BIOACTIVATION OF THE ANTICANCER AGENT CPT-11 TO SN-38 BY HUMAN HEPATIC-MICROSOMAL CARBOXYLESTERASES AND THE IN-VITRO ASSESSMENT OF POTENTIAL-DRUG INTERACTIONS

Human hepatic microsomes were used to investigate the carboxylesterase -mediated bioactivation of CPT-11 to the active metabolite, SN-38, SN- 38 formation velocity was determined by HPLC over a concentration rang e of 0.25-200 mu M CPT-11. Biphasic Eadie Hofstee plots were observed in seven donors, suggesting that two isoforms catalyzed the reaction. Analysis by nonlinear least squares regression gave K-M estimates of 1 29-164 mu M with a V-max of 5.3-17 pmol/mg/min for the low affinity is oform. The high affinity isoform had K-M estimates of 1.4-3.9 mu M wit h V-max of 1.2-26 pmol/mg/min. The low K-M carboxylesterase may be the main contributor to SN-38 formation at clinically relevant hepatic co ncentrations of CPT-11. Using standard incubation conditions, the effe cts of potential inhibitors of carboxylesterase-mediated CPT-11 hydrol ysis were evaluated at concentrations greater than or equal to 21 mu M . Positive controls bis-nitrophenylphosphate (BNPP) and physostigmine decreased CPT-11 hydrolysis to 1.3-3.3% and 23% of control values, res pectively. Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethano l, dexamethasone, 5-fluorouracil, loperamide, and prochlorperazine had no statistically significant effect on CPT-11 hydrolysis. Small decre ases were observed with metoclopramide (91% of control), acetaminophen (93% of control), probenecid (87% of control), and fluoride (91% of c ontrol). Of the compounds tested above, based on these in vitro data, only the potent inhibitors of carboxylesterase (BNPP, physostigmine) h ave the potential to inhibit CPT-11 bioactivation if administered conc urrently. The carboxylesterase-mediated hydrolysis of alpha-naphthyl a cetate (alpha-NA) was used to determine whether CPT-11 was an inhibito r of hydrolysis of high turnover substrates of carboxylesterases. Inhi bition of alpha-NA hydrolysis by CPT-11 was determined relative to pos itive controls BNPP and NaF. Incubation with microsomes pretreated wit h CPT-11 (80-440 mu M) decreased alpha-naphthol formation to approxima tely 80% of control at alpha-NA concentrations of 50-800 mu M. The inh ibitors BNPP (360 mu M) and NaF (500 mu M) inhibited alpha-naphthol fo rmation to 9-10% of control and to 14-20% of control, respectively. Th erefore, CPT-11-sensitive carboxylesterase isoforms may account for on ly 20% of total alpha-NA hydrolases. Thus, CPT-11 is unlikely to signi ficantly inhibit high turnover, nonselective substrates of carboxylest erases.