Study On The Metabolism Of Imrecoxib In Rat Liver Microsomes

1. BackgroundImrecoxib, [4-(4-methane-sulfonyl-phenyl)-1-propyl-3-p-tolyl-1,5-dihydro-pyrrol-2-one], is a novel and moderately selective inhibitor of COX-2, an inducible isoform of cyclooxygenase that plays a key role in inflammatory processes. The drug inhibits COX-1 and COX-2 with IC₅₀ value of 115 ± 28 nmol/L and 18 ± 4 nmol/L, respectively. It possesses the anti-inflammatory effect by inhibiting COX-II mRNA expression. Imrecoxib has been introduced into clinical trials in China to treat acute and chronic inflammatory disease. Our previous study has found that imrecoxib was extensively metabolized after i.v. administration with less than 2% of the dose recovered in urine or feces as parent drug. The major route of metabolism appears to be methyl hydroxylation to form 4'-hydoxymethylimrecoxib (M4), followed by further oxidation to 4'-carboxylic acid imrecoxib (M2). The metabolism of imrecoxib in human is similar with that in rat. M2 and M4 are the major metabolites of imrecoxib and circulated in blood in human.2. AimsThe primary objectives of the present study were to characterize cytochrome P450 (CYP450) enzyme(s) involved in the metabolism of imrecoxib and evaluate the effects of imrecoxib and its major circulating metabolites, M2 and M4, on CYP450. It is useful in the further understanding of imrecoxib metabolic mechanism, the predicton of pharmacokinetic drug-drug interactions and interpatient variability in drug exposure.3. MethodsLiver microsomes and cytosol of male Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of imrecoxib was studied by incubation with rat liver microsomes. To characterize the CYP450 isozymes involved in the 4'-methyl hydroxylation of imrecoxib, the effects of typical CYP450 inducers (such as dexomethasone, isoniazid and β-naphthoflavone) and of CYP450 inhibitors (such as ketoconazole, quinine, α-naphthoflavone, methylpyrazole, and cimetidine) on the formation rate of 4'-hydroxy-methyl imrecoxib were investigated.The formation of M2 was studied in rat liver cytosol in the absence or presence of liver microsomes using imrecoxib or M4 as substrate. The effect of cofactors, NADH and NADPH, on the formation of M2 was studed. In addition, to characterize the CYP450 isozymes involved in M2 formation, the effects of typical CYP450 inhibitors on the formation rate of M2 from M4 were investigated.Groups of male Wistar rats were orally administered imrecoxib, M2, and M4 at 50 mg/kg/day for 7 days, followed by assays to detect changes in the levels of liver microsomal protein, CYP450, and drug metabolizing enzyme activities. The inhibitory effects of imrecoxib, M2, and M4 on CYP450 were also investigated in the present study. The following CYP450 isoform specific marker reactions were measured: phenacetion O-deethylase (CYP 1A), tolbutamide hydroxylase (CYP 2C), codeine O-demethylase (CYP 2D), anline hydroxylase (CYP 2E), and nifedipine dehydrogenase (CYP 3A).4. ResultsThree phase I metabolites of imrecoxib were detected in rat liver microsomal incubates with 200 |amol/L of imrecoxib and 1 h of incubation. The identities of them were confirmed by chromatographic and mass spectra comparison with reference substances. These metabolites were identified as 4'-hydroxymethyl-5-hydroxyl metabolite (M3), 4'-hydroxy-methyl metabolite (M4), and 4'-hydroxymethyl-5-carbonyl (M5) metabolite. No carboxylic acid metabolite (M2) was found in this study. The formation of M3, M4, and M5 was enzymatic and NADPH-dependent. NADH as cofactor had no catalytic effect on the metabolism of imrecoxib.M4 was produced to the greatest extent by microsomes from dexamethasone-induced rats. On the other hand, ketoconazole markedly lowered the metabolic rate of imrecoxib in a concentration-dependent manner. Moreover, a significant inhibitory effect of quinine on the formation of M4 was observed in microsomes obtained from control rats, isoniazid-induced rats, /?-naphthoflavone-induced rats and phenobarbital-induced rats. In contrast, a-naphthoflavone, cimetidine, and methylpyrazole had no inhibitory effects on this metabolic pathway. Over the imrecoxib concentration range studied (5-600 ^mol/L), the rate of 4'-methy hydroxy-lation conformed to monophasic Michaelis-Menten kinetics.In the study to investigate the formation of M2, no M2 formed from imrecoxib or M4 wasdetected in rat liver cytosol, either NADPH or NADH alone, or both NADPH and NADH was used as cofactors. M4 was metabolized to M2 in rat liver microsomes with NADPH as cofactor. A little of M2 was formed in dexamethasone-induced rat liver microsomes with imrecoxib as substrate when a higher substrate concentration (600 ^mol/L imrecoxib) and a longer incubation time (2 h) were used.Enzyme kinetic study demonstrated that the formation rate of M2 conformed to monophasic Michaelis-Menten kinetics. Additional experiments showed that the formation of M2 was induced significantly by dexamethasone and lowered by ketoconazole strongly and concentration-dependently. By comparison, other inhibitors, such as or-naphthoflavone, cimetidine, quinine, and methylpyrazole had no inhibitory effects on this metabolic pathway.No increases in the concentrations of liver microsomal protein, CYP450, and the enzymatic activities were observed after a 7-day administration of imrecoxib and its major metabolites.IC50 values of imrecoxib, M2, and M4 were determined for each substrate mentioned above at respective Km concentratons. The potent inhibition was seen for the CYP2D-dependent O-demethylation of codeine, where an IC50 value of 19.4 \imo\IL was observed. Therefore, additional codeine concentrations (5, 20, and 60 jimol/L) were tested. Imrecoxib was a compentative inhibitor of the reaction with a K, value of 9.0 nmol/L The K, value falls in the range of the total plasma concentrations of imrecoxib observed in rat in vivo, and is a 10-times higher than the concentrations in human. These results suggested that co-administraton of high doses of imrecoxib with drugs that are primarily metabolized by CYP 2D may result in significant drug interactions.Minimal inhibition of CYP 2D activity by M4 was observed with an IC50 value of 1430.9 nmol/L Compared with M4 peak plasma concentration below 0.63 jamol/L in human after administration of 200 mg imrecoxib twice daily for 11 days, the 1000-times higher IC50 value however suggests that inhibition of CYP 2D by M4 is without any clinical relevance. M2 did not show inhibitory effect on CYP450 activities.5. Conclusion:This biotransformation study of imrecoxib in rat liver microsomes showed that imrecoxib is metabolized via 4'-methyl hydroxylation in rat liver microsomes. The reaction is mainlycatalyzed by CYP 3A. CYP 2D also played a role in control rats, isoniazid-induced rats /?-naphthoflavone-induced rats and phenobarbital-induced rats. The formation of M2 was completely dependent on rat liver microsomes and NADPH. The biotransformation of M2 from M4 was mainly catalyzed by CYP 3A in untreated rats and in dexamethasone-induced rats. Imrecoxib did not induce the activities of CYP450, however significantly inhibited the activity of CYP 2D. M2 and M4 had little effect on CYP450.