Oxidative metabolism of bupivacaine into pipecolylxylidine in humans is mainly catalyzed by CYP3A

Bupivacaine is used to provide prolonged anesthesia and postoperative analg esia. The human cytochrome P450 (CYP) involved in bupivacaine degradation i nto pipecolylxylidine (PPX), its major metabolite, has, to our knowledge, n ever been described. Microsome samples were prepared from six human livers and incubated in the presence of bupivacaine. The concentrations of PPX in the microsomal suspensions were assessed, and K-m and V-max values were cal culated. Bupivacaine incubations were then performed with specific CYP subs trates and inhibitors. For each sample of hepatic microsomes, the correlati on between the rate of PPX formation and the corresponding erythromycin N-d emethylase activity was analyzed. Finally, an immunoinhibition study using an anti-rabbit CYP3A6 antibody and assays with cDNA-expressed human CYP wer e conducted. The apparent K-m and V-max values of bupivacaine were, respect ively, 125 mu M and 4.78 nmol/min/mg of microsomal protein. The strongest i nhibition of bupivacaine metabolism was obtained for troleandomycin (-95% a t 50 mu M), a specific CYP3A inhibitor. The correlation between PPX formati on and erythromycin N-demethylase activity showed an R value of 0.99 wherea s anti-rabbit CYP3A6 antibody inhibited the degradation of bupivacaine into PPX by 99%. Finally, CYP1A2 and CYP2E1 cDNA-expressed forms of human CYP d id not allow PPX formation, CYP2C19 and CYP2D6 produced only small amounts whereas CYP3A4 most efficiently metabolized bupivacaine into PPX. These res ults demonstrated that bupivacaine degradation into PPX was mediated in hum ans by CYP3A.