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 KM estimates of 129–164 μM with a Vmax of 5.3–17 pmol/mg/min for the low affinity isoform. The high affinity isoform hadKM estimates of 1.4–3.9 μM withVmax of 1.2–2.6 pmol/mg/min. The lowKM 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.
Footnotes
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Send reprint requests to: Dr. J. Greg Slatter, Drug Metabolism Research, Pharmacia and Upjohn Co., 301 Henrietta St., Kalamazoo MI 49007. E-mail: john.g.slatter{at}am.pnu.com.
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↵1 Present address: Department of Biopharmaceutical Sciences, University of California at San Francisco.
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A preliminary account of this work was given at the 4th International ISSX Meeting, Abstracts #88, 218 and 219. Seattle WA, Aug 27–31, 1995.
- Abbreviations used are::
- CPT
- camptothecin
- CPT-11
- irinotecan hydrochloride trihydrate
- EtOH
- ethanol
- ASA
- acetylsalicylic acid
- HMB
- hydroxymercuribenzote
- CMB
- chloromercuribenzoate
- HPLC
- high performance liquid chromatography
- KM
- Michaelis constant
- 5-FU
- 5-fluorouracil
- MeOH
- methanol
- BNPP
- bis-nitrophenylphosphate
- α-NA
- alpha naphthyl acetate
- SN-38
- active antineoplastic metabolite of CPT-11
- DMSO
- dimethylsulfoxide
- UPACS
- Upjohn Physical and Analytical Chemistry System
- V
- reaction velocity
- QC
- quality control standard
- [S]
- substrate concentration
- Received March 28, 1997.
- Accepted June 5, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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