CATALYTIC ANTIBODIES FOR REDUCTION OF COCAINE USE

这项工作的目标是开发基于抗体的试剂，这种试剂可以 用于人道治疗可卡因成瘾。这些试剂会 通过以下方式提供针对可卡因药效作用的保护 迅速降低血浆中可卡因浓度，从而最大限度地减少 可卡因的阳性强化。它们将与以下内容一起使用 其他涉及吸毒行为的治疗方式。他们会 在动物体内的可卡因的药理研究中也有用处。小鼠 将开发出催化水解酶的单抗。 从可卡因到非精神活性产品。试验性的方法将 涉及两个水解反应--(1)可卡因的水解制依古宁 甲酯和(2)可卡因的水解制得苯甲酰ecGonine。过渡 到目前为止，这些反应将被合成并耦合到状态类似物(TSA) 载体蛋白。小鼠将被免疫，杂交瘤细胞株 分泌抗TSA抗体将通过有限稀释法进行克隆。 将测试抗TSA单抗的催化活性 体外培养。将对催化抗体基因进行测序，并对其性质 催化抗体将通过定点突变得到增强。 具有足够反应性的催化抗体或抗体片段 体外实验将在小鼠模型中测试它们是否有能力修改 体内药代动力学和降低可卡因的精神活性作用。 体内活跃的抗体或抗体片段将是 为可能用于人类而设计的。 拟议的工作是多学科性质的，将使用 传统的化学、免疫化学、药理学和 分子生物学。其中包括过渡态类似物的合成和 结合物、小鼠免疫和杂交瘤的生产、检测 杂交瘤细胞培养上清液的酶联免疫吸附试验 腹水中的单抗，使用化学方法检测 测定底物到产物的转化率、体外动力学和体内动力学 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。将建立一种灵敏的免疫-聚合酶链式反应半抗原检测方法 重组抗体的结合和/或催化活性测试 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.