(T0930-02-10) PK/PD Relationship of a Promising Protein Drug Candidate (CocH5-Fc(M6)) and Its Efficacy in Blocking Cocaine Discrimination in Rats

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. There is still no FDA-approved pharmacotherapy specific for cocaine addiction or overdose. It is highly desired to develop novel therapeutic strategies [1]. We have successfully designed and discovered a long-acting cocaine hydrolase using novel computational enzyme redesign methodologies by introducing mutations into human butyrylcholinesterase (hBChE) and linking it to the N-terminus of human immunoglobulin G1 (IgG1) Fc fragment. The leading drug candidate CocH5-Fc(M6) (i.e. A199S/F227A/P285A/S287G/A328W/Y332G mutant of hBChE: k_{cat = 13,800 min}⁻¹ and K_M = 3.89 μM) with ~4000-fold improved in vitro catalytic efficiency against cocaine compared to wild-type BChE [2,3] is capable of rapidly hydrolyzing cocaine into pharmacologically inactive metabolites. In this study, we aimed to characterize the PK/PD relationship of CocH5-Fc(M6) and its efficacy in inhibiting cocaine interoceptive effects.

Methods: A stable cell line was developed for large-scale production of CocH5-Fc(M6). The purified CocH5-Fc(M6) protein was tested in SD rats for its potency in accelerating cocaine metabolism and efficacy in inhibiting cocaine discriminative stimulus effects. The pharmacokinetic (PK) profile of CocH5-Fc(M6) was determined at different intravenous (IV) dose levels: 0.075, 1.5, 3, and 10 mg/kg by using a sensitive radiometric assay [4]. Meanwhile, the pharmacodynamic (PD) profile of CocH5-Fc(M6) was generated by administering 5 mg/kg cocaine (IV) to the rats on day 15 & day 30 after protein injection with the above-mentioned various doses of CocH5-Fc(M6). Blood samples at various time points were taken and analyzed to determine the blood concentrations of cocaine, BE, and EME by using our previously developed LC-MS/MS protocol [5]. To determine its PK/PD relationship, plots of the obtained cocaine/BE/EME AUCs vs the measured CocH5-Fc(M6) concentration were fitted to E_max model with a baseline effect parameter in GraphPad Prism to calibrate the enzyme effects. The E_max model is based on the equation: E=E₀ ± E_MAX*C/(EC₅₀+C) where E is AUCs of cocaine or its metabolites; E₀ is E value when the enzyme concentration C=0; EC₅₀ is half maximal effective concentration; E_MAX is the maximal effect (AUC) of cocaine or its metabolites. Additionally, long-term effects of CocH5-Fc(M6) on the discriminative stimulus effects of cocaine were determined by injecting different doses of CocH5-Fc(M6) (0.3, 1.0, or 3.0 mg/kg; IV) into three groups of trained rats (n = 6–7/group). Cocaine discrimination tests with cumulative doses of 10, 32, and 56 mg/kg cocaine (IP) were carried out daily until rats allocated at least 80% of their responding on the cocaine-appropriate lever following that dose. Furthermore, a PD experiment was conducted to map the effects of CocH5-Fc(M6) on cocaine metabolism to understand the behavioral outcomes. The concentrations of cocaine and its hydrolytic metabolite EME were analyzed targeting when the plasma CocH5-Fc(M6) at the threshold levels, i.e., 28.7, 9.38, and 2.07 mg/L by using the LC-MS/MS method.

Results: According to the in vivo data, CocH5-Fc(M6) indeed had a markedly prolonged biological half-life (T_1/2 = 229 ± 5 h) and had a linear PK profile. 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). The cocaine discrimination experiment showed that the duration of CocH5-Fc(M6) in blocking cocaine discrimination was dependent on cocaine dose and CocH5-Fc(M6) plasma concentration. A dose of 3 mg/kg CocH5-Fc(M6) effectively attenuated discriminative stimulus effects of 10 mg/kg cocaine, cumulative doses of 10 and 32 mg/kg cocaine, and cumulative doses of 10, 32 and 56 mg/kg cocaine by ≥ 20% for 41, 19, and 10 days, respectively, and completely blocked the discriminative stimulus effects for 30, 13, and 5 days with corresponding threshold plasma CocH5-Fc(M6) concentrations of 2.07, 9.38, and 28.7 mg/L, respectively, under which blood cocaine concentration was negligible [6].

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 long-lasting in vitro and in vivo activity, which is a truly promising therapeutic protein candidate as a treatment for 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, F Zheng, Z Jin, J Deng, X Chen, X Zheng, G Wang, K Kim, L Shang, Z Zhou, C.-G. 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
[6] Long-lasting blocking of interoceptive effects of cocaine by a highly efficient cocaine hydrolase in rats, H Wei, JE LeSaint, Z Jin, CG Zhan, F Zheng, Scientific Reports 14 (1), 927, 2024

Acknowledgements: This work was supported in part by the National Institutes of Health (NIH grants U01 DA051079, UH2/UH3 DA041115, UG3/UH3 NS134920, R01 DA056646, U18 DA052319, R01 DA035552, R01 DA032910, and R01 DA013930). There is no conflict of interest for this study.