Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase

Abstract

Aims Celecoxib is a novel selective cyclooxygenase-2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib. Methods Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1-3) was analysed by spectrophotometric monitoring of NADH generation from NAD + . Results The intrinsic clearance of celecoxib hydroxylation was significantly lower for yeast-expressed CYP2C9.3 (0.14 ml min − 1 nmol − 1 enzyme) compared with CYP2C9.1 (0.44 ml min − 1 nmol − 1 enzyme). In human liver microsomes, a significant 2-fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9 * 1/ * 3 samples compared with CYP2C9 * 1/ * 1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9 * 3/ * 3 . However, the CYP2C9 * 2 samples did not differ significantly from CYP2C9 * 1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD + . Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent K m values of 42 µ M and 10 µ M , respectively, whereas ADH3 showed no activity. Conclusions The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9 * 3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.

The Impact of Cyp2c9 Genetics and Oral Contraceptives on Cytochrome P450 2c9 Phenotype

“…For example, the clearance of warfarin was recently shown to vary 12-fold in 54 patients homozygous for the CYP2C9*1 allele (Scordo et al, 2002). A similar variation in CYP2C9 enzyme activity has been demonstrated in human liver samples incubated with different CYP2C9 substrates in vitro (Yamazaki et al, 1998;Yasar et al, 2001b;Sandberg et al, 2002). Moreover, results from a study on patients treated with phenytoin showed that only 15% of the variation in dose-response could be explained by the *1-, *2-, or *3-polymorphisms of the CYP2C9 gene (Kerb et al, 2001).…”

Bioequivalence assessment of two formulations of celecoxib: Open label, single dose and two-way cross over study in healthy human male volunteers

“…Chemical structure of CEL is 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazole-1-yl] benzenesulfonamide ( Manzoori et al, 2005) as shown in Figure 1. Metabolism of CEL takes place in liver by the enzymes cytochrome P450 2C9 (Cyp 2C9) (Sandberg et al, 2002;Sweetman et al, 2007), a cytochrome P450 isoform that is known to exist as several genetic variants (Stormer et al, 2003). CEL eliminated primarily by metabolism and about 3% is recovered in urine and faeces as unchanged compound (Stempak et al, 2002).…”

Pharmacogenetics of nonsteroidal anti-inflammatory drugs

“…25 An in vitro study utilizing human liver microsomes in addition to yeast microsomes expressing a number of CYP polymorphisms showed that CYP2C9*3 had a lower intrinsic clearance of celecoxib when compared with the wild-type CYP2C9*1 samples. 26 There are conflicting results about whether CYP2C9*2 has a significant decrease in intrinsic clearance. 26,27 The most marked decrease in metabolism of celecoxib was noted in enzymes expressing homozygous polymorphism CYP2C9*3/*3.…”

“…26 There are conflicting results about whether CYP2C9*2 has a significant decrease in intrinsic clearance. 26,27 The most marked decrease in metabolism of celecoxib was noted in enzymes expressing homozygous polymorphism CYP2C9*3/*3. 26,27 A corresponding increase in celecoxib's AUC would be expected in individuals expressing CYP2C9*3 allele, and this could result in an increase in dose-related adverse reactions due to accumulation of celecoxib.…”

“…29 These data help to support the hypothesis that CYP2C9 polymorphisms affect the variability in the pharmacokinetics of celecoxib. 26 Celecoxib exerts its effect by inhibition of COX-2, which is encoded by the prostaglandinendoperoxide synthase 2/COX-2 (PTGS2) gene. In a small study of 20 healthy individuals, Skarke et al 31 examined the effect of a SNP (_765G_C) in the PTGS2 gene on the inhibitory effect of celecoxib or the expression of COX-2 enzyme.…”