白缀合物，得到均一的蛋白质缀合物。通过离子喷雾质谱法测定每个全运铁蛋白分子的青蒿琥酯分子数。
尽管已经解释和描述了本发明的优选实施方案，但是可以理解可以对其中进行各种改变而不会脱离本发明的实质和范围。
实施例2
本实施例描述了本发明有代表性的组合物的制备方法，该组合物含有与血红蛋白分子共价连接的一个或多个青蒿素相关性内过氧化物分子。
为了用青蒿素衍生物共价修饰非糖基化的蛋白质，诸如血红蛋白，首先将青蒿素的羧酸衍生物、诸如artelinic acid活化为N-羟基琥珀酰亚胺(HOSu)酯，以便与蛋白质表面上的赖氨酸残基反应。将artelinicacid(4.2mg，0.01mmol)溶于二甲基甲酰胺(DMF)(0.5mL)中，并将该溶液在冰浴中冷却。向该溶液中加入N-乙基-N′-二甲氨基乙基碳二亚胺(EDC)(1.5mg，0.01mmol)和HOSu(1.1mg，0.01mmol)。将该反应混合物在0℃下保持搅拌2小时以完全形成artelinic acid的HOSu酯。
将血红蛋白(10mg)溶于7.0的0.1M磷酸盐缓冲液(5mL)。在0℃下将artelinicacid HOSu酯的DMF溶液缓慢加入到血红蛋白溶液中。将该反应混合物在0℃下保持搅拌2小时并在室温下再保持搅拌2小时。然后使该反应混合物上样到用pH 7的0.1M磷酸盐缓冲液平衡的Sephadex G-25柱上，并用相同的缓冲液洗脱该柱。经修饰的血红蛋白以外水体积洗脱下来。合并含有血红蛋白的级分，然后通过疏水相互作用HPLC纯化。通过离子喷雾质谱法测定与蛋白质连接的青蒿素衍生物数目。
1.包含与携带铁的蛋白质共价连接的青蒿素相关性内过氧化物的共价缀合物。
2.权利要求1所述的共价缀合物，其中所述青蒿素相关性内过氧化物包括阿替林酯。
3.权利要求1所述的共价缀合物，其中所述青蒿素相关性内过氧化物是酰肼衍生物。
4.权利要求2所述的共价缀合物，其中所述青蒿素相关性内过氧化物具有如下结构：
其中n＝1-3，m＝0-3，Ar＝芳基，且Y＝-(C＝O)NH-、-NH-或-O-。
5.权利要求1所述的共价缀合物，其中所述携带铁的蛋白质选自全运铁蛋白、全乳铁蛋白、血红蛋白、血红素结合蛋白和与中性明胶酶相关的脂质运载蛋白组成的组。
6.权利要求5所述的共价缀合物，其中所述携带铁的蛋白质为全运铁蛋白。
7.权利要求5所述的共价缀合物，其中所述携带铁的蛋白质为全乳铁蛋白。
8.权利要求5所述的共价缀合物，其中所述携带铁的蛋白质为哺乳动物蛋白。
9.权利要求5所述的共价缀合物，其中所述携带铁的蛋白质为人蛋白。
10.权利要求1所述的共价缀合物，其中包括阿替林酯和全运铁蛋白。
11.权利要求1所述的共价缀合物，其中包括阿替林酯和全乳铁蛋白。
12.权利要求1所述的共价缀合物，其中包括阿替林酯和血红蛋白。
13.一种组合物，其中包括与携带铁的蛋白质共价连接的青蒿素相关性内过氧化物的共价缀合物，以及药物上可接受的载体。
14.包含与携带铁的蛋白质共价连接的青蒿素相关性内过氧化物的共价缀合物在制备用于治疗癌症的药物中的用途。
15.权利要求14所述的用途，其中所述青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。
16.权利要求14所述的用途，其中所述携带铁的化合物为全运铁蛋白。
17.权利要求14所述的用途，其中所述药物被制成经非胃肠道给药。
18.含有青蒿素相关性内过氧化物与携带铁的蛋白质的共价缀合物在制备用于治疗被病原体感染的受试者的药物中的用途，其中所述的病原体表达该携带铁的蛋白质的受体。
19.权利要求18所述的用途，其中所述的青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。
20.权利要求18所述的用途，其中所述的携带铁的蛋白质选自全运铁蛋白、全乳铁蛋白、血红蛋白、血红素结合蛋白和与中性明胶酶相关的脂质运载蛋白组成的组。
21.权利要求18所述的用途，其中所述的病原体包括幽门螺杆菌(Helicobacter pylori)，其中所述的携带铁的蛋白质包括人乳铁蛋白，且其中所述的青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。
22.权利要求18所述的用途，其中所述的病原体包括脑膜炎奈瑟氏球菌(Neisseria meningitidis)，其中所述的携带铁的蛋白质包括人运铁蛋白，且其中所述的青蒿素相关性内过氧化物选自阿替林酯和二氢青蒿素组成的组。
Covalent conjugates between artemisinin-related endoperoxides and iron-carrying proteins and methods of use
In one aspect, the invention provides covalent conjugates between artemisinin related endoperoxides and iron-carrying proteins. In some embodiments, the covalent conjugates comprise artelinate and holotransferrin. In another aspect, the invention provides methods for administering the covalent conjugates of the invention to treat cancer and infections by pathogens that bind iron-carrying proteins.
Arteannuin dependency endoperoxide and carry covalent conjugates and using method thereof between the protein of ferrum
The cross reference of related application
The application requires the interests of the U.S. Provisional Application submitted on June 6th, 2002 according to 35U.S.C.119 number 60/386,928.
Invention field
The present invention relates to covalent conjugates between arteannuin (artemisinin) dependency endoperoxide (endoperoxide) and the protein that carries ferrum and these conjugates in the treatment cancer with by can be in conjunction with the application in the infection that the proteinic pathogen of carrying ferrum causes.
Background of invention
Arteannuin is an isolating sesquiterpene lactones class from plant Artemisia annua (Artemisia annua L), and the extract of this plant has been used for the treatment of malaria at least 1600 years.The arteannuin molecule contains the endoperoxides bridge, and it and iron atom reaction form free radical.The malaria effect of arteannuin is to form free radical cell death is caused because of the haemachrome in itself and the parasite reacts.Cancerous cell has and is significantly higher than Normocellular flowing molten iron and goes into.Therefore, confirmed that arteannuin and analog of artemisinin have cytotoxicity (for example, referring to (1993) such as Woerdenbag " natural product magazine " (J.Nat.Prod.) 56 (6): 849-56 to the tumor set up and tumor cell line; Lai ﹠amp; Singh (1995) " cancer communication " (Cancer Lett.) 91:41-6; Efferth etc. (2001) " international oncology's magazine " are 18:767-73 (Int.J.Oncol.); Li etc. (2001) " communication of bioorganic pesticide thing chemistry " are 11:5-8 (Bioorg.Med.Chem.Lett.); Singh﹠amp; Lai (2001) " life sciences " (Life Sci.) 70:49-56; Efferth etc. (2002) " biochemistry and pharmacology " are 64:617-23 (Biochem.Pharmacol.); Efferth etc. (2002) " hemocyte, molecule and disease " (Blood Cells, Molecules ﹠amp; Diseases) 28 (2): 160-8; Sadava etc. (2002) " cancer communication " (Cancer Lett.) 179:151-6).
The chemical compound that contains endoperoxides bridge (endoperoxide bridge) of having described many analog of arteannuin and other biologically active is (for example, referring to U.S. Pat 5,180,840; U.S. Pat 5,216,175; U.S. Pat 5,225,427; Cumming etc. (1998) " pharmaceutical chemistry magazine " (J Med.Chem.) 41 (6): 952-64; Posner etc. (1999) " pharmaceutical chemistry magazine " (J Med.Chem.) 42:300-4; Li etc. (2001) " communication of bioorganic pesticide thing chemistry " are 11:5-8 (Bioorg.Med.Chem.Lett.); Wu etc. (2001) " European pharmaceutical chemistry magazine " are 36:469-79 (Eur.J.Med.Chem.); Posner etc. (2003) " pharmaceutical chemistry magazine " (J Med.Chem.) 46:1060-5).The analog of artemisinin that has been used for the treatment of malaria comprises dihydroartemisinine, Artemether, artesunate, arteether, dihydroartemisinin propyl carbonic ester and artelinic acid.
Arteannuin is comparatively safe medicine, even also has only few and humble side effect under high dose.6 days 70mg/kg/ days oral dose is used for the treatment of people's malaria.After with artesunate treatment cancer patient, do not observe tangible adverse side effect (oral dose 50mg/ days; Intramuscular dosage 60mg/ days; 9 months by a definite date) (Singh ﹠amp; Verma (2002) " oncology's journal " (Arch.Oncol.) 10 (4): 279-80).Also arteannuin and analog of artemisinin are used for the treatment of dermatosis, such as psoriasis, foaming characteristic dermatosis, viral verruca, condyloma subcutaneum (mulluscum contagiosum) and hemorrhoid (for example, referring to U.S. Pat 4,978,676; U.S. Pat 5,219,880).Also arteannuin and analog of artemisinin are used for prevention of malaria.
The verified protein ferritin for the national games (holotransferrin) that gives iron salt or carry ferrum has increased the sensitivity (Lai of the tumor of cancerous cell and implantation to arteannuin and analog thereof; Singh (1995) " cancer communication " (Cancer Lett.) 91:41-46; Moore etc. (1995) " cancer communication " (Cancer Lett.) 98:83-7; Singh ﹠amp; Lai (2001) " life sciences " (Life Sci.) 70:49-56; Sadava etc. (2002) " cancer communication " (Cancer Lett.) 1179:151-6).
Confirm that also some pathogen is by obtaining ferrum in conjunction with the host protein that carries ferrum.For example, the pathogen Neisseria meningitidis of bacterial meningitis is expressed the cell surface receptor of the chemical compound that carries ferrum, such as transferrin (transferrin) and lactoferrin (lactoferrin) (Evans ﹠amp; Oakhill (2002) " biochemistry association journal " (Biochem.Soc.Trans.) 30 (4): 705-7).At present, still not having vaccine, to can be used for Neisseria meningitidis Type B bacterial strain be most popular bacterial strain in the Western countries.In addition, the pathogen helicobacter pylori of confirmer's gastritis, gastric duodenal ulcer and adenocarcinoma (Helicobacter pylori) can (Husson etc. (1993) " infectious immunity " (Infect.Immun.) 61 (6): 2694-7) by obtain ferrum in conjunction with human lactoferrin.
Still need the arteannuin compositions that render a service to increase in this area, to be used for the treatment of cancer and by the disease that can cause in conjunction with the pathogen of the host protein that carries ferrum.Also need to be used for the treatment of cancer and by the method that can obtain the infection that pathogen caused of ferrum by the host protein that ferrum is carried in absorption.The present invention has satisfied these demands.
Summary of the invention
Noval chemical compound is provided among the present invention in one aspect and comprises arteannuin dependency endoperoxide and carry the compositions of the covalent conjugates between the protein of ferrum.Arteannuin dependency endoperoxide can be connected with the chemical compound that carries ferrum by hydrazides part, hydrazine part or aminooxy group part.In certain embodiments, described arteannuin dependency endoperoxide has following structure:
N=1-3 wherein, m=0-3, the Ar=aryl, and Y=-(C=O) NH-,-NH-or-O-.The representational arteannuin dependency endoperoxide that is present in the covalent conjugates of the present invention comprises artelinate (artelinate) and dihydroartemisinine.
The representational protein that carries ferrum that is present in the covalent conjugates of the present invention comprise transferrin family in the protein, the lipocalin protein relevant with neutral gelatinase (neutralgelatinaseassociated lipocalin, NGAL), hemoprotein and other carry the protein of ferrum.Therefore, described covalent conjugates can comprise: the conjugate of artelinate and ferritin for the national games, artelinate and full milk ferritin (hololactoferrin) or artelinate and hemoglobin for example.The present invention also provides the compositions that comprises covalent conjugates of the present invention and pharmaceutically acceptable carrier.Covalent conjugates of the present invention can be used for treating cancer and by can be in conjunction with the infection that proteinic pathogen caused of carrying ferrum.
The present invention provides the method that the experimenter of needs is given covalent conjugates of the present invention in one aspect of the method.The typical covalent conjugates that is suitable for administration in the present invention is aspect this comprises: for example between artelinate and the ferritin for the national games, between artelinate and the full milk ferritin and the covalent conjugates between artelinate and the hemoglobin.
In certain embodiments, the invention provides the treatment method for cancer, by the compositions that has this human or animal experimenter who needs to give effective dose is carried out, said composition comprises arteannuin dependency endoperoxide and carries covalent conjugates between the protein of ferrum.Can give described covalent conjugates by part or general, maybe they directly can be injected tumor.Can give described covalent conjugates separately or with one or more other therapeutic agents.For example, can give described covalent conjugates with the activating agent that for example can increase to intracellular iron transfer by the proteinic cell surface receptor number that carries ferrum in the increase conjugate.
The present invention also provides treatment by can be in conjunction with the method for the infection that proteinic pathogen caused of carrying ferrum, by the compositions that has this human or animal experimenter who needs to give effective dose is carried out, said composition comprises arteannuin dependency endoperoxide and carries covalent conjugates between the protein of ferrum.In one embodiment, described pathogen comprises helicobacter pylori, and the protein that wherein carries ferrum comprises human lactoferrin, and wherein said arteannuin dependency endoperoxide is selected from the group of artelinate and dihydroartemisinine composition.In other typical embodiment, described pathogen comprises Neisseria meningitidis, and the wherein said protein that carries ferrum comprises human transferrin, and wherein said arteannuin dependency endoperoxide is selected from the group of artelinate and dihydroartemisinine composition.
The method of treatment helicobacter pylori infections is provided in other embodiments, by the compositions that has this human or animal experimenter who needs to give effective dose is carried out, said composition comprises arteannuin dependency endoperoxide and carries covalent conjugates between the protein of ferrum.
Detailed description of the preferred embodiments
The compositions of the covalent conjugates between the protein that comprises arteannuin dependency endoperoxide and carry ferrum is provided among the present invention in one aspect.Term used herein " covalent conjugates " refers to wherein arteannuin dependency endoperoxide and the covalently bound chemical compound together of protein that carries ferrum.Term " arteannuin dependency endoperoxide " refers to the chemical compound that contains interior peroxide bridge (endoperoxide bridge), and it can form free radical with the iron atom reaction, causes cell death.Arteannuin dependency endoperoxides compounds can also form free radical under the situation that has copper and manganese to exist.Representational arteannuin dependency endoperoxide is as described herein, and but, obviously other endoperoxide also can be used for this purpose.
In general, arteannuin dependency endoperoxide is selected from by sesquiterpene lactones class and alcohols, carbonates, esters, ethers and sulfonic acid esters, arteflene, 1,2,4-three alkanes and 1,2,4, the group that 5-four alkanes are formed.Arteannuin dependency endoperoxide can have following structure:
Wherein R is Or , R 1For hydrogen, hydroxyl, alkyl or have following general formula:
-O-R 2, Or
R wherein 2For alkyl or aryl and n are 1-6; Perhaps its pharmaceutically acceptable salt.Term used herein " alkyl " refers to the low alkyl group that contains 1-6 carbon atom, preferred 1-4 carbon atom.Alkyl of the present invention can be the straight or branched group.Term " aryl " refers to and contains 4-14 main chain carbon or heteroatomic monocycle and polycyclic aromatic group, comprises isocyclic aryl and heterocyclic aryl.Isocyclic aryl is the aryl that wherein all annular atomses are carbon.Heterocyclic aryl contains 1-4 hetero atom as annular atoms, and remaining annular atoms is a carbon.Representational aryl comprises for example phenyl and benzyl.Pharmaceutically acceptable salt comprises alkali metal or alkali salt, preferred sodium or potassium salt.
For example, arteannuin dependency endoperoxide of the present invention comprises: arteannuin, wherein R is
Dihydroartemisinine (R 1=-OH); Artesunicacid (R 1=-OCO (CH 2) 2CO 2H); And artesunate; Artemether (R 1=-OCH 3); And arteether (R 1=-OC 2H 5).Other representational endoperoxides compounds of the present invention comprises artelinicacid, dihydroartemisinin propyl carbonic ester, arteflene (Ro.42-1611) and analog thereof (Biirgen etc. (1994) Sixth Int.Cong.Infect.Dis.Abst.427, p.152, Prague), 1,2,4-three alkanes (Peters etc. (1993) Ann.Trop.Med.Parasit.87 (1): 9-16) with 1,2,4,5-four alkanes (Vennerstrom etc. (1992) " pharmaceutical chemistry magazine " (J Med Chem.) 35 (16): 3023-3027).The analog that other of arteannuin is suitable is described in for example U.S. Pat 5,216,175 and US5,180,840, (1998) " pharmaceutical chemistry magazine " (J Med Chem.) 41 (6) such as Cumming: among 952-64 and PCT patent application WO 97/01548, WO 99/33461 and the WO 00/42046.
The source of arteannuin dependency endoperoxide can be natural (for example separating from plant), synthetic or semisynthetic.For example, can (for example produce free radical generating agent by the enzyme of in microbial hosts, expressing in the relevant route of synthesis, referring to (2003) such as Martin " natural biological technology " (Nature Biotechnol.), online open: on June 1st, 2003, doi:10.1038/nbt833).
Term used herein " carries the protein of ferrum " and refers to and is suitable for the protein of iron transfer to cell.The protein that carries ferrum in the covalent conjugates of the present invention can be mammalian proteins, for example human protein.The protein that typically carries ferrum comprise transferrin family in the protein, the lipocalin protein relevant with neutral gelatinase (neutral gelatinase-associatedlipocalin, NGAL), hemoprotein and other protein in conjunction with ferrum.Ferrum plays a part crucial in the cell growth.Transferrin family in the protein comprises transferrin, lactoferrin, ovum transferrin (ovotransferrin) and bad luck ferritin (melanotransferrin) (Aisen ﹠amp; Harris (1989) is at (ed.Loehr, VCH, New York) 273-320 page or leaf described in " siderophore and ferritin " (Iron Carriers and IronProteins); Baker (1994) " senior inorganic chemistry " is 41:389-463 (Adv.Inorg.Chem.)).All these protein are the strand glycoprotein of structural dependence, contain 670-690 aminoacid.Transferrin is transported into cell with ferrum and haemachrome from the circulation transfer.The ferrum form (ferritin for the national games) of carrying of transferrin is in conjunction with the transferrin receptor on the cell surface, and is ingested in the cell by receptor-mediated endocytosis, discharges ferrum at this.Lactoferrin has pivotal role in the process that absorbs ferrum from human milk.The another kind of function of lactoferrin is to stop the ferrum contagium.Be different from transferrin, lactoferrin can be under low pH stably in conjunction with ferrum and be absorbed in the intestinal.
NGAL is and the irrelevant protein that carries ferrum of transferrin family, infers that it can be delivered to ferrum the epithelial cell place (Kaplan (2002) " cell " is 111:603-6 (Cell)) that is breaking up.Hemoprotein is for to carry the protein of haemachrome as prothetic group such as this class of hemoglobin, Myoglobin, Hemopexin, cytochrome, catalase and peroxidase.For example, Hemopexin is the seroglycoid of 60kDa, it is with high affinity chelating haemachrome from blood flow, and it is transported to liver (Baker etc. (2003) " NAS's journal " (Proc.Natl.Acad Sci.US.A.) 100 (7): 3579-83).
In covalent conjugates of the present invention, arteannuin dependency endoperoxide is connected with the protein that carries ferrum, and ways of connecting can be the two any way of activity that can keep the activity of proteins of carrying ferrum and arteannuin dependency endoperoxide.For example, as described in example 1 above, can through the following steps arteannuin dependency endoperoxide be connected with the glycosylated protein that carries ferrum: at first prepare the hydrazide derivatives of arteannuin dependency endoperoxide, it is connected with one or more oxidation of polysaccharides groups on the protein that carries ferrum.In order to prepare the hydrazide derivatives of arteannuin dependency endoperoxide, can form the ester of arteannuin dependency endoperoxide at first through the following steps: I-hydroxybenzotriazole (HOBt) is joined in the arteannuin dependency endoperoxide, add ethyl dimethyl ethyl carbodiimide subsequently.Make HOBt ester and hydrazine reaction generate hydrazide derivatives then.Make this hydrazide derivatives covalently bound subsequently, wherein used the oxidized polysaccharide group of oxidant (such as sodium metaperiodate) with the protein that carries ferrum.Any containing-(C=O), the arteannuin dependency endoperoxide of NH-group all can be used to prepare the hydrazide derivatives that can be connected with transferrin or other glycoprotein that carries ferrum (for example, referring to (1998) such as Gumming " pharmaceutical chemistry magazine " (J Med.Chem.) 41 (6): 952-64).
Hydrazine that can also be by at first preparing arteannuin dependency endoperoxide or aminooxy group derivant and arteannuin dependency endoperoxide and the glycosylated protein that carries ferrum are coupled together.For hydrazine or the aminooxy group derivant for preparing arteannuin dependency endoperoxide, form the halogenide of arteannuin dependency endoperoxide at first through the following steps: halohydrin is joined in the arteannuin dependency endoperoxide, add etherate of trifluoroboron subsequently.The reaction of described halogenide or arteannuin dependency endoperoxide and hydrazine or hydroxylamine be can make then and hydrazine or aminooxy group derivant generated respectively.The halo group can and be used for reaction by p-toluenesulfonyl or mesyl replacement.Can make hydrazine or aminooxy group derivant covalently bound then, wherein use the oxidized polysaccharide group of oxidant (such as sodium metaperiodate) with the protein that carries ferrum.The NH-of any containing-(C=O) ,-NH-or-the arteannuin dependency endoperoxide of O-group all can be used to prepare the hydrazine derivate or the aminooxy group derivant that can be connected with transferrin or other glycoprotein that carries ferrum.
The arteannuin dependency endoperoxide that is suitable for connecting in this manner comprise can therefrom prepare hydrazides, hydrazine or aminooxy group derivant and do not destroy in the active chemical compound of peroxide bridge.In general, by at interval basic (such as hydrocarbon chain) interior peroxide bridge and hydrazides, hydrazine or aminooxy group are separated.Can also be with ethers, esters, amide-type, sulfide and disulphide as basic at interval.For example, can with phenyl ring with the endoperoxides bridge with keep apart as the hydrazides on the artelinate, hydrazine or aminooxy group.Therefore, be applicable to the endoperoxide of arteannuin dependency endoperoxide of the present invention including, but not limited to following structure:
N=1-3 wherein, m=0-3, the Ar=aryl, for example phenyl, naphthyl, pyrenyl, pyridine radicals or pyrimidine radicals, and Y=-(C=O) NH-,-NH-or-O-.The representational arteannuin dependency endoperoxide that is present in the covalent conjugates of the present invention comprises artelinate and dihydroartemisinine.
Be used for the method that arteannuin dependency endoperoxide is connected with the glycoprotein that carries ferrum is comprised borate mediation with the glycoprotein that carries ferrum on company's key (for example, referring to U.S. Pat 5,919,708) of saccharide residue.Therefore, contain the boric acid ester group arteannuin dependency endoperoxide will with 1 of saccharide residue on the protein surface, the reaction of 2-glycol moiety.
With regard to the nonglycosylated protein that carries ferrum, can be as described in example 2 above by using amino acid side chain that arteannuin dependency endoperoxide is connected with this protein.For example, artesunate and artelinate have can use N-hydroxy-succinamide (for example, referring to (1994) such as Lewis " bioconjugates chemistry " (Bioconj.Chem.) 5 (6): 655-76) with water-soluble carbodiimide reagent, activate into free carboxy acid's base of active ester such as N-ethyl (etheyl)-N '-dimethylaminoethyl carbodiimides.Can be with this ester with nonglycosylated proteinic aqueous solution of carrying ferrum and arteannuin dependency endoperoxide is connected with Lys residue on this protein surface.
Can be by the standard method in this area, for example by using gel filtration chromatography, ion exchange and the anti-phase or described covalent conjugates of hydrophobic interaction high pressure lipuid chromatography (HPLC) (HPLC) purification.Can be by using the standard method in this area, ionspray mass spectrometry is for example measured the quantity of the arteannuin dependency endoperoxide molecule that combines with the protein that carries ferrum of a molecule.Arteannuin dependency endoperoxide depends on used protein that carries ferrum and the method that forms conjugate with the proteinic ratio of carrying ferrum in the covalent conjugates.For example, the ferritin for the national games that uses the method described in the embodiment 1 to obtain per molecule contains the covalent conjugates of the arteannuin dependency endoperoxide of 1-10 molecule.
The present invention also provides the compositions that comprises covalent conjugates of the present invention.In certain embodiments, as described in example 1 above, compositions of the present invention comprises the covalent conjugates between artelinate and the people's ferritin for the national games.In other embodiments, described compositions comprises the covalent conjugates between artelinate and the people's full milk ferritin.
Covalent conjugates of the present invention can be used for treating cancer.Because most of cancerous cell divides fast, so they have the ferrum uptake rate higher than normal cell, and the concentration of the cell surface transferrin receptor of expressing also is higher than normal cell.Verifiedly give iron salt or ferritin for the national games has increased sensitivity (Moore etc. (1995) " cancer communication " (Cancer Lett.) 98:83-7 of cancerous cell to arteannuin and analog thereof; Singh ﹠amp; Lai (2001) " life sciences " is 70:49-56 (LifeSci.); Sadava etc. (2002) " cancer communication " (Cancer Lett.) 1179:151-6).Therefore, by with arteannuin dependency endoperoxide and the protein, covalently bound that carries ferrum such as ferritin for the national games, can increase effect and the selectivity of arteannuin dependency endoperoxide, because endoperoxide and ferrum are transported to simultaneously or transport into same cell to cancer.
Covalent conjugates of the present invention also can be used for treating the infection that pathogenic organisms body with the proteinic receptor that carries ferrum described in the covalent conjugates is caused.In order to set up successful infection, pathogen must overcome the strict ferrum restriction that the host applies.In order to overcome this restriction, many pathogen obtain ferrum (for example, referring to Cornelissen, (2003) Front.Biosci.8:D836-47) from the host protein that carries ferrum.For example, the pathogen Neisseria meningitidis of bacterial meningitis is expressed and carries the protein-transferrin of ferrum and the cell surface receptor (Evans﹠amp of lactoferrin; Oakhill (2002) " biochemistry association journal " (Biochem.Soc.Trans.) 30 (4): 705-7), (Husson etc. (1993) " infectious immunity " (Infect.Immun.) 61 (6): 2694-7), and staphylococcus aureus is expressed hemoglobin receptor (Mazmanian etc. (2003) " science " are 299:906-9 (Science)) to the receptor of the pathogen helicobacter pylori expressing human lactoferrin of people's gastritis and peptic ulcer.Therefore, covalent conjugates of the present invention can be used for killing the pathogenic organisms body that wherein has the proteinic receptor that carries ferrum described in the covalent conjugates.For example, the covalent conjugates between arteannuin dependency endoperoxide and human transferrin or the people's full milk ferritin can be used for the treatment of gastritis and the peptic ulcer disease that bacterial meningitis that Neisseria meningitidis causes or helicobacter pylori cause.
According to method of the present invention, when to the cancerous cell administration, arteannuin dependency endoperoxide and the covalent conjugates of carrying between the protein of ferrum have the cellular cytoxicity activity that protein reached that is better than giving arteannuin dependency endoperoxide respectively and carries ferrum.In addition, covalent conjugates of the present invention can effectively be killed and wounded the pathogen that has the proteinic receptor that carries ferrum.
The present invention provides in aspect second there being this experimenter who needs to give method for compositions, and said composition comprises arteannuin dependency endoperoxide and carries covalent conjugates between the protein of ferrum.Arteannuin dependency endoperoxide and the proteinic covalent conjugates of carrying ferrum are as mentioned above.For example, as described in example 1 above, some embodiment provides the covalent conjugates of artelinate and ferritin for the national games.
These methods are suitable for any animal experimenter, such as the human body experimenter.For example, the experimenter who needs the compositions of the covalent conjugates between the protein that comprises arteannuin dependency endoperoxide and carry ferrum can be the cancer patient.As mentioned above, the cell of fast breeding, generally have higher cell surface transferrin receptor concentration such as cancerous cell.The mechanism that this method provides selectivity to send endoperoxide part and ferrum, it can produce to the cell of fast breeding, such as cancerous cell reacts.Therefore, the invention provides the treatment method for cancer, by the chemical compound that has this human or animal experimenter who needs to give effective dose is carried out, this chemical compound comprises arteannuin dependency endoperoxide and carries covalent conjugates between the protein of ferrum.The propagation that there is cell hyperproliferation in other and can brings out after including, but not limited to restenosis, autoimmune disease, arthritis, transplant rejection, inflammatory bowel or medical care precess with the disease of covalent conjugates of the present invention treatment.In certain embodiments, described method comprises the step that gives chemical compound, and this chemical compound comprises the covalent conjugates between artelinate and the people's ferritin for the national games.
Can also comprise arteannuin dependency endoperoxide and carry the chemical compound and the compositions of the covalent conjugates between the protein of ferrum, so that treatment is by expressing the infection that pathogen caused of carrying the proteinic cell surface receptor of ferrum described in the covalent conjugates.Term used herein " infection that the treatment pathogen causes " refers to and suppresses pathogenic growth and/or prevention or improve the disease symptoms relevant with this infection.
Have the host protein that carries ferrum receptor typical pathogen as mentioned above, comprise the helicobacter pylori of the receptor of the Neisseria meningitidis of receptor of expressing human transferrin and expressing human lactoferrin.Confirmed that at present staphylococcus aureus expresses hemoglobin receptor (Mazmanian etc. (2003) " science " are 299:906-9 (Science)), Listeria monocytogenes (Listeria monocytogenes) and Bacillus anthracis (Bacillus anthracis) are also expressed similar protein (Cabanes etc. (2002) " microbiology trend " (Trends.Microbiol.) 10 (5): 238-45).It is as shown in table 1 that the typical pathogen example of receptor of host protein of ferrum is carried in expression.In case this protein that carries ferrum combines with the receptor that pathogen is expressed, then ferrum or haemachrome generally discharge from the protein that carries ferrum, and are gone into cell (for example, referring to Gray-Owens ﹠amp by transhipment; Schryyers (1996) " microbiology trend " (Trends.Microbiol.) 4 (5): 185-91; Wandersman ﹠amp; Stojiljkovic (2000) " up-to-date microbiology viewpoint " is 3:215-20 (Curer.Op.Microbiol.)).
Carry the proteinic receptor of ferrum in the table 1. humans and animals pathogen
| Pathogen | The host | Disease | Receptor |
| Ox catarrhalis morazella catarrhalis lacuna catarrhalis Neisseria meningitidis Diplococcus gonorrhoeae actinobacillus actinomycetem comitans (Actinobacillus actinomycetecomitans) actinobacillus equuli Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) Haemophilus agni bird pasteurella haemophilus influenzae haemophilus paragallinarum (Haemophilus paragallinarum) Haemophilus somnus (Haemophilus somnus) haemophilus parasuis haemophilus ducreyi | Ox everybody everybody the pig sheep poultry people of forces poultry ox pig people | Keratoconjunctivitis tympanitis keratoconjunctivitis meningitis gonorrhoea juvenile periodontitis septicemia pneumosepticemia sinusitis meningitis, the sick genitals canker of tympanitis infective rhinitis's thromboembolia type meningoencephalitis Karl-Heinz Grasser | Transferrin, lactoferrin 1Transferrin, lactoferrin 1Transferrin, lactoferrin 1Transferrin, lactoferrin 1Hemoglobin 1，19Transferrin, lactoferrin, hemoglobin 1，18Transferrin 1Transferrin 1Transferrin 1Transferrin 1Transferrin 1Transferrin, hemoglobin 1，20Transferrin 1Transferrin 1Transferrin 1Hemoglobin 15 |
| Pathogen | The host | Disease | Receptor |
| Haemolysis pasteurella multocida staphylococcus aureus MRSE, (Staphyloccus epidermidis) streptococcus pneumonia Leishmania chagasi bacillus coli K88 Tritrichonomas foetus Spirochaeta pallida Mycoplasma pneumoniae Bordetella pertussis Trichonomas vaginalis aeromonas salmonicida helicobacter pylori yersinia enterocolitica Vibrio vulnificus porphyromonas gingivalis, (Porphyromonas gingivalis) | Ox; Sheep, everybody eel (eel) people of everybody people mermaid of goat ox everybody everybody pig ox | Shipping fever, the hemorrhagic septicemia pneumonia, the septicemia bacteremia, pneumonia, endocarditis, septic arthritis, osteomyelitis, deep abscess, the alimentary toxicosis endocarditis, endophthalmitis, septicemia, the cystitis pneumonia, meningitis, bacteremia, otitis media leishmaniasis enteropathy generation trichomonacide syphilis pneumonia pertussis vaginosis furunculosis gastritis, gastric duodenal ulcer, adenocarcinoma of stomach, lymphoma enteritis alimentary toxicosis, septicemia, the traumatic infection periodontal disease | Transferrin 1Transferrin 1The transferrin hemoglobin 2，13Transferrin 2Lactoferrin 3Transferrin, lactoferrin 4Transferrin 5The lactoferrin hemoglobin 6Lactoferrin 7Lactoferrin 8Lactoferrin 9Lactoferrin 10Transferrin, lactoferrin 11Lactoferrin 12Hemoglobin, Myoglobin, Hemopexin, catalase, albumin-haemachrome 14Hemoglobin 16Hemoglobin 17 |
1 Gray-Owen & Schryvers(1996)Trends Microbiol.4(5)：185-91
2 Modun et al.(1998)Infect.Immun.66(8)：3591-6
3 Hammerschmidt et al.(1999)Infect.Immun.67(4)：1683-7
4 Britigan et al.(1998)Adv.Exp.Med.Biol.443：135-40
5 Grange et al.(1997)Adv.Exp.Med.Biol.412：357-61
6 Tachezy et al.(1996)Exp.Parasitol.83(2)：216-28
7 Alderete et al.(1988)Genitourin.Med.64(6)：359-63
8 Tryon & Baseman(1987)Microb.Pathog.3(6)：437-43
9 Redhead et al.(1987)J.Gen.Microbiol.133(4)：891-8
10 Peterson & Alderete(1984)J.Exp.Med.160(2)：398-410
11 Chart & Trust(1983)J.Bacteriol.156(2)：758-64
12 Dhaenens et al.(1997)Infect.Immun.65(2)：514-8
13 Mazmanian et al.(2003)Science 299：906-9
14 Bracken et al.(1999)J.Bacteriol.181(19)：6063-72
15 Al-Twafiq et al.(2000)J.Infect.Dis.181(3)：1049-54
16 Fouz et al.(1997)Microbiol.Lett.156(2)：187-91
17 Simpson et al.(2000)J.Bacteriol.182(10)：5737-48
18 Chen et al.(1996)Infect.Immun.64：5008-14
19 Stojiljkovic et al.(1996)J.Bacteriol.179(15)：4670-78
20 Frangipane et al.(1994)FEMS Microbiol.Lett.118：243-8
The mechanism that the method for this aspect of the present invention provides selectivity to send endoperoxide part and ferrum, it can produce directly reaction with the proteinic receptors bind of carrying ferrum to the cell membrane of pathogenic organisms body by making covalent conjugates.According to method of the present invention, the endoperoxide part in the bonded covalent conjugates and ferrum that from the protein that carries ferrum, discharges or haemachrome reaction, thus near pathogen, produce harmful free radical.Therefore, the invention provides the method for the disease that causes of treatment helicobacter pylori, by the compositions that comprises the covalent conjugates between arteannuin dependency endoperoxide and the people's full milk ferritin that has this human body experimenter who needs to give effective dose is carried out.The present invention also provides the method for the treatment disease that Neisseria meningitidis causes, by the compositions that comprises the covalent conjugates between arteannuin dependency endoperoxide and the people's ferritin for the national games that has this human body experimenter who needs to give effective dose is carried out.
Other typical infection of the combination treatment that comprises covalent conjugates of the present invention that can be by giving effective dose comprises localized bacterial infection, such as gingivitis, skin and ocular infection.
The effective dose of described covalent conjugates generally can reach maximum tolerated dose, but concentration and non-key and can extensively change.Certainly, the accurate amount of clinicist's use changes with chemical compound, route of administration, patient's body situation and other factors.Dosage every day maybe can be able to be divided into the multidose administration as single dose administration.
The consumption of the actual conjugate of the present invention that gives should be the treatment effective dose, and the required consumption of substantial beneficial effect represented to produce in this term used herein.Can be according to by the extrapolated effective dose of dose-response curve external or that animal model test system obtains.Generally also animal model is used for determining ideal dosage range and route of administration.Then this category information is used to be identified for people or other mammiferous useful dosage and route of administration.The mensuration of effective dose also belongs to those skilled in the art's limit of power.Therefore, the actual consumption that gives depends on the individuality that treatment is used, and preferably optimizes consumption, does not have remarkable side effect so that obtain required effect.
Can in cell culture or laboratory animal, measure the therapeutic efficiency of covalent conjugates of the present invention and possible toxicity (ED for example by standard pharmacy step 50, promptly effectively treat the dosage of 50% colony; And LD 50Even, the lethal dosage of 50% colony).Dosage between treatment and the toxic action can be expressed as LD with it than for therapeutic index 50With ED 50The ratio.The covalent conjugates that shows high therapeutic index is particularly suitable for implementing method of the present invention.The data that obtain can be used to prepare people or the employed dosage range of other mammal from cell culture test and zooscopy.The dosage of this class conjugate preferably belongs to and comprises almost not having or avirulent ED 50The circulation composition scope in.Dosage generally changes in this scope, and this depends on used dosage form, experimenter's sensitivity and route of administration.Therefore, optimum amount can change according to the difference of medication, and is generally the consumption with the medicament commonly used of same or similar form administration.
Can be with covalent conjugates of the present invention separately or with one or more other therapeutic activity agent administrations.For example, in the process of treatment cancer, can be with this conjugate and therapeutic agent coupling, described therapeutic agent is including, but not limited to inhibitor for androgen, such as flutamide and luprolide; Antiestrogen is such as tamoxifen; Antimetabolite and cytotoxic agent are such as daunorubicin, fluorouracil, floxuridine, interferon-ALPHA, methotrexate, plicamycin, purinethol, thioguanine, doxorubicin, carmustine, lomustine, cytosine arabinoside, cyclophosphamide, doxorubicin, estramustine, altretamine, hydroxyurea, ifosfamide, procarbazine, Mutamycin, busulfan, mitoxantrone, carboplatin, cisplatin, streptozocin, bleomycin, D actinomycin D and idarubicin; Hormone is such as medroxyprogesterone, estramustine, ethinylestradiol, estradiol, leuproside, megestrol, octreotide, diethylstilbestrol, chlorotrianisene, etoposide, podophyllotoxin and goserelin; Nitrogen mustard derivatives replaces group such as melphalan, chlorambucil, methlorethamine and plug; Steroid is such as betamethasone; With other antineoplastic agent, such as cattle on the hoof mycobacteria (Mycobacterium bovis), dicarbazine, asparaginase, folinic acid, mitotane, vincristine, vinblastine and taxotere.Suitable consumption in each case changes with the difference of particular active agent, and those skilled in the art are easy to learn or are easy to and measure by normal experiment.
Covalent conjugates of the present invention can also be increased the activating agent administering drug combinations of ferrum to intracellular transhipment with for example increasing the proteinic cell surface receptor quantity of carrying ferrum in the conjugate.For example, confirmed that insulin, insulin-like growth factor I and epidermal growth factor can cause that the transferrin receptor quantity on the cell surface increases (for example, referring to (1987) " journal of biological chemistry " (J Biol.Chem.) 261 (19): 8708-11 such as Davis; Davis etc. (1986) " journal of biological chemistry " (J Biol.Chem.) 262 (17): 13126-34).Therefore, in certain embodiments, the present invention is contained covalent conjugates and insulin, insulin-like growth factor I or the epidermal growth factor administering drug combinations of transferrin.
Can be by any some effective, for example by non-intestinal or orally give covalent conjugates of the present invention.Medication comprises part (for example skin patch), sucks, sucks, in the intra-arterial, subcutaneous, marrow, intravenous, intranasal, internal rectum, eye drops and other usual way.For example, described covalent conjugates directly can be injected tumor, inject near the tumor or inject blood vessel tumor feeding.
Covalent conjugates of the present invention can be mixed with compositions, contain suitable pharmaceutically acceptable carrier in the said composition in addition, comprise that excipient and other help mammalian subject is given other chemical compound of this covalent conjugates.About preparation and the technology more specifically of administration can find in latest edition " RemingtonShi pharmaceutical science " (" Remington ' s PharmaceuticalSciences ") (Maack Publishing Co, Easton PA).
Can use pharmaceutically acceptable carrier preparation well known in the art to be used for liquid preparations for oral administration, its dosage is suitable for oral administration.This class carrier can be mixed with the compositions that contains covalent conjugates of the present invention the tablet that is suitable for experimenter picked-up, pill, lozenge, capsule, liquid, gel, syrup, serosity, suspension etc.The compositions of oral application can be prepared with solid excipient, optional grinding gained mixture, and if desired, after adding other suitable chemical compound, this granulate mixture is processed, to make tablet or lozenge medicated core.Suitable excipient comprises carbohydrate or protein filler.They including, but not limited to: saccharide comprises lactose, sucrose, mannitol or sorbitol; Starch from corn, Semen Tritici aestivi, rice, Rhizoma Solani tuber osi or other plant; Cellulose is such as methylcellulose, hydroxypropyl emthylcellulose or sodium carboxymethyl cellulose; And natural gum, such as arabic gum and tragacanth gum; And protein, such as gelatin and collagen protein.If desired, can add disintegrating agent or solubilizer, such as crospolyvinylpyrrolidone, agar, alginic acid or its salt, such as sodium alginate.
Give ingot core enclose suitable coatings, such as priming, it can also contain Radix Acaciae senegalis, Talcum, polyvinylpyrrolidone, carbomer gel, Polyethylene Glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.Can in tablet or lozenge coatings, add dyestuff or pigment to be used for product identification or to characterize reactive compound consumption (being dosage).
For example, the covalent conjugates that is used for oral administration can be mixed with the sucking fit formula capsule made by gelatin and by gelatin and the sealing soft capsule formed such as glycerol or this class coating material of sorbitol.Sucking fit formula capsule can contain covalent conjugates, wherein is mixed with: filler or binding agent, such as lactose or starch; Lubricant is such as Talcum or magnesium stearate; With optional stabilizing agent.In soft capsule, covalent conjugates can be dissolved in or be suspended in suitable containing or do not contain in the liquid of stabilizing agent, such as fatty oil, liquid paraffin or liquid macrogol.
Parenterai administration comprises a kind of aqueous solution of or covalent conjugates of the present invention with compositions.In order to inject, can be with the buffer of physical compatibility, covalent conjugates of the present invention is mixed with aqueous solution such as Hank ' s solution, Ringer's solution or physiological buffer saline.Aqueous injection suspension can contain the material that increases this suspension viscosity, such as sodium carboxymethyl cellulose, sorbitol or glucosan.In addition, the suspension of covalent conjugates can be prepared into suitable oily injection suspension.Suitable lipophilic solvent or carrier comprise: fatty oil, such as Oleum sesami; Or synthetic fatty acid ester, such as ethyl oleate or triglyceride; Or liposome.This suspension can also be chosen the reagent that contains suitable stabilizers or increase the covalent conjugates dissolubility wantonly so that prepare highly concentrated solution.
In order to carry out part or nasal administration, the penetrating agent that generally will be suitable for penetrating particular barrier is used for preparation.Their example is 2-Pyrrolidone, N-N-methyl-2-2-pyrrolidone N-, dimethyl acetylamide, dimethyl formamide, propylene glycol, methanol or isopropyl alcohol, dimethyl sulfoxine and Laurel nitrogen ketone.Preparation can also comprise other reagent so that can be accepted in beauty treatment.Their example is fat, wax, oil, dyestuff, spice, antiseptic, stabilizing agent and surfactant.Can also comprise all those cutin distintegrants as known in the art.Example is salicylic acid and sulfur.In order to carry out topical, compositions can be the percutaneous ointment that is used for the described chemical compound of systemic delivery or the form of patch, (for example can prepare in a conventional manner, referring to Barry, " dermatosis preparation " (Dermatological Formulations) (Drugs and the PharmaceuticalSciences--Dekker); Harrys Cosmeticology (Leonard Hill Books).
In order to carry out rectally, can give compositions with the form of suppository or retention enema.Can prepare this based composition by covalent conjugates is mixed with suitable nonirritant excipient, described nonirritant excipient is a solid at normal temperatures, and is liquid under rectal temperature, fusing and discharge medicine in rectum thus.Suitable excipient is including, but not limited to cocoa butter and polyethylene glycols.
These dissimilar additives consumption separately will be apparent to those skilled in the art, and optimum amount is identical with other known formulations that is designed for the same type administration.For example, the horny layer penetration enhancer generally is about 15% concentration of about 0.1%-.
Can according to this area in like the known class mode prepare the compositions that contains covalent conjugates of the present invention mixing, dissolving, granulating, system ingot, grinding, emulsifying, encapsulation, embedding or the lyophilization of routine (for example, by).(for example coating) changes compositions one-tenth so that suitable release characteristics to be provided by conventional methods, and for example slow release or targeting discharge.
Form that can salt provides the compositions that comprises covalent conjugates, and can with many sour salifies, including, but not limited to hydrochloric acid, sulphuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid etc.Salt tends to more be soluble in aqueous or other proton solvent than corresponding free alkali form.
Prepared make this based composition that contains covalent conjugates and acceptable carrier after, they can be put into suitable containers and labelling and use.
The following example has only been explained and has been become known at present implementing best mode of the present invention, but shall not be applied to restriction the present invention.
Embodiment 1
Present embodiment has been described the representational preparation of compositions method of the present invention, and said composition contains one or more and the covalently bound arteannuin dependency endoperoxide molecule of ferritin molecule for the national games.
Synthetic (the ART-NH-NH of Artelinic acid hydrazide 2
the trial of the hydrazide derivatives of preparation artesunate is also unsuccessful, because of cyclization causes forming dihydroartemisinine.As mentioned above by the synthetic artelinate (Shrimali etc. (1998) " India's The Chemicals " (Indian J Chem.) 37B:1161-1163) of dihydroartemisinine.(0.1g 0.24mmol) is dissolved in anhydrous acetonitrile (0.48mL) with artelinic acid.In this solution, add I-hydroxybenzotriazole (HOBt) (0.038g, 0.29mmol), add subsequently ethyl dimethyl ethyl carbodiimides (0.055g, 0.29mmol).At room temperature stir this reactant mixture and, all change into the HOBt ester up to all acid by thin layer chromatography (TLC) monitoring.
the trial of the hydrazide derivatives of preparation artesunate is also unsuccessful, because of cyclization causes forming dihydroartemisinine.As mentioned above by the synthetic artelinate (Shrimali etc. (1998) " India's The Chemicals " (Indian J Chem.) 37B:1161-1163) of dihydroartemisinine.(0.1g 0.24mmol) is dissolved in anhydrous acetonitrile (0.48mL) with artelinic acid.In this solution, add I-hydroxybenzotriazole (HOBt) (0.038g, 0.29mmol), add subsequently ethyl dimethyl ethyl carbodiimides (0.055g, 0.29mmol).At room temperature stir this reactant mixture and, all change into the HOBt ester up to all acid by thin layer chromatography (TLC) monitoring.
With hydrazine (0.46mL, 0.48mmol) solution in acetonitrile (0.48mL) is cooled to 0 ℃, and above-mentioned reactant mixture is joined in this system, simultaneously with temperature maintenance at 0-10 ℃.As what measure, complete at 10 minutes afterreactions by TLC.With in this reactant mixture impouring water (5mL), (the salt water washing is also used in 3 * 10mL) extractions with ethyl acetate.Separate organic facies, with anhydrous sodium sulfate drying and be evaporated to dried.Obtain pure product (0.078g) by the silica gel chromatography purification crude product that uses methanol/chloroform gradient, productive rate is 76%.
Artelinate-ferritin conjugate for the national games synthetic: at room temperature use the 10mmol/L sodium metaperiodate will be dissolved in the ferritin for the national games (2mg) of sodium acetate of 1ml 0.1mol/L pH 5.5 the horizontal oxidation of its polysaccharide 30 minutes.With sample on this solution to the short Sephadex G-25 post that the UV monitor has been installed.With this post of sodium acetate eluting of 0.1mol/L pH 5.5 and collect the protein fraction.In the ferritin for the national games of oxidation, add excessive ART-NH-NH 2Solution also at room temperature keeps this reaction system to spend the night, jolting gently simultaneously.Then with sample on artelinate-ferritin conjugate solution for the national games to Sephadex G-25 post, to remove excessive ART-NH-NH 2With this post of 0.1mol/L Tris-HCl buffer solution elution of pH 7.5, and collect the protein fraction.Conjugate is stored under 4 ℃.
By hydrophobic interaction HPLC purification artelinate-ferritin conjugate for the national games, obtain the protein conjugate of homogeneous.Measure the artesunate molecular number of each ferritin molecule for the national games by ionspray mass spectrometry.
Although explained and described the preferred embodiments of the invention, be appreciated that and can not break away from the spirit and scope of the invention to wherein carrying out various changes.
Embodiment 2
Present embodiment has been described the representational preparation of compositions method of the present invention, and said composition contains the one or more arteannuin dependency endoperoxide molecules covalently bound with haemoglobin molecule.
In order to use the nonglycosylated protein of artemisinin derivative covalent modification, such as hemoglobin, at first with the carboxylic acid derivates of arteannuin, activation is N-hydroxy-succinamide (HOSu) ester such as artelinic acid, so as with protein surface on the lysine residue reaction.With artelinicacid (4.2mg, 0.01mmol) be dissolved in dimethyl formamide (DMF) (0.5mL) in, and this solution cooled off in ice bath.In this solution, add N-ethyl-N '-dimethylaminoethyl carbodiimide (EDC) (1.5mg, 0.01mmol) and HOSu (1.1mg, 0.01mmol).This reactant mixture is kept down stirring 2 hours to be completed into the HOSu ester of artelinic acid at 0 ℃.
Hemoglobin (10mg) is dissolved in 7.0 0.1M phosphate buffer (5mL).DMF solution with artelinicacid HOSu ester under 0 ℃ slowly joins in the hemoglobin solutions.This reactant mixture is kept down stirring 2 hours and at room temperature keeps stirring 2 hours again at 0 ℃.Make on this reactant mixture sample to on the equilibrated Sephadex G-25 of the 0.1M phosphate buffer post of pH 7 then, and with this post of identical buffer solution elution.Modified hemoglobin elutes with void volume.Merge the fraction that contains hemoglobin, then by hydrophobic interaction HPLC purification.Measure the artemisinin derivative number that is connected with protein by ionspray mass spectrometry.
1. the covalent conjugates that comprises the arteannuin dependency endoperoxide covalently bound with carrying the protein of ferrum.
2. the described covalent conjugates of claim 1, wherein said arteannuin dependency endoperoxide comprises artelinate.
3. the described covalent conjugates of claim 1, wherein said arteannuin dependency endoperoxide is a hydrazide derivatives.
4. the described covalent conjugates of claim 2, wherein said arteannuin dependency endoperoxide has following structure:
N=1-3 wherein, m=0-3, the Ar=aryl, and Y=-(C=O) NH-,-NH-or-O-.
5. the described covalent conjugates of claim 1, the wherein said protein that carries ferrum are selected from the group that ferritin for the national games, full milk ferritin, hemoglobin, Hemopexin and the lipocalin protein relevant with neutral gelatinase are formed.
6. the described covalent conjugates of claim 5, the wherein said protein that carries ferrum is ferritin for the national games.
7. the described covalent conjugates of claim 5, the wherein said protein that carries ferrum is the full milk ferritin.
8. the described covalent conjugates of claim 5, the wherein said protein that carries ferrum is mammalian proteins.
9. the described covalent conjugates of claim 5, the wherein said protein behaviour albumen that carries ferrum.
10. the described covalent conjugates of claim 1 is comprising artelinate and ferritin for the national games.
11. the described covalent conjugates of claim 1 is comprising artelinate and full milk ferritin.
12. the described covalent conjugates of claim 1 is comprising artelinate and hemoglobin.
13. a compositions, comprising with the covalent conjugates of the covalently bound arteannuin dependency endoperoxide of the protein that carries ferrum, and pharmaceutically acceptable carrier.
14. the covalent conjugates that comprises the arteannuin dependency endoperoxide covalently bound with carrying the protein of ferrum is used for the treatment of purposes in the medicine of cancer in preparation.
15. the described purposes of claim 14, wherein said arteannuin dependency endoperoxide are selected from the group of artelinate and dihydroartemisinine composition.
16. the described purposes of claim 14, the wherein said chemical compound that carries ferrum is a ferritin for the national games.
17. the described purposes of claim 14, wherein said medicine is made into through parenteral administration.
Be used for the treatment of by the purposes in the experimenter's of pathogenic infection the medicine in preparation with the proteinic covalent conjugates of carrying ferrum 18. contain arteannuin dependency endoperoxide, wherein said pathogen is expressed this proteinic receptor that carries ferrum.
19. the described purposes of claim 18, wherein said arteannuin dependency endoperoxide are selected from the group of artelinate and dihydroartemisinine composition.
20. the described purposes of claim 18, the wherein said protein that carries ferrum are selected from the group that ferritin for the national games, full milk ferritin, hemoglobin, Hemopexin and the lipocalin protein relevant with neutral gelatinase are formed.
21. the described purposes of claim 18, wherein said pathogen comprises helicobacter pylori (Helicobacter pylori), the wherein said protein that carries ferrum comprises human lactoferrin, and wherein said arteannuin dependency endoperoxide is selected from the group of artelinate and dihydroartemisinine composition.
22. the described purposes of claim 18, wherein said pathogen comprises Neisseria meningitidis (Neisseria meningitidis), the wherein said protein that carries ferrum comprises human transferrin, and wherein said arteannuin dependency endoperoxide is selected from the group of artelinate and dihydroartemisinine composition.
0 Comments
Recommended Comments
There are no comments to display.