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.

Several-fold differences have been observed among patients in the biotransformation of cyclophosphamide. The aim of this study was to investigate the contribution of CYP2C9 and CYP2C19 and their polymorphisms to the variability of cyclophosphamide activation. The formation of 4-hydroxycyclophosphamide was studied in microsomes from a total of 32 different genotyped human livers, as well as in yeast microsomes expressing different genetic variants of CYP2C9 and CYP2C19. The kinetic data obtained in the yeast system revealed that the intrinsic clearance (V(max)/K(m)) of cyclophosphamide by CYP2C9.2 and CYP2C9.3 samples was approximately threefold lower than that by CYP2C9.1. However, in liver microsomes, there were no statistically significant differences in the intrinsic clearance of 4-hydroxycyclophosphamide formation between the group of seven CYP2C9*1/*1 livers and the remaining nine with one or two variant CYP2C9 alleles ( P>0.7). We found a statistically significant correlation ( r(s)=0.65, P=0.003) between 4-hydroxylation of cyclophosphamide and 5'-hydroxylation of R-omeprazole, a measure of CYP2C19 activity in human liver microsomes ( n=19). No correlation was found between 4-hydroxylation of cyclophosphamide and the formation rate of hydroxycelecoxib, mainly catalysed by CYP2C9 ( r(s)=0.17, P=0.55, n=32). In conclusion, based on the correlation with the formation of R-5'-hydroxyomeprazole, CYP2C19 may partly contribute to the bioactivation of cyclophosphamide in human liver microsomes, while the role of CYP2C9 appears minor.

The urinary losartan to E3174 metabolic ratio after a 25-mg losartan dose was found to be a safe and useful phenotyping assay for CYP2C9 activity in vivo. CYP2C9*3 variant allele is a major determinant of the enzyme activity, and it decreases losartan metabolism significantly, while CYP2C9*2 allele has less impact on enzyme function.