Abstract
Using human liver microsomes (HLMs) and recombinant cytochrome P450s (CYP450s), we characterized the CYP450 isoforms involved in the primary metabolic pathways of cisapride and documented the ability of cisapride to inhibit the CYP450 system. In HLMs, cisapride was N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and to 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). Formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS in HLMs exhibited Michaelis-Menten kinetics (Km: 23.4 ± 8.6, 32 ± 11, and 31 ± 23 μM; Vmax: 155 ± 91, 52 ± 23, and 31 ± 23 pmol/min/mg of protein, respectively). The average in vitro intrinsic clearance (Vmax/Km) revealed that the formation of NORCIS was 3.9- to 5.9-fold higher than that of the two hydroxylated metabolites. Formation rate of NORCIS from 10 μM cisapride in 14 HLMs was highly variable (range, 4.9–133.6 pmol/min/mg of protein) and significantly correlated with the activities of CYP3A (r = 0.86,P = .0001), CYP2C19, and 1A2. Of isoform-specific inhibitors, 1 μM ketoconazole and 50 μM troleandomycin were potent inhibitors of NORCIS formation from 10 μM cisapride (by 51 ± 9 and 44 ± 17%, respectively), whereas the effect of other inhibitors was minimal. Of 10 recombinant human CYP450s tested, CYP3A4 formed NORCIS from 10 μM cisapride at the highest rate (V = 0.56 ± 0.13 pmol/min/pmol of P450) followed by CYP2C8 (V = 0.29 ± 0.08 pmol/min/pmol of P450) and CYP2B6 (0.15 ± 0.04 pmol/min/pmol of P450). The formation of 3-F-4-OHCIS was mainly catalyzed by CYP2C8 (V = 0.71 ± 0.24 pmol/min/pmol of P450) and that of 4-F-2-OHCIS by CYP3A4 (0.16 ± 0.03 pmol/min/pmol of P450). Clearly, recombinant CYP2C8 participates in cisapride metabolism, but when the in vitro intrinsic clearances obtained were corrected for abundance of each CYP450 in the liver, CYP3A4 is the dominant isoform. Cisapride was a relatively potent inhibitor of CYP2D6, with no significant effect on other isoforms tested, but theKi value derived (14 ± 16 μM) was much higher than the clinically expected concentration of cisapride (<1 μM). Our data suggest that CYP3A is the main isoform involved in the overall metabolic clearance of cisapride. Cisapride metabolism is likely to be subject to interindividual variability in CYP3A expression and to drug interactions involving this isoform.
Footnotes
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Send reprint requests to: Zeruesenay Desta, Ph.D., Division of Clinical Pharmacology, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Med-Dent Bldg. Room SE408, Washington, DC 20007. E-mail: gebreegz{at}gusun.georgetown.edu
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This work was funded in part by Grant T32-GM08386 from the National Institute of General Medical Sciences, Bethesda, MD.
- Abbreviations used are::
- CYP450
- cytochrome P450
- NORCIS
- norcisapride
- 3-F-4-OHCIS
- 3-fluoro-4-hydroxycisapride
- 4-F-2-OHCIS
- 4-fluoro-2-hydroxycisapride
- HLMs
- human liver microsomes
- Clint
- intrinsic clearance
- Received November 4, 1999.
- Accepted April 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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