title: Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions.
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 microM 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 KM estimates of 129-164 microM with a Vmax of 5.3-17 pmol/mg/min for the low affinity isoform. The high affinity isoform had KM estimates of 1.4-3.9 microM with Vmax of 1.2-2.6 pmol/mg/min. The low KM 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 microM. 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 alpha-naphthyl acetate (alpha-NA) was used to determine whether CPT-11 was an inhibitor of hydrolysis of high turnover substrates of carboxylesterases. Inhibition of alpha-NA hydrolysis by CPT-11 was determined relative to positive controls BNPP and NaF. Incubation with microsomes pretreated with CPT-11 (80-440 microM) decreased alpha-naphthol formation to approximately 80% of control at alpha-NA concentrations of 50-800 microM. The inhibitors BNPP (360 microM) and NaF (500 microM) inhibited alpha-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 alpha-NA hydrolases. Thus, CPT-11 is unlikely to significantly inhibit high turnover, nonselective substrates of carboxylesterases.
authors.affiliation: Drug Metabolism Research, Pharmacia and Upjohn Co., Kalamazoo, MI 49007, USA. john.g.slatter@am.pnu.com
authors.forename: J G
authors.initials: JG
authors.lastname: Slatter
authors.forename: P
authors.initials: P
authors.lastname: Su
authors.forename: J P
authors.initials: JP
authors.lastname: Sams
authors.forename: L J
authors.initials: LJ
authors.lastname: Schaaf
authors.forename: L C
authors.initials: LC
authors.lastname: Wienkers
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D001711:Biotransformation; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D004347:Drug Interactions; D006801:Humans; D000077146:Irinotecan; D008862:Microsomes, Liver / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D000077146:Irinotecan; D002265:Carboxylic Ester Hydrolases; D002166:Camptothecin