Cocaine blocks dopamine uptake via dopamine transporter (DAT) on plasma membrane of neuron cells and, as a result, produces the high and induces DAT trafficking to plasma membrane which contributes to the drug seeking or craving. In this study, we first examined the dose dependence of cocaine-induced DAT trafficking and hyperactivity in rats, demonstrating that cocaine at an intraperitoneal dose of 10 mg/kg or higher led to redistribution of most DAT to the plasma membrane while inducing significant hyperactivity in rats. However, administration of 5-mg/kg cocaine (ip) did not significantly induce DAT trafficking or hyperactivity in rats. So the threshold (intraperitoneal) dose of cocaine that can significantly induce DAT trafficking or hyperactivity should be between 5 and 10 mg/kg. These data suggest that when a cocaine dose is high enough to induce significant hyperactivity, it can also significantly induce DAT trafficking to the plasma membrane. Further, the threshold brain cocaine concentration required to induce significant hyperactivity and DAT trafficking was estimated to be $2.0 ± 0.8 μg/g. Particularly, for treatment of cocaine abuse, previous studies demonstrated that an exogenous cocainemetabolizing enzyme, for example, CocH3-Fc(M3), can effectively block cocaineinduced hyperactivity. However, it was unknown whether an enzyme could also effectively block cocaine-induced DAT trafficking to the plasma membrane. This study demonstrates, for the first time, that the enzyme is also capable of effectively blocking cocaine from reaching the brain even with a lethal dose of 60-mg/kg cocaine (ip) and, thus, powerfully preventing cocaine-induced physiological effects such as the hyperactivity and DAT trafficking.

Dysregulation of dopamine transporters (DAT) within the dopaminergic system is an important biomarker of cocaine exposure. Depending on cocaine amount in-taken, one-time exposure in rats could lead to most (>95% of total) of DAT translocating to plasma membrane of the dopaminergic neurons compared to normal DAT distribution ($5.7% on the plasma membrane). Without further cocaine exposure, the time course of striatal DAT distribution, in terms of intracellular and plasma membrane fractions of DAT, represents a recovery process of the dopaminergic system. In this study, we demonstrated that after an acute cocaine exposure of 20 mg/kg (i.p.), the initial recovery process from days 1 to 15 in rats was relatively faster (from >95% on day 1 to $35.4% on day 15). However, complete recovery of the striatal DAT distribution may take about 60 days. In another situation, with repeated cocaine exposures for once every other day for a total of 17 doses of 20 mg/kg cocaine (i.p.) from days 0 to 32, the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which represents a consequence of chronic cocaine use. Further, we demonstrated that a highly efficient Fc-fused cocaine hydrolase, CocH5-Fc(M6), effectively blocked cocaine-induced hyperactivity and DAT trafficking with repeated cocaine exposures by maintaining a plasma CocH5-Fc (M6) concentration ≥58.7 ± 2.9 nM in rats. The cocaine hydrolase protected dopaminergic system and helped the cocaine-altered DAT distribution to recover by preventing the dopaminergic system from further damage by cocaine.

It is particularly challenging to develop a truly effective pharmacotherapy for cocaine use disorder (CUD) treatment. Accelerating cocaine metabolism via hydrolysis at cocaine benzoyl ester using an efficient cocaine hydrolase (CocH) is known as a promising pharmacotherapeutic approach to CUD treatment. Preclinical and clinical studies on our first CocH (CocH1), in its human serum albumin-fused form known as TV-1380, have demonstrated the promise of a general concept of CocH-based pharmacotherapy for CUD treatment. However, the biological half-life of TV-1380 (t 1/2 = 8 h in rats, associated with t 1/2 = 43−77 h in humans) is not long enough for practical treatment of cocaine dependence, which requires enzyme injection for no more than once weekly. Through protein fusion of a human butyrylcholinesterase mutant (denoted as CocH5) with a mutant (denoted as Fc(M6)) of Fc from human IgG1, we have designed, prepared, and tested a new fusion protein (denoted as CocH5-Fc(M6)) for its pharmacokinetic profile and in vivo catalytic activity against (−)-cocaine. CocH5-Fc(M6) represents the currently most efficient long-acting cocaine hydrolase with both the highest catalytic activity against (−)-cocaine and the longest elimination half-life (t 1/2 = 229 ± 5 h) in rats. As a result, even at a single modest dose of 3 mg/kg, CocH5-Fc(M6) can significantly and effectively accelerate the metabolism of cocaine in rats for at least 60 days. In addition, ∼70 nM CocH5-Fc(M6) in plasma was able to completely block the toxicity and physiological effects induced by intraperitoneal injection of a lethal dose of cocaine (60 mg/kg).

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