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 metabolism. Studies with (-)-, (+)-, and (+/-)-cisapride were undertaken in human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s) to determine the stereoselective metabolism and enantiomer-enantiomer interaction. Each enantiomer and racemic cisapride were N-dealkylated to norcisapride (NORCIS) and hydroxylated to 3-fluoro-4-hydroxycisapride (3-F-4-OHCIS) and 4-fluoro-2-hydroxycisapride (4-F-2-OHCIS). The kinetics for the formation of NORCIS from (-)-cisapride (Km = 11.9 +/- 4.8 microM; Vmax = 203 +/- 167 pmol/min/mg of protein) or (+)-cisapride (Km = 18.5 +/- 4.7 microM; Vmax = 364 +/- 284 pmol/min/mg of protein) in HLMs exhibited simple Michaelis-Menten kinetics, while a sigmoidal model characterized those of 3-F-4-OHCIS and 4-F-2-OHCIS. In vitro, NORCIS appears to be the major metabolite of both enantiomers. NORCIS and 3-F-4-OHCIS were preferentially formed from (+)-cisapride rather than (-)-cisapride, but that of 4-F-2-OHCIS was the reverse, suggesting regio- and stereoselective metabolism. The formation rate of each metabolite from each enantiomer (20 microM) in 18 HLMs was highly variable (e.g., NORCIS, >35-fold) and correlated with the activity of CYP3A (r = 0.6-0.85; p < 0.05). Coincubation of troleandomycin (50 microM) with cisapride enantiomers (15 microM) in HLMs resulted in potent inhibition of NORCIS formation (by 75-80%), while other inhibitors showed negligible effect. Of 10 recombinant human P450s tested, CYP3A4 catalyzed the formation of NORCIS, 3-F-4-OHCIS, and 4-F-2-OHCIS from each enantiomer and racemic cisapride (15 microM) with the highest specific activity (Km values close to those in HLMs). We noted that the rate of racemic cisapride metabolism by HLMs and recombinant human CYP3A4 is slower compared with equimolar concentrations of each enantiomer. When incubated simultaneously in HLMs, the enantiomers inhibit each other's metabolism. In conclusion, our data demonstrate for the first time the stereoselective metabolism and enantiomer-enantiomer interaction of cisapride. Provided that the potency or the response of the enantiomers differ, understanding the factors that control their disposition as opposed to that of racemic cisapride may better predict adverse drug interactions and the resulting prokinetic efficacy and cardiac safety of cisapride.