In vitro inhibition of CYP1A2 by model inhibitors, anti-inflammatory analgesics and female sex steroids: predictability of in vivo interactions.

The cytochrome P450 enzyme CYP1A2 is crucial for the metabolism of many drugs, for example, tizanidine. As the effects of several non-steroidal anti-inflammatory drugs (NSAID) and female sex steroids on CYP1A2 activity in vitro are unknown, their effects on phenacetin O-deethylation were studied and compared with the effects of model inhibitors in human liver microsomes, followed by prediction of their interaction potential with tizanidine in vivo. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently inhibited CYP1A2 [the 50% inhibitory concentration (IC(50)) < 10 microM]. Ethinyloestradiol, celecoxib, desogestrel and zolmitriptan were moderate (IC(50) 20-200 microM), and etodolac, ciprofloxacin, etoricoxib and gestodene weak inhibitors of CYP1A2 (IC(50) > 200 microM). At 100 microM, the other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Pre-incubation increased the inhibitory effects of rofecoxib, progesterone and desogestrel. Using the free portal plasma inhibitor concentration and the competitive inhibition model, the effect of fluvoxamine and the lack of effects of tolfenamic acid and celecoxib on tizanidine pharmacokinetics in human beings were well predicted. However, the effects of ciprofloxacin, rofecoxib and oral contraceptives were greatly underestimated even when the predictions were based on their total portal plasma concentration. Besides rofecoxib, and possibly mefenamic acid, other NSAIDs were predicted not to significantly inhibit CYP1A2 in human beings. The type of enzyme inhibition, particularly metabolism-dependent inhibition, free inhibitor concentration and accumulation of the inhibitor into the hepatocytes should be considered in extrapolations of in vitro results to human beings.

Celecoxib is a CYP1A2 inhibitor in vitro but not in vivo.

BACKGROUND AND OBJECTIVE: We recently discovered that rofecoxib is a potent mechanism-based inhibitor of CYP1A2. The effect of the widely used cyclo-oxygenase-2 selective non-steroidal anti-inflammatory drug celecoxib on CYP1A2 activity has not been reported. METHODS: The effect of celecoxib on CYP1A2 activity (phenacetin O-deethylation) was first studied in vitro using human liver microsomes. This was followed by a randomized, placebo-controlled, cross-over study in which 12 healthy volunteers were given celecoxib (200 mg twice daily) or placebo for 4 days. On day 3, a caffeine test was performed. On day 4, the subjects ingested 2 mg tizanidine. Plasma samples for the measurement of the concentrations of tizanidine, its metabolites and celecoxib were collected up to 24 h post-administration. Pharmacodynamic variables (e.g. blood pressure, subjective drowsiness and drug effect) were recorded up to 12 h post-adm. RESULTS: Celecoxib was found to be a moderately potent competitive inhibitor of CYP1A2 in vitro with a K(i) (inhibitor constant) of 25.4 microM. However, in vivo, celecoxib did not affect the caffeine test, or the peak concentration, time to peak concentration, area under the concentration-time curve or half-life of tizanidine. The pharmacodynamic variables of tizanidine also remained unchanged. CONCLUSIONS: Unlike rofecoxib, celecoxib does not clinically to significantly inhibit CYP1A2. The lack of significant in vivo inhibition of CYP1A2 can be correctly predicted on the basis of in vitro K(i) data and the free peripheral or portal plasma concentration of celecoxib.

Tolfenamic acid is a potent CYP1A2 inhibitor in vitro but does not interact in vivo: correction for protein binding is needed for data interpretation.

OBJECTIVE: Our aim was to correlate the in vitro and in vivo CYP1A2 inhibition potential of tolfenamic acid, an NSAID highly (99.7%) bound to plasma proteins, to study the significance of protein binding of inhibitor in metabolic drug interactions. METHODS: The effect of tolfenamic acid on CYP1A2 (phenacetin O-deethylation) was studied using human liver microsomes, with and without albumin (0-10 mg/ml). In a randomized, crossover study, 10 volunteers took 200 mg tolfenamic acid or placebo t.i.d. for 3 days. On day 2, a caffeine test was performed. On day 3, each ingested 4 mg of the CYP1A2 substrate tizanidine. Plasma tizanidine, its metabolites (M) and tolfenamic acid, and pharmacodynamic variables were measured. RESULTS: Tolfenamic acid strongly inhibited phenacetin-O-deethylation in vitro (IC(50) 1.8 microM without albumin). Albumin decreased its inhibitory effect in a concentration-dependent manner; the IC(50) exceeded 100 microM with 10 mg/ml of albumin. Tolfenamic acid had no effect on the area under the concentration-time curve (AUC(0-oo)), peak concentration, time of peak concentration or half-life of tizanidine or M-3; only the AUC(0-oo) of secondary metabolite M-4 was slightly decreased (13%, P = 0.004). The caffeine test and the pharmacodynamic effects of tizanidine were unchanged. CONCLUSIONS: Tolfenamic acid potently inhibits CYP1A2 in vitro when studied without albumin, but not in vivo. This apparent discrepancy is due to the high protein binding of tolfenamic acid. To avoid overestimation of the interaction potential, the inhibitory effect of highly albumin-bound compounds should also be studied in vitro with albumin, or their exact unbound plasma concentration should be used in predictions.

Rofecoxib is a potent, metabolism-dependent inhibitor of CYP1A2: implications for in vitro prediction of drug interactions.

Rofecoxib was recently found to greatly increase plasma concentrations of the CYP1A2 substrate drug tizanidine in humans, but there are no published in vitro studies on the CYP1A2-inhibiting effects of rofecoxib. Our objective was to investigate whether rofecoxib is a direct-acting or metabolism-dependent inhibitor of CYP1A2 in vitro. The effect of rofecoxib on the O-deethylation of phenacetin (20 microM) was studied using human liver microsomes. The effect of preincubation time on the inhibitory potential of rofecoxib was also studied, and the inhibitor concentration that supports half the maximal rate of inactivation (KI) and the maximal rate of inactivation (kinact) were determined. Rofecoxib moderately inhibited phenacetin O-deethylation (IC50 23.0 microM), and a 30-min preincubation with microsomes and NADPH considerably increased its inhibitory effect (IC50 4.2 microM). Inactivation of CYP1A2 by rofecoxib required NADPH, and was characterized by a KI of 4.8 microM and a kinact of 0.07 min(-1). Glutathione, superoxide dismutase, mannitol, or dialysis could not reverse the inactivation of CYP1A2 caused by rofecoxib. Fluvoxamine decreased the rofecoxib-caused inactivation of CYP1A2 in a concentration-dependent manner. In conclusion, rofecoxib is a potent, metabolism-dependent inhibitor of CYP1A2, a cytochrome P450 form contributing to rofecoxib metabolism. The results provide a mechanistic explanation for the interactions of rofecoxib with CYP1A2 substrates and may partially explain its nonlinear pharmacokinetics.

Rofecoxib is a potent inhibitor of cytochrome P450 1A2: studies with tizanidine and caffeine in healthy subjects.

AIMS: Case reports suggest an interaction between rofecoxib and the CYP1A2 substrate tizanidine. Our objectives were to explore the extent and mechanism of this possible interaction and to determine the CYP1A2 inhibitory potency of rofecoxib. METHODS: In a randomized, double-blind, two-phase cross-over study, nine healthy subjects took 25 mg rofecoxib or placebo daily for 4 days and, on day 4, each ingested 4 mg tizanidine. Plasma concentrations and the urinary excretion of tizanidine, its metabolites (M) and rofecoxib, and pharmacodynamic variables were measured up to 24 h. On day 3, a caffeine test was performed to estimate CYP1A2 activity. RESULTS: Rofecoxib increased the area under the plasma concentration-time curve (AUC(0-infinity)) of tizanidine by 13.6-fold [95% confidence interval (CI) 8.0, 15.6; P < 0.001), peak plasma concentration (C(max)) by 6.1-fold (4.8, 7.3; P < 0.001) and elimination half-life (t(1/2)) from 1.6 to 3.0 h (P < 0.001). Consequently, rofecoxib markedly increased the blood pressure-lowering and sedative effects of tizanidine (P < 0.05). Rofecoxib increased several fold the tizanidine/M-3 and tizanidine/M-4 ratios in plasma and urine and the tizanidine/M-5, tizanidine/M-9 and tizanidine/M-10 ratios in urine (P < 0.05). In addition, it increased the plasma caffeine/paraxanthine ratio by 2.4-fold (95% CI 1.4, 3.4; P = 0.008) and this ratio correlated with the tizanidine/metabolite ratios. Finally, the AUC(0-25) of rofecoxib correlated with the placebo phase caffeine/paraxanthine ratio (r = 0.80, P = 0.01). CONCLUSIONS: Rofecoxib is a potent inhibitor of CYP1A2 and it greatly increases the plasma concentrations and adverse effects of tizanidine. The findings suggest that rofecoxib itself is also metabolized by CYP1A2, raising concerns about interactions between rofecoxib and other CYP1A2 substrate and inhibitor drugs.