Reply to Curry and Coombs: Benzoic acid is formed predominantly from the benzoyl ester hydrolysis in the presence of cocaine hydrolase.

As is well known, and also pointed out correctly by Curry and Coombs (1), cocaine has three metabolic pathways associated with butyrylcholinesterase (BChE), carboxylesterase, and microsomal cytochrome P450 3A4, which convert cocaine to ecgonine methyl ester (EME) + benzoic acid (BA), benzoylecgonine (BE) + methanol, and norcocaine (NC), respectively. However, in contrast to what Curry and Coombs describe, BA cannot be formed from EME. Instead, further hydrolysis of EME can only produce ecgonine and methanol. Therefore, further metabolism of EME does not contribute to BA formation at all. The questions raised by Curry and Coombs (1) are based on amisunderstanding of the metabolic pathway via EME. Concerning cocaine metabolites from the remaining pathways, it is well-known that BE is extremely stable and, therefore, has a very long half-life in the body. In fact, BE is a commonly used cocaine biomarker for cocaine addiction diagnosis in human drug tests. Theoretically, NC may be hydrolyzed to form BA. However, under the experimental conditions described in our study (2), the NC pathway is actually negligible. As is well known, when a highly efficient cocaine-metabolic enzyme, such as CocH3 or CocH3-Fc(M3) (2), is available to considerably accelerate cocaine hydrolysis to EME + BA in the body, cocaine metabolism via the other pathways are significantly reduced (3–5). In fact, we tried to measure the concentrations of other metabolites (including EME, NC, BE, and ecgonine) in the blood samples collected in our study (2), and concluded that there were no detectable NC concentrations in the blood samples. Even without administration of an exogenous enzyme, the detected highest NC concentration generated by intravenous injection of 5 mg/kg cocaine was 4.9 ng/mL, less than 1% of the highest cocaine concentration of ∼1,700 ng/kg. Hence, the NC concentrations were negligible compared with the corresponding cocaine and BE concentrations under the experimental conditions used in our study in rats (2). Therefore, both BA and EME are formed predominantly and equally from the hydrolysis of the cocaine benzoyl ester group (cocaine→ EME + BA). It is reasonable to detect either EME or BA for analyzing the effects of CocH3-Fc(M3) on cocaine metabolism via the benzoyl ester hydrolysis, though it would not hurt to detect all of the metabolites individually, as we have done more recently. The other two cocaine-metabolizing enzymes [i.e., RBP-8000 (6) and TV-1380 (3, 7)] mentioned by Curry and Coombs (1) are not Fc-fused enzymes. Actually, RBP-8000 is our previously reported thermally stable T172R/G173Q mutant (8) of a bacterial cocaine esterase, and TV-1380 is a human serum albumin-fused BChE mutant: that is, our previously designed, discovered, and reported A199S/S287G/A328W/Y332Gmutant of human BChE (9, 10). CocH3-Fc(M3) reported in our study (2) is the first Fc-fusion enzyme (anticocaine catalytic antibody analog) capable of catalyzing cocaine hydrolysis.