D. Sutton et al., ROLE OF CYP3A4 IN HUMAN HEPATIC DILTIAZEM N-DEMETHYLATION - INHIBITION OF CYP3A4 ACTIVITY BY OXIDIZED DILTIAZEM METABOLITES, The Journal of pharmacology and experimental therapeutics, 282(1), 1997, pp. 294-300
The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabo
lism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ m
etabolites in serum occurs during prolonged therapy and leads to decre
ased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhi
bition. This study assessed the role of human CYPs in microsomal DTZ o
xidation and the capacity of DTZ metabolites to inhibit specific CYP a
ctivities. DTZ N-demethylation varied 10-fold in microsomal fractions
from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was c
orrelated with testosterone GP-hydroxylation (r = 0.82) and, to a less
er extent, tolbutamide hydroxylation (r = 0.59) but not with activitie
s mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsom
es catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also activ
e (similar to 20% and 10% of the activity supported by CYP3A4); seven
other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4
inhibitors ketoconazole and troleandomycin decreased microsomal DTZ ox
idation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produ
ced no inhibition. Some inhibition was produced by alpha-naphthoflavon
e, a chemical that inhibits CYP1As and also interacts with CYP3A4. In
further experiments, the capacities of DTZ and three metabolites to mo
dulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vi
tro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selective
ly inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibito
ry. The IC50 value of DTZ against GYP3A4-mediated testosterone 6 beta-
hydroxylation (substrate concentration, 50 mu M) was 120 mu M The N-de
smethyl (IC50 = 11 mu M) and N,N-didesmethyl (IC50 = 0.6 mu M) metabol
ites were 11 and 200 times, respectively, more potent. From kinetic st
udies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive
inhibitors of CYP3A4 (K-i = similar to 2 and 0.1 mu M, respectively).
CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent
biotransformation in NADPH-supplemented hepatic microsomes in vitro,
supporting the contention that inhibitory metabolites may be generated
in situ. These findings suggest that N-demethylated metabolites of DT
Z may contribute to CYP3A4 inhibition in vivo, especially under condit
ions in which N-desmethyl DTZ accumulates, such as during prolonged DT
Z therapy.