IDENTIFICATION OF HUMAN LIVER CYTOCHROME-P450 ISOFORMS INVOLVED IN THE IN-VITRO METABOLISM OF CYCLOBENZAPRINE

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricycli c structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver micro somes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoc onazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies d irected against CYP1A1/2 and CYP3A4 inhibited the demethylation reacti on whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did no t show any inhibitory effects. When a panel of microsomes prepared fro m human B-lymphoblastoid cells that expressed specific human cytochrom e P450 isoforms were used, only microsomes containing cytochromes P450 1A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylatio n catalyzed by these recombinant cytochromes P450 can be completely in hibited with selective inhibitors at concentrations as low as 1 to 20 mu M. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 bet a-hydroxylation (3A4), but not with dextromethorphan O-demethylation ( 2D6) in human liver microsomes. To further determine the involvement o f cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes f rom a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occu rred in the incubation with this microsome. These results suggested th at cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-dem ethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenz aprine metabolism in human liver microsomes. Due to the minimum involv ement of CYP2D6 in the in vitro metabolism of cyclobenzaprine, the pol ymorphism of cytochrome P4502D6 in man should not be of much concern i n the clinical use of cyclobenzaprine.