LETTER LETTER REPLY TO CURRY AND COOMBS: Benzoic acid is formed predominantly from the benzoyl ester hydrolysis in the presence of cocaine hydrolase

Fang Zhenga,b,1 and Chang-Guo Zhana,b,1

As is well known, and also pointed out correctly by the detected highest NC concentration generated by Curry and Coombs (1), cocaine has three metabolic intravenous injection of 5 mg/kg cocaine was 4.9 ng/mL, pathways associated with butyrylcholinesterase (BChE), less than 1% of the highest cocaine concentration carboxylesterase, and microsomal cytochrome P450 of ∼1,700 ng/kg. Hence, the NC concentrations were 3A4, which convert cocaine to ecgonine methyl ester negligible compared with the corresponding co- (EME) + benzoic acid (BA), benzoylecgonine (BE) + caine and BE concentrations under the experimental methanol, and norcocaine (NC), respectively. However, conditions used in our study in rats (2). Therefore, both in contrast to what Curry and Coombs describe, BA BA and EME are formed predominantly and equally cannot be formed from EME. Instead, further hydrolysis from the hydrolysis of the cocaine benzoyl ester group of EME can only produce ecgonine and methanol. (cocaine → EME + BA). It is reasonable to detect either Therefore, further metabolism of EME does not contrib- EME or BA for analyzing the effects of CocH3-Fc(M3) ute to BA formation at all. The questions raised by Curry on cocaine metabolism via the benzoyl ester hydroly- and Coombs (1) are based on a misunderstanding of the sis, though it would not hurt to detect all of the me- metabolic pathway via EME. tabolites individually, as we have done more recently. Concerning cocaine metabolites from the remain- The other two cocaine-metabolizing enzymes [i.e., ing pathways, it is well-known that BE is extremely RBP-8000 (6) and TV-1380 (3, 7)] mentioned by Curry stable and, therefore, has a very long half-life in the and Coombs (1) are not Fc-fused enzymes. Actually, body. In fact, BE is a commonly used cocaine bio- RBP-8000 is our previously reported thermally stable marker for cocaine addiction diagnosis in human T172R/G173Q mutant (8) of a bacterial cocaine ester- drug tests. Theoretically, NC may be hydrolyzed to ase, and TV-1380 is a human serum albumin-fused form BA. However, under the experimental condi- BChE mutant: that is, our previously designed, discov- tions described in our study (2), the NC pathway is ered, and reported A199S/S287G/A328W/Y332G mu- actually negligible. As is well known, when a highly tant of human BChE (9, 10). CocH3-Fc(M3) reported efficient cocaine-metabolic enzyme, such as CocH3 in our study (2) is the first Fc-fusion enzyme (antico- or CocH3-Fc(M3) (2), is available to considerably ac- caine catalytic antibody analog) capable of catalyz- celerate cocaine hydrolysis to EME + BA in the body, ing cocaine hydrolysis. cocaine metabolism via the other pathways are signif- icantly reduced (3–5). In fact, we tried to measure the Acknowledgments concentrations of other metabolites (including EME, This work was supported in part by the National NC, BE, and ecgonine) in the blood samples collected Institutes of Health (NIH Grants UH2 DA041115, R01 in our study (2), and concluded that there were no DA035552, R01 DA032910, R01 DA013930, and R01 detectable NC concentrations in the blood samples. DA025100) and the National Science Foundation Even without administration of an exogenous enzyme, (NSF Grant CHE-1111761).

1 Curry SH, Coombs SE (2016) Benzoic acid is not the only important product of accelerated metabolism of cocaine. Proc Natl Acad Sci USA 113:E2101. 2 Chen X, et al. (2016) Long-acting cocaine hydrolase for addiction therapy. Proc Natl Acad Sci USA 113(2):422–427.

aMolecular Modeling and Biopharmaceutical Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536; and bDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536 Author contributions: F.Z. and C.-G.Z. designed research, performed research, analyzed data, and wrote the paper. The authors declare no conflict of interest. 1To whom correspondence may be addressed. Email: [email protected] or [email protected].

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