Development of a Human Hydrolase to Treat Cocaine Abuse and Overdose: Rat Models

描述（由申请人提供）：一种治疗可卡因滥用的新概念设想从根本上加速可卡因的代谢，作为减少觅药行为的一种手段。我们提出了一项研究的“代谢疗法”可卡因过量和可卡因成瘾的两个关键阶段：稳态维持药物自我管理，药物引发的复发或恢复药物寻求行为后，一段时间的禁欲。我们的治疗剂是一种人血浆胆碱酯酶，通过结构驱动的诱变转化为可卡因水解酶（CocH），该水解酶以异常的速度代谢可卡因，并且比细菌可卡因酯酶稳定得多。注射这种蛋白质可以加速药物消除100倍或更多，防止致命剂量的可卡因在12小时后产生毒性，甚至在惊厥发作开始后也能拯救过量的大鼠。在初步试验中，这种水解酶阻止了先前自我给药的大鼠恢复可卡因寻求行为。这些发现表明，长期递送CocH可能有助于处理可卡因滥用及其相关毒性。该研究计划以三个目标来解决这一前景。首先是研究扩展了令人惊讶的发现，即可卡因过量的毒性在注射CocH后几秒钟内减弱。可卡因激发大鼠新纹状体多巴胺瞬变的广泛药代动力学和机制研究将检验癫痫发作的恢复反映了大脑和血浆之间陡峭的可卡因扩散梯度的建立这一假设。第二是测试CocH对大鼠可卡因自我给药的动机，这些大鼠在几个剂量的药物的渐进比例时间表下工作。还将用CocH预处理大鼠以进一步研究其阻断可卡因引发的可卡因寻求行为的恢复的能力。三是CocH基因转移的研究。第一代腺病毒基因转移载体的初步结果显示，肝脏中可卡因水解酶的高水平转导阻断了新纹状体中delta-FosB的诱导，这是可卡因成瘾的分子信号。我们现在计划测试先进的辅助依赖性腺病毒载体，这种载体应该能在外周和大脑中持续表达数月。沿着病毒载体，我们也将研究通过修饰的干细胞传递水解酶。我们的研究计划是基于这样的前提，即一种或多种基因转移方法将抑制成瘾大鼠的药物寻求行为，并降低复发的倾向。如果这一假设得到证实，以后可能会扩展到人类患者。以前试图阻止这种行为，其中涉及多巴胺能奖励回路的激活，使用多巴胺受体拮抗剂。与这些药物的许多副作用相反，我们希望我们提出的治疗方法在抑制药物寻求的剂量下不会产生不良反应。我们的研究结果应该增加对滥用生物学的理解，并测试可卡因滥用可能通过阻止药物进入大脑靶点的方法有效治疗的概念。项目叙述：蛋白质工程的最新进展导致了一种人类酶，它可以迅速破坏可卡因，阻止它到达心脏或大脑。我们的初步研究结果表明，这种酶可以拯救药物过量后致命的癫痫发作，也可以防止以前上瘾的大鼠在获得可卡因时复发。这项应用将研究是否直接治疗或基因治疗与这种酶可以减少药物寻求行为的大鼠作为一个模型，可卡因成瘾的人。

DESCRIPTION (provided by applicant): A novel concept for treatment of cocaine abuse envisages radically accelerated metabolism of cocaine as a means of reducing drug-seeking behavior. We propose a study of "metabolic therapy" for cocaine overdose and two critical stages of cocaine addiction: steady-state maintenance of drug self-administration, and drug-primed relapse or reinstatement of drug-seeking behavior after a period of abstinence. Our therapeutic agent is a human plasma cholinesterase converted by structure-driven mutagenesis into a cocaine hydrolase (CocH) that metabolizes cocaine with exceptional speed and is far more stable than bacterial cocaine esterase. Injections of this protein hastened drug elimination 100-fold or more, prevented toxicity from a lethal dose of cocaine given up to 12 hr later, and rescued rats from overdose even after convulsive seizures commenced. In preliminary trials this hydrolase prevented reinstatement of cocaine-seeking behavior in rats that had previously self-administered the drug. These findings indicate that long-term delivery of CocH might aid in dealing with cocaine abuse and its associated toxicity. The research plan addresses this prospect with three aims. FIRST are studies to extend the surprising finding that toxicity from cocaine overdose abates in seconds after CocH injection. Extensive pharmacokinetics and mechanistic studies of dopamine transients in the neostriatum of cocaine-challenged rats will test the hypothesis that recovery from seizures reflects the creation of steep cocaine diffusion gradients between brain and plasma. SECOND are tests of CocH on the motivation for cocaine self-administration in rats working under a progressive ratio schedule for several doses of drug. Rats will also be pretreated with CocH to further investigate its ability to block cocaine-primed reinstatement of cocaine-seeking behavior. THIRD are studies on CocH gene transfer. Preliminary results with a first-generation adenoviral gene transfer vector showed that high-level transduction of cocaine hydrolase in liver blocked the induction of delta-FosB in neostriatum, a molecular signal of cocaine addiction. We now plan to test advanced helper-dependent adenoviral vectors that should sustain expression for months in the periphery and the brain. Along with viral vectors we will also examine the delivery of hydrolase by modified stem cells. Our Research Plan is based on the premise that one or more of these gene-transfer approaches will suppress drug-seeking behavior in addicted rats and also reduce the propensity for relapse. This hypothesis, if substantiated, might later be extended to human patients. Previous attempts to block such behavior, which involves activation of dopaminergic reward circuitry, have used dopamine receptor antagonists. In contrast to the many side effects of those agents, we expect our proposed treatment to lack adverse effects at doses that suppress drug seeking. Our results should increase understanding of the biology of abuse and test the concept that cocaine abuse might be effectively treated by methods that prevent drug access to targets in the brain. Program Narrative: Recent advances in protein engineering have led to a human enzyme that destroys cocaine rapidly enough to prevent it from reaching the heart or brain. Our preliminary results show that this enzyme rescues rats from lethal seizures after drug overdose and also prevent formerly addicted rats from relapsing when they get access to cocaine. This application will investigate whether direct treatment or gene therapy with this enzyme can reduce drug-seeking behavior in rats as a model for cocaine addiction in humans.