In vitro transesterification of cocaethylene (ethylcocaine) in the presence of ethanol: Esterase-mediated ethyl ester exchange

This study reports that cocaethylene undergoes an esterase-mediated ethyl ester exchange with ethanol, resulting in an increase in the apparent in vitro t( 1/4 ), compared with control conditions. Homogenized liver from male Sprague Dawley rats in pH 7.4 phosphate buffer was centrifuged at 9000g, and the resulting supernatant (S9) fraction was collected. Tubes containing the rat S9 fraction and 50 μM cocaethylene plus aqueous buffer (control), 50 mM ethanol, or 51.3 mM ²H₆-ethanol were incubated at 37°C for 4 hr. Samples were collected from the incubation tubes at various times, extracted with a solid-phase extraction system, and assayed for cocaethylene and ²H₅- cocaethylene by GC/MS. Concentration-time profiles were constructed and kinetic parameters were determined. The experiment was repeated in the presence of specific and nonspecific esterase inhibitors. Enzyme kinetic parameters were also determined. Cocaethylene underwent ethyl ester exchange, being converted to ²H₆-cocaethylene in the presence of ²H₆-ethanol. The average apparent in vitro t( 1/4 ) value for cocaethylene (13.0 ± 1.4 min) incubated with the S9 fraction and buffer only was increased ~5-fold (67.8 ± 0.3 min) in the presence of ethanol. Formation of ²H₆-cocaethylene was totally blocked with the addition of bis-(p-nitrophenyl)phosphate but was unaffected by physostigmine. The intrinsic metabolite formation clearance of ²H₆-cocaethylene from cocaethylene and ²H₆-ethanol (1.92 ± 0.03 μl/min- mg protein) was several times greater than the corresponding value for cocaethylene formation from cocaine and ethanol (0.94 ± 0.01 μl/min-mg protein) or ²H₆-ethanol (0.87 ± 0.04 μl/min-mg protein).