CATALYTIC ANTIBODIES FOR REDUCTION OF COCAINE USE

这项工作的目标是开发基于抗体的试剂， 用于人道治疗可卡因成瘾。 这些试剂将 通过以下方式提供针对可卡因药效学作用的保护： 快速降低血浆中可卡因的浓度，从而最大限度地减少 可卡因的正强化它们将与以下方面结合使用： 治疗吸毒行为的其他方法。他们会 也可用于动物体内可卡因的药理学研究。鼠 将开发单克隆抗体，其催化 可卡因和非精神药物实验方法将 包括两个水解反应：（1）可卡因水解为芽子碱 甲酯和（2）可卡因水解为苯甲酰芽子碱。过渡 这些反应的状态类似物（TSA）将被合成并偶联到 载体蛋白将对小鼠进行免疫，并将杂交瘤细胞系 分泌抗TSA抗体的细胞将通过有限稀释法克隆。 将检测单克隆抗TSA抗体的催化活性， 体外催化抗体基因将被测序， 通过定点诱变将增强催化抗体。 催化性抗体或抗体片段，其具有足够的反应性， 体外将在小鼠模型中测试它们修饰 药物代谢动力学和减少可卡因在体内的精神作用。 在体内有活性的抗体或抗体片段将被 可能用于人类。 拟议的工作是多学科性质的，将使用 常规的化学、免疫化学、药理学技术， 分子生物学这些包括过渡态类似物的合成， 缀合物，小鼠免疫和杂交瘤的产生， 通过ELISA纯化杂交瘤上清液， 腹水单克隆抗体，使用化学方法 测量底物向产物的转化，体外动力学和体内 药代动力学程序，运动活性测试，基因克隆， 基因测序，抗体片段的产生，和位点特异性 诱变将开发一种灵敏的半抗原免疫PCR检测方法， 重组抗体的结合和/或催化活性的测试，以及 抗体片段。

The goal of this work is to develop antibody-based reagents which can be used in humane for treatment of cocaine addiction. These reagents would provide protection against the pharmacodynamic action of cocaine by rapidly reducing the concentration of cocaine in plasma, thus minimizing positive reinforcement by cocaine. They would be used in conjunction with other treatment modalities dealing with drug-taking behavior. They would also find use in pharmacological studies of cocaine in animals. Murine monoclonal antibodies will be developed which catalyze the hydrolysis of cocaine to nonpsychoactive products. The experimental approach will involve two hydrolysis reactions-(1) hydrolysis of cocaine to ecgonine methyl ester and (2) hydrolysis of cocaine to benzoylecgonine. Transition state analogs (TSA) far these reactions will be synthesized and coupled to carrier proteins. Mice will be immunized, and hybridoma cell lines secreting anti-TSA antibodies will be cloned by limiting dilution. Monoclonal anti-TSA antibodies will be tested for catalytic activity in vitro. Catalytic antibody genes will be sequenced and properties of catalytic antibodies will be enhanced by site-directed mutagenesis. Catalytic antibodies or antibody fragments with sufficient reactivity in vitro will be tested in a mouse model for their ability to modify the pharmacokinetics and reduce the psychoactive effects of cocaine in vivo. Antibodies or antibody fragments which are active in vivo will be engineered for possible use in humans. The proposed work is multidisciplinary in nature and will be done using conventional techniques of chemistry, immunochemistry, pharmacology, and molecular biology. These include synthesis of transition state analogs and conjugates, immunization of mice and production of hybridomas, testing of hybridoma supernatants by ELISA, purification of high concentrations of monoclonal antibodies from ascites fluids, the use of chemical methods for measuring conversion of substrate to product, in vitro kinetic and in vivo pharmacokinetic procedures, tests of locomotor activity, gene cloning, gene sequencing, production of antibody fragments, and site-specific mutagenesis. A sensitive immuno-PCR assay for haptens will be developed to test for binding and/or catalytic activity of recombinant antibodies and antibody fragments.