Abstract

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 range of 0.25–200 μM 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 129–164 μM with a V_max of 5.3–17 pmol/mg/min for the low affinity isoform. The high affinity isoform hadK_M estimates of 1.4–3.9 μM withV_max of 1.2–2.6 pmol/mg/min. The lowK_M carboxylesterase may be the main contributor to SN-38 formation at clinically relevant hepatic concentrations of CPT-11.

Using standard incubation conditions, the effects of potential inhibitors of carboxylesterase-mediated CPT-11 hydrolysis were evaluated at concentrations ≥ 21 μM. Positive controls bis-nitrophenylphosphate (BNPP) and physostigmine decreased CPT-11 hydrolysis to 1.3–3.3% and 23% of control values, respectively. Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethanol, dexamethasone, 5-fluorouracil, loperamide, and prochlorperazine had no statistically significant effect on CPT-11 hydrolysis. Small decreases were observed with metoclopramide (91% of control), acetaminophen (93% of control), probenecid (87% of control), and fluoride (91% of control). Of the compounds tested above, based on these in vitro data, only the potent inhibitors of carboxylesterase (BNPP, physostigmine) have the potential to inhibit CPT-11 bioactivation if administered concurrently.

The carboxylesterase-mediated hydrolysis of α-naphthyl acetate (α-NA) was used to determine whether CPT-11 was an inhibitor of hydrolysis of high turnover substrates of carboxylesterases. Inhibition of α-NA hydrolysis by CPT-11 was determined relative to positive controls BNPP and NaF. Incubation with microsomes pretreated with CPT-11 (80–440 μM) decreased α-naphthol formation to approximately 80% of control at α-NA concentrations of 50–800 μM. The inhibitors BNPP (360 μM) and NaF (500 μM) inhibited α-naphthol formation to 9–10% of control and to 14–20% of control, respectively. Therefore, CPT-11-sensitive carboxylesterase isoforms may account for only 20% of total α-NA hydrolases. Thus, CPT-11 is unlikely to significantly inhibit high turnover, nonselective substrates of carboxylesterases.