Research Assistant University of Kentucky Lexington, Kentucky, United States
Purpose: As most cocaine users drink alcohol, it is interesting to understand how alcohol affects the metabolism and toxicity of cocaine. It is known that concurrent use of cocaine and alcohol produces additional metabolites and that multiple metabolites of cocaine are even more toxic than cocaine itself.[1] It is essential to know how alcohol changes the metabolism and toxicity of cocaine for the development of a truly effective therapeutic enzyme to detoxify cocaine when co-administered with alcohol. In this report, we investigated the effects of alcohol on cocaine metabolism and toxicity. Our previous studies demonstrated that a cocaine hydrolase (denoted as CocH5) designed and discovered by us can rapidly reverse the cocaine toxicity whenever the enzyme is given to living rats.[2] The effectiveness of the Fc-fused cocaine hydrolase (denoted as CocH5-Fc(M6)) for treatment of cocaine toxicity in combination with alcohol was evaluated in this study. Methods: The cocaine toxicity in rats with or without alcohol co-administration is characterized by not only the commonly used LD50, but also the average times for the appearance of convulsion and death as well as total toxicity level (TTL) in the blood. The pharmacokinetic (PK) profiles of cocaine (including the time courses of cocaine and its metabolites) in rats were determined by LC-MS/MS analysis. The PK parameters for cocaine and its metabolites were calculated by using the MATLAB software and the non-compartmental model coded in the Phoenix WinNonlin software (Certara, Princeton, NJ).
CocH5-Fc(M6) is an Fc-fusion protein with CocH5 (the A199S/F227A/P285A/S287G/A328W /Y332G mutant of human butyrylcholinesterase) fused with the sextuple-mutated (A1V/M38Y/S40T/T42E/D142E/L144M) Fc region of human IgG1. In order to produce the protein, the cDNA for the CocH5-Fc(M6) was first constructed via site-directed mutagenesis by using the QuikChange method. A lentivirus-based method (using the pCSC-SP-PW lentiviral vector) which we described previously [3, 4] was used to generate a stable (CHO-S) cell pool expressing CocH5-Fc(M6). The obtained stable CHO-S cell pool was kept frozen before use for protein production. The CocH5-Fc(M6) protein production was performed in an agitated bioreactor BioFlo/CelliGen 115 (Eppendorf, Hauppauge, NY). The protein was purified by using the protein A affinity chromatography with an ÄKTA Avant 150 system (GE Healthcare Life Sciences, Pittsburgh, PA). The purified protein was dialyzed in storage buffer and stored at −80°C before use.
Protection experiment was performed by pretreatment of rats (n=10) with 1 mg/kg CocH5-Fc(M6) (IV) 1 min before administration of 180 mg/kg cocaine (IP) with 1 g/kg alcohol (IP). Recuse experiment with 1 mg/kg CocH5-Fc(M6) (IV) was also performed to the rats (n=10) once convulsion started. Following the cocaine/enzyme administration, rats were immediately placed in containers for observation. Results: The animal data revealed that co-administration of alcohol markedly decreased the LD50 of cocaine from 73 mg/kg to 56 mg/kg (~23% decrease) and shortened the average times for the appearance of death and/or convulsion after cocaine administration. According to the obtained PK data, the alcohol-enhanced toxicity of cocaine is mainly attributed to two additional metabolites (cocaethylene and norcocaethylene) that are more toxic than cocaine itself. When co-administered with 1 g/kg alcohol and 180 mg/kg cocaine (IP), all rats (n=10) started convulsion at ~2 min and died at ~3 min. 1 mg/kg CocH5-Fc(M6) (IV) fully protected or rescued all the rats from the acute toxicity of 180 mg/kg cocaine with 1 g/kg alcohol; no convulsion or death were observed in any rats administered with CocH5-Fc(M6). Conclusion: All these toxicity data have consistently demonstrated that co-administration of alcohol makes cocaine more toxic. and that the alcohol-enhanced toxicity of cocaine is mainly attributed to the observed two additional metabolites (cocaethylene and norcocaethylene) that are more toxic than cocaine itself. So, evaluation of the substance TTL should account for the blood levels of not only cocaine itself, but also all its toxic metabolites. With CocH5-Fc(M6), all the rats were protected or rescued from the lethality of combined 180 mg/kg cocaine and 1 g/kg alcohol; cocaine and other toxic metabolites were rapidly and completely converted to physiologically inactive metabolites according to our LC-MS/MS analysis. References: 1. Zheng, X., et al., In vivo characterization of toxicity of norcocaethylene and norcocaine identified as the most toxic cocaine metabolites in male mice. Drug Alcohol Depend, 2019. 204: p. 107462. 2. Zheng, F., et al., A highly efficient cocaine-detoxifying enzyme obtained by computational design. Nat Commun, 2014. 5: p. 3457. 3. Chen, X., et al., Long-acting cocaine hydrolase for addiction therapy. Proc Natl Acad Sci U S A, 2016. 113(2): p. 422-7. 4. Xue, L., et al., Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine. Biochem J, 2013. 453(3): p. 447-54.
Acknowledgements: This work was supported in part by the National Institutes of Health (NIH grants U01 DA051079, UH2/UH3 DA041115, U18 DA052319, R01 DA035552, R01 DA032910, and R01 DA013930) and the National Science Foundation (NSF grant CHE-1111761).
