Characterization of CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2.

7-Ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (irinotecan; CPT-11) is a prodrug activated by carboxylesterase enzymes. We characterized the hydrolysis of CPT-11 by two recently identified human carboxylesterase (hCE) enzymes, hCE-1 and hCE-2. Km and Vmax for hCE-1 and hCE-2 are 43 microM and 0.53 nmol/min/mg protein and 3.4 microM and 2.5 nmol/min/mg protein, respectively. hCE-2 has a 12.5-fold higher affinity for CPT-11 and a 5-fold higher maximal rate of CPT-11 hydrolysis when compared with hCE-1. In cytotoxicity assays, incubation of 1 microM CPT-11 with hCE-2 (3.6 microg/ml) resulted in a 60% reduction in survival of SQ20b cells. No significant reduction in cell survival was observed after incubation of CPT-11 with hCE-1. These data indicate that hCE-2 is a high-affinity, high-velocity enzyme with respect to CPT-11. hCE-2 likely plays a substantial role in CPT-11 activation in human liver at relevant pharmacological concentrations.

Lack of evidence for a polymorphism at codon 160 of human O6-alkylguanine-DNA alkyltransferase gene in normal tissue and cancer.

O6-benzylguanine (BG) is a potent, specific inactivator of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which enhances sensitivity to nitrosoureas in cells and tumor-bearing animals. BG is presently undergoing clinical trials for development as an agent to enhance the therapeutic index of alkylating agent chemotherapy. It has been reported that a polymorphism exists in the human agt gene, with about 15% of the Japanese population having arginine at codon 160 instead of glycine on the polypeptide (Y. Imai et al., Carcinogenesis, 16: 2441-2445, 1995). The resultant mutant AGT protein is equally effective against both methylated DNA as compared with wild type protein. However, this mutant AGT protein was less sensitive to inactivation by BG with a 20-fold increase in the ED50 value. This observation raised the possibility that a subpopulation of patients may be resistant to BG due to a single base change. We have demonstrated that this alteration also reduces the sensitivity to O6-benzyl-8-oxoguanine, an equally potent, yet much longer-lived human metabolite of BG. To test the possibility that this germ-line mutation of the agt gene might explain resistance to BG and O6-benzyl-8-oxoguanine of patients on our Phase I clinical trials, we evaluated the DNA from lymphocytes of 18 patients. The G160R mutation was not found in any of the 18 patients. To determine the frequency of this mutation in the United States population, DNA from 181 healthy individuals were investigated and, again, the mutation was not observed in this cohort. Therefore, if the mutation exists, it is in statistically <1.6% of the United States noncancerous population. To investigate the possibility that this mutation might be somatic, we evaluated genomic DNA samples from 94 human primary cancers of four different histological subtypes (brain, colon, esophageal, and head and neck). Again, none were found to have the G160R mutation.