Abstract
RP 73401 is a potent inhibitor of cyclic nucleotide phosphodiesterase type IV. RP 73401 is metabolized by human liver microsomes almost exclusively by transhydroxylation of the cyclopentyl group to RPR 113406. Liquid chromatography/mass spectrometry/mass spectrometry analysis of plasma from patients given RP 73401 also revealed a molecular ion and fragmentation consistent with RPR 113406. Thus, the objective was to determine the oxidative enzyme(s) responsible for RP 73401 hydroxylation. Kinetic constants of RP 113406 formation ranged from 8 to 26 μM and 0.83 to 5.99 nmol/min/mg protein forKm and Vmax , respectively (n = 3). Enzyme activity varied 23-fold among 15 human liver microsome samples and correlated with CYP2A6-catalyzed coumarin hydroxylase (r2 = 0.85, P < .01) and CYP2B6-catalyzed 7-ethoxytrifluoromethylcoumarin O-deethylase (r2 = 0.82, P < .01) activities. Chemical inhibition studies showed a 63% decrease in RP 73401 hydroxylation by 500 μM orphenadrine. Coumarin (10 μM), however, did not inhibit RP 73401 hydroxylation. Also, anti-CYP2B1 IgG produced 85% inhibition of RP 73401 hydroxylation, but only a negligible decline in coumarin hydroxylase activity. Of the 10 expressed P450 forms studied, only CYP2B6 catalyzed RP 73401 hydroxylation. Finally, expressed CYP2B6 showed a high affinity (K m = 22.5 μM) for RP 73401 hydroxylation, similar to the human liver microsome studies.
Footnotes
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Send reprint requests to: Jeffrey C. Stevens, Ph.D., Department of Drug Metabolism and Pharmacokinetics, Rhône-Poulenc Rorer, Mail Stop NW12, 500 Arcola Road, Collegeville, PA 19426.
- Abbreviations:
- PDE
- phosphodiesterase
- 7-EFC
- 7-ethoxytrifluoromethylcoumarin
- 7-HFC
- 7-hydroxytrifluoromethyl coumarin
- G6PDH
- glucose-6-phosphate dehydrogenase
- TAO
- troleandomycin
- LC
- liquid chromatography
- MS
- mass spectrometry
- HPLC
- high-performance liquid chromatography
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- FMO
- flavin monooxygenase
- Received January 6, 1997.
- Accepted May 19, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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