Hepatic metabolism of diclofenac: Role of human CYP in the minor oxidativepathways

The aim of this study was to re-examine the human hepatic metabolism of dic lofenac, with special focus on the generation of minor hydroxylated metabol ites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and engineered cells expressing single human CYP (cytochromes P450). Human hep atocytes formed 3'-hydroxy-, 4'-hydroxy-, 5-hydroxy-4',5-dihydroxy-, and N, 5-dihydroxydiclofenac, as well as several lactams. Formation of 4'- and 5-h ydroxydiclofenac by human liver microsomes followed a Michaelis-Menten kine tics (K-m 9 +/- 1 mu M; V-max 432 +/- 15 pmol/min/mg and K-m 43 +/- 5 mu M; and V-max 15.4 +/- 0.6 pmol/min/mg, respectively). Secondary metabolites w ere detected after incubation of 5-hydroxydiclofenac with human liver micro somes, yielding 4',5-dihydroxydiclofenac (K-m 15 +/- 1 mu M; V-max 96 +/- 3 pmol/min/mg) and small amounts of N,5-dihydroxydiclofenac (non-Michaelis-M enten kinetics). Based on microsome studies and the incubations with human hepatocytes and engineered cells, we estimated that in vivo CYP2C9 would be exclusively responsible for the 4' hydroxylation of diclofenac (>99.5%) as well as 5-hydroxydiclofenac (>97%). CYP2C9 was exclusively responsible for the formation of 3'-hydroxydiclofenac. Multiple regression analysis eviden ced that the rate of production of 5-hydroxydiclofenac in human microsomes followed the algorithm: 0.040 x S-mephenytoin 4'-hydroxylation + 0.083 x to lbutamide methylhydroxylation, (multiple correlation coefficient = 0.969). However, the incubation of diclofenac with cell lines expressing different human CYP suggested that 7 isoforms could be involved. Comparison of data o btained with CYP-expressing cells and human hepatocytes suggests that CYP2C 8 > CYP2C19 congruent to CYP2C18 much greater than CYP2B6 are the isoforms implicated in the 5-hydroxylation of diclofenac in vivo. (C) 1999 Elsevier Science Inc.