Monica R. Brzezinski

Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine.

The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.

Gender-specific differences in activity and protein levels of cocaine carboxylesterase in rat tissues

The gender-specific differences in the content of cocaine methyl esterase and ethyl transferase activities are examined in rat tissues and related to differences in hydrolase A protein in rat liver, lung, and kidney reported previously. The rat hydrolase A catalyzes the conversion of cocaine to benzoylecgonine and the ethyl transesterification of cocaine to form cocaethylene. An HPLC assay was used to quantitate and compare cocaine esterase activities in male and female rat tissues. The cocaine methyl esterase and ethyl transferase activities are 1.4 to 2.5 fold greater in male than in female liver and slightly greater in female than in male lung. No gender-specific differences were detected in the kidney. Gel electrophoresis was used to separate three non-specific carboxylesterases (hydrolases A, B, and C) in rat tissues and the isoenzymes were visualized with a hydrolase activity stain using 4-methylumbelliferyl acetate as substrate. The activity of cocaine methyl esterase and content of hydrolase A protein are not consistently different in the lung or the kidney of male versus female rats. Activity of hydrolase A in gels of male liver is greater than in female liver. Similarly, the content of the corresponding hydrolase A immunoreactive protein in male liver is 1.6 fold greater than in female liver. In contrast to hydrolase A, hydrolase C activity is greater in gels of female than male liver extracts. The greater content of cocaine methyl esterase and ethyl transferase activity in male versus female rat livers suggests that there may be gender-specific differences in pharmacokinetics of cocaine metabolism and extent of cocaine-induced hepatotoxicity in rats.

CONVENIENT SYNTHESIS OF BENZOYLECGONINE ETHYL ESTER, A HOMOLOG OF COCAINE

Abstract Benzoylecgonine reacted with tetramethylethylenediamine to form a lipophilic ion pair, which was alkylated in the absence of water. The ethyl ester was readily recrystallized for pharmacological studies.