CONVERSION OF IRINOTECAN (CPT-11) TO ITS ACTIVE METABOLITE, 7-ETHYL-10-HYDROXYCAMPTOTHECIN (SN-38), BY HUMAN LIVER CARBOXYLESTERASE

We have investigated the conversion of the novel anti-topoisomerase I agent CPT-11 (irinotecan; -piperidino)-1-piperidino]carbonyloxycamptot hecin) to its active metabolite, SN-38 (7- ethyl-10-hydroxycamptotheci n), by human liver carboxylesterase (HLC). Production of SN-38 was rel atively inefficient and was enzyme deacylation rate-limited with a ste ady-state phase occurring after 15-20 min of incubation. This later ph ase followed Michaelis-Menten kinetics with an apparent K-m, of 52.9 /- 5.9 mu M and a specific activity of 200 +/- 10 mu mol/sec/mol. Howe ver, the total enzyme concentration estimated from the intercept conce ntrations of SN-38 was much lower than that estimated directly from th e titration of active sites with paraoxon (0.65 vs. 2.0 mu M, respecti vely). Because deacylation rate-limiting kinetics result in the accumu lation of inactive acyl-enzyme complex, we postulated that incubation of CPT-11 with HLC would result in an inhibition of the HLC-catalysed hydrolysis of p-nitrophenylacetate (p-NPA), an excellent substrate for this enzyme. Indeed, this was found to be the case although complete inhibition could not be attained. Analysis of possible kinetic schemes revealed that the most likely explanation for the disparity in estima ted enzyme concentrations and the incomplete inhibition of p-NPA hydro lysis is that CPT-11 also interacts at a modulator site on the enzyme, which profoundly reduces substrate hydrolysis. Furthermore, loperamid e, a drug often used for the treatment of CPT-11-associated diarrhea, was found to inhibit both CPT-11 and p-NPA HLC-catalysed hydrolysis, m ost likely by a similar interaction. These observations have direct im plications for the clinical use of CPT-11.