COMPETITIVE-INHIBITION OF HUMAN LIVER MICROSOMAL CYTOCHROME-P450 3A-DEPENDENT STEROID 6-BETA-HYDROXYLATION ACTIVITY BY CYCLOPHOSPHAMIDE ANDIFOSFAMIDE IN-VITRO
M. Murray et al., COMPETITIVE-INHIBITION OF HUMAN LIVER MICROSOMAL CYTOCHROME-P450 3A-DEPENDENT STEROID 6-BETA-HYDROXYLATION ACTIVITY BY CYCLOPHOSPHAMIDE ANDIFOSFAMIDE IN-VITRO, The Journal of pharmacology and experimental therapeutics, 270(2), 1994, pp. 645-649
The prodrugs cyclophosphamide (CP) and ifosfamide (IF) are oxidized by
hepatic cytochrome P450 (P450) to the active cytotoxic species, phosp
horamide mustard. Acrolein (prop-2-enal) is also formed during CP and
IF activation in rat liver and has been associated with P450 destructi
on. Analogous inactivation of human liver P450s by CP or IF could lead
to pharmacokinetic interactions with coadministered drugs. The presen
t study investigated the susceptibilities of human hepatic P450s to in
hibition and inactivation by CP and IF in vitro. Unlike the situation
in rat liver microsomes, total P450 was not decreased after incubation
of CP or IF with NADPH and human fractions. However, CP and IF inhibi
ted testosterone 6 beta-hydroxylation mediated by P450s 3A but not P45
0 1A2-dependent 7-ethylresorufin O-deethylation, P450 2C-dependent tol
butamide methyl hydroxylation or P450 2E1-mediated N-nitrosodimethylam
ine N-demethylation. Kinetic analysis indicated that the drugs were re
versible (competitive) inhibitors of testosterone 6 beta-hydroxylation
(K-m, 94 +/- 8 mu M) in human liver microsomes (K(l)s, 510 +/- 20 mu
M and 490 +/- 40 mu M for CP and IF, respectively). Time-dependent int
ensification of the inhibition of the activity by CP or IF did not occ
ur; this supports the observation that P450 was refractory to inactiva
tion. The rates of acrolein formation from CP and IF in human hepatic
microsomes (0.76 +/- 0.23 and 0.19 +/- 0.07 nmol min(-1) mg(-1) of pro
tein, respectively) were only 18% and 10% of the rates estimated in fr
actions from untreated rat liver (4.20 +/- 0.04 and 1.96 +/- 0.12 nmol
min(-1) mg(-1) of protein, respectively). Direct addition of acrolein
to human microsomes at a similar concentration to that formed from CP
in vitro (similar to 23 mu M) did not result in P450 loss but concent
rations similar to those produced from CP in microsomes from phenobarb
ital-induced rat liver (similar to 390 mu M) led to the loss of about
35% of total P450. In summary, these findings establish that CP and IF
are reversible and preferential inhibitors of human hepatic P450s 3A.
The absence of microsomal P450 destruction during NADPH-mediated CP o
r IF metabolism seems related to the generation of insufficient quanti
ties of acrolein in situ. Possible pharmacokinetic interactions with C
P and IF are therefore unlikely to be attributable to P450 inactivatio
n by acrolein.