Stereoselective metabolism of cisapride and enantiomer-enantiomer interaction in human cytochrome P450 enzymes: Major role of CYP3A

Cisapride is a chiral molecule that is marketed as a racemate consisting of two optical isomers, but little is known about its stereoselective metabol ism. Studies with (-)-, (+)-, and (+/-)-cisapride were undertaken in human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s) to determi ne the stereoselective metabolism and enantiomer-enantiomer interaction. Ea ch enantiomer and racemic cisapride were N-dealkylated to norcisapride (NOR CIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and 4-fl uoro-2-hydroxycisapride (4-F-2-OHCIS). The kinetics for the formation of NO RCIS from (-)-cisapride (K-m = 11.9 +/-4.8 muM; V-max = 203 +/- 167 pmol/mi n/mg of protein) or (+)-cisapride (K-m = 18.5 +/-4.7 muM; V-max = 364 +/- 2 84 pmol/min/mg of protein) in HLMs exhibited simple Michaelis-Menten kineti cs, while a sigmoidal model characterized those of 3-F4-OHCIS and 4-F-2-OHC IS. In vitro, NORCIS appears to be the major metabolite of both enantiomers . NORCIS and 3-F-4-OHCIS were preferentially formed from (+)-cisapride rath er than (-)-cisapride, but that of 4-F-2-OHCIS was the reverse, suggesting regio- and stereoselective metabolism. The formation rate of each metabolit e from each enantiomer (20 muM) in 18 HLMs was highly variable (e.g., NORCI S, >35-fold) and correlated with the activity of CYP3A (r = 0.6-0.85; p < 0 .05). Coincubation of troleandomycin (50 <mu>M) with cisapride enantiomers (15 muM) in HLMs resulted in potent inhibition of NORCIS formation (by 75-8 0%), while other inhibitors showed negligible effect. Of 10 recombinant hum an P450s tested, CYP3A4 catalyzed the formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS from each enantiomer and racemic cisapride (15 muM) with the h ighest specific activity (Km values close to those in HLMs). We noted that the rate of racemic cisapride metabolism by HLMs and recombinant human CYP3 A4 is slower compared with equimolar concentrations of each enantiomer. Whe n incubated simultaneously in HLMs, the enantiomers inhibit each other's me tabolism. In conclusion, our data demonstrate for the first time the stereo selective metabolism and enantiomer-enantiomer interaction of cisapride. Pr ovided that the potency or the response of the enantiomers differ, understa nding the factors that control their disposition as opposed to that of race mic cisapride may better predict adverse drug interactions and the resultin g prokinetic efficacy and cardiac safety of cisapride.