(W1030-10-68) In Vitro and In Vivo Potency of a Highly Efficient Cocaine Hydrolase and Its Long-Term Stability

Purpose: Cocaine use disorder, including cocaine addiction and overdose, continues to impose major burdens on affected individuals and communities. It is a grand challenge to develop a truly effective pharmacotherapy to tackle this chronic relapsing disease. Traditional pharmacodynamic (PD) approaches that aim to antagonize the action of cocaine have been very elusive. There is still no FDA-approved pharmacotherapy specific for cocaine addiction or overdose. It is highly desired to develop novel therapeutic strategies [1]. Our previous studies have successfully designed and discovered a highly efficient cocaine hydrolase, denoted as CocH5-Fc(M6) (with k_cat = 13,800 ± 750 min^-1 and K_M = 3.89 ± 0.76 μM against (-)-cocaine at 37 ^oC) [2,3] capable of rapidly hydrolyzing cocaine into pharmacologically inactive metabolite(s). In this study, we aimed to characterize CocH5-Fc(M6) for its in vitro and in vivo potency in accelerating cocaine metabolism and its long-term stability as a therapeutic candidate.

Methods: A stable cell line was developed for large-scale production of CocH5-Fc(M6). Using the stable cell line, the purified CocH5-Fc(M6) protein was characterized in vitro, as well as in vivo in Sprague-Dawley rats for its potency and stability in accelerating cocaine hydrolysis at the benzoyl ester moiety. The pharmacokinetic (PK) profile of CocH5-Fc(M6) was determined at five different dose levels ̶ 0.075, 1.5, 3, and 10 mg/kg, intravenously (IV), by using a sensitive radiometric assay based on toluene extraction of [³H](-)-cocaine labeled on its benzene ring [4] Rats were also challenged by IV administration of 5 mg/kg cocaine at various plasma concentrations of CocH5-Fc(M6) to determine its PK/PD relationship. Blood samples were collected at 13 time points within 0-180 minutes after the IV administration of cocaine. Blood samples collected from rats after the cocaine administration were analyzed by using our previously developed LC-MS/MS protocol capable of simultaneously determine the time-dependent concentrations of cocaine and its metabolites in the whole blood [5]. Additionally, the in vitro and in vivo stability of CocH5-Fc(M6) was tested after the CocH5-Fc(M6) protein was stored at various temperatures (-80, -20, 4, 25, and 37 ^oC) for 1, 6, 12, and 18 months. The in vitro stability was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and in vitro catalytic activity for cocaine hydrolysis was measured by the radiometric assay. The in vivo stability of CocH5-Fc(M6) was determined by comparing PK profiles of 0.075 mg/kg CocH5-Fc(M6) at day 0 to those at 18-month after stored under different temperature conditions.

Results: According to the in vivo activity data, CocH5-Fc(M6) indeed had a markedly prolonged biological half-life (t_1/2 = 229 ± 5 h), and the biological/elimination half-life was independent of the dose. Concerning the in vivo potency of CocH5-Fc(M6) against cocaine, a PK/PD relationship was established, which indicates that CocH5-Fc(M6) at a plasma concentration of ~0.15 mg/L was able to decrease the 5 mg/kg cocaine’s presence by 50% in blood; when the enzyme concentration was 0.5 mg/L or higher, cocaine was rapidly eliminated with a negligible area under curve (AUC) compared to the baseline cocaine AUC (when C = 0). Further, according to the in vitro and in vivo evaluations, CocH5-Fc(M6) is a dimer with very stable in vitro properties at all the storage temperatures tested, except for 37 ^oC. No significant catalytic activity or structure change was observed when the protein was stored at -80, -20, 4, or 25 ^oC for 18 months.

Conclusion: CocH5-Fc(M6) represents the currently most efficient long-acting cocaine hydrolase with both the highest catalytic activity against the naturally occurring (-)-cocaine and the longest elimination half-life (t_1/2 = 229±5 h) in rats. CocH5-Fc(M6) has the desired in vivo potency in accelerating cocaine metabolism and high in vitro and in vivo stability as a truly promising therapeutic protein candidate for treatment of cocaine use disorder.

References: [1] Cocaine hydrolases designed from butyrylcholinesterase
F Zheng, CG Zhan
“Biologics to Treat Substance Use Disorders: Vaccines, Monoclonal Antibodies, and Enzymes”,
Springer International Publishing, 2016, pp187-225.
[2] A highly efficient cocaine-detoxifying enzyme obtained by computational design
F Zheng, L Xue, S Hou, J Liu, M Zhan, W Yang, CG Zhan
Nature Communications 5, 3457, 2014
[3] Development of a highly efficient long-acting cocaine hydrolase entity to accelerate cocaine metabolism
Fang Zheng, Zhenyu Jin, Jing Deng, Xiabin Chen, Xirong Zheng, Guojun Wang, Kyungbo Kim, Linyue Shang, Ziyuan Zhou, Chang-Guo Zhan
Bioconjugate Chemistry 33 (7), 1340-1349, 2022
[4] Long-acting cocaine hydrolase for addiction therapy
X Chen, L Xue, S Hou, Z Jin, T Zhang, F Zheng, CG Zhan
Proceedings of the National Academy of Sciences 113 (2), 422-427, 2016
[5] A quantitative LC–MS/MS method for simultaneous determination of cocaine and its metabolites in whole blood
X Chen, X Zheng, K Ding, Z Zhou, CG Zhan, F Zheng
Journal of Pharmaceutical and Biomedical Analysis 134, 243-251, 2017

Acknowledgements: This work was supported in part by the National Institutes of Health (NIH grants U01 DA051079, UH2/UH3 DA041115, R01 DA056646, U18 DA052319, R01 DA035552, R01 DA032910, and R01 DA013930). There is no conflict of interest in this study.