The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.