Evaluation in Primates of Cocaine Esterase for the Treatment of Cocaine Toxicity

描述（申请人提供）：古柯酯酶（CocE）是生长在南美洲古柯植物根际的红球菌属细菌的产物。这种细菌利用CocE分解可卡因，以提供其唯一的碳和氮来源。CocE是迄今为止观察到的最有效的可卡因酯酶。我们在啮齿动物体内和体外研究了它，发现它能够立即逆转这些动物中致命剂量的可卡因的影响。虽然CocE似乎是可卡因过量的极好治疗方法，但其作为可卡因滥用治疗的有用性受到其非常短的半衰期的限制。一项资助的赠款（DA021416）目前正在开发具有更大热稳定性和更长作用持续时间的CocE突变体，并在可卡因毒性的啮齿动物模型中对其进行测试。本申请的目的是评价非人灵长类动物中的天然CocE，作为使这种酶可用于治疗人类可卡因过量的过程中的一个步骤。该项目的每一年都将评估单一版本的CocE，从天然酶开始，并通过突变体进行，这些突变体已被证明具有更长的作用时间。每年将对每种酶依次进行三种类型的研究。第一个将确定有效剂量的酶，以减少可卡因对心血管的影响，并从恒河猴血浆中清除一系列剂量的可卡因。第二项研究将侧重于测量每种酶的免疫反应，当它以最大限度地提高滴度的间隔重复给药时。在该过程中还将研究CocE滴度发展对酶降低可卡因作用的能力的影响。第三项研究涉及确定CocE及其长效突变体防止可卡因强化作用和改变自我服用可卡因的心血管效应的能力。将重复获得每种酶的剂量反应函数，并分析血液样品的滴度发展，以便监测对CocE作用的耐受性。

DESCRIPTION (provided by applicant): Cocaine esterase (CocE) is a product of the bacterium Rhodococcus sp. which grows in the rhizospheres of coca plants in South America. The bacterium uses CocE to break down cocaine to provide its sole source of carbon and nitrogen. CocE is the most efficient cocaine esterase yet observed. We have studied it in vitro and in vivo in rodents and found that it is able to reverse immediately the effects of lethal doses of cocaine in these animals. Although CocE appears to be a superb treatment for cocaine overdose, its usefulness as a treatment for cocaine abuse is limited by its very short half life. A funded grant (DA021416) is currently developing mutants of CocE that have greater thermostability and longer durations of action and testing them in rodent models of cocaine toxicity. The purpose of the current application is to evaluate native CocE in non-human primates as a step in the process of making this enzyme available for treatment of cocaine overdose in humans. Each year of the project will evaluate a single version of CocE, starting with the native enzyme, and proceeding through mutants that have been shown to have substantially longer durations of action. Three types of studies will be conducted sequentially in each year with each enzyme. The first will identify effective doses of the enzyme for reducing the cardiovascular effects of cocaine and clearing a range of doses of cocaine from the plasma of rhesus monkeys. The second study will focus on measuring immunological responses to each enzyme when it is administered repeatedly at intervals that should maximize development of titers. The effect of CocE titer development on the ability of the enzyme to reduce the effects of cocaine will also be studied in this process. The third study involves determination of the ability of CocE and its longer-acting mutants to prevent the reinforcing effects of cocaine and to modify the cardiovascular effects of self-administered cocaine. Dose-response functions for each enzyme will be obtained repeatedly and blood samples analyzed for titer development so that tolerance to the effects of CocE can be monitored.