Analysis of the human intestinal carboxylesterase and development of specific inhibitors to ameliorate the delayed diarrhea associated with CPT-ll administration.

5526 Carboxylesterases (CE) are members of the esterase class of enzymes and are involved in the metabolism of a broad range of xenobiotic substrates, including activation of the potent anticancer prodrug CPT-11. However, the high level expression of an intestinal CE in the gut appears to be partially responsible for the dose limiting toxicity (delayed diarrhea) for this drug. Therefore the development of specific inhibitors of this enzyme that prevent CPT-11 to SN-38 conversion in the intestine may serve to reduce the toxicity associated with CPT-11. The work reported here describes the purification, kinetic analysis, and biochemical characterization of a human intestinal CE (hiCE). This includes the analysis of the metabolism of clinically relevant substrates including CPT-11, heroin, and cocaine, as well as the development of specific hiCE inhibitors. Media containing secreted hiCE was obtained from baculovirus-infected Sf21 cells and the enzyme purified by ultrafiltration followed by ion-exchange and size exclusion chromatography. The resulting hiCE was determined to be greater than 98% pure by SDS-PAGE. We have determined the kinetic parameters for CPT-11 with the recombinant protein and they are in agreement with the enzyme purified from human liver. In addition, this CE converts cocaine to its inactive metabolite methylecgonine and heroin to 6-monoacetylmorphine. Selective inhibition of hiCE has also been achieved using a variety of dione-based inhibitors and we have demonstrated that these compounds can inhibit CEs intracellularly. Since we have hypothesized that the delayed diarrhea that occurs following CPT-11 administration results from activation of the drug in the gut epithelium, potentially these inhibitors may be utilized in the amelioration of this dose limiting toxicity. This work was supported in part by the American Lebanese Syrian Associated Charities.