In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase.

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (- 80, - 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including - 80, - 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

Catalytic activities of a highly efficient cocaine hydrolase for hydrolysis of biologically active cocaine metabolites norcocaine and benzoylecgonine.

Cocaine is a widely abused, hepatotoxic drug without an FDA-approved pharmacotherapy specific for cocaine addiction or overdose. It is recognized as a promising therapeutic strategy to accelerate cocaine metabolism which can convert cocaine to pharmacologically inactive metabolite(s) using an efficient cocaine-metabolizing enzyme. Our previous studies have successfully designed and discovered a highly efficient cocaine hydrolase, denoted as CocH5-Fc(M6), capable of rapidly hydrolyzing cocaine at the benzoyl ester moiety. In the present study, we determined the kinetic parameters of CocH5-Fc(M6) against norcocaine (kcat = 9,210 min-1, KM = 20.9 µM, and kcat/KM = 1.87 × 105 min-1 M-1) and benzoylecgonine (kcat = 158 min-1, KM = 286 µM, and kcat/KM = 5.5 × 105 min-1 M-1) for the first time. Further in vivo studies have demonstrated that CocH5-Fc(M6) can effectively accelerate clearance of not only cocaine, but also norcocaine (known as a cocaine metabolite which is more toxic than cocaine itself) and benzoylecgonine (known as an unfavorable long-lasting metabolite with some long-term toxicity concerns) in rats. Due to the desired high catalytic activity against norcocaine, CocH5-Fc(M6) is capable of quickly detoxifying both cocaine and its more toxic metabolite norcocaine after intraperitoneally administering lethal dose of 60 or 180 mg/kg cocaine. In addition, the ability of CocH5-Fc(M6) to accelerate clearance of benzoylecgonine should also be valuable for the use of CocH5-Fc(M6) in treatment of cocaine use disorder.