J. L. Goldman, L. Van Haandel, J. Leeder, R. Pearce; Children's Mercy Hospital, Kansas City, MO
BACKGROUND: We recently identified N-acetyl cysteine (NAC) adducts of trimethoprim (TMP) metabolites in urine of children taking TMP-sulfamethoxazole, raising the possibility that TMP bioactivation to reactive metabolites (RM) may contribute to adverse drug reactions (ADR). TMP-NAC adducts were also formed by human liver microsomes (HLM), suggesting cytochrome P450 enzymes (CYPs) catalyze TMP bioactivation. The objective of this study was to identify the CYPs involved in the formation of TMP primary metabolites, with a focus on the formation of 3- and 4-desmethyl-TMP (suspected RM precursors) and Cα-NAC-TMP (a RM adduct).
METHODS: Recombinant human CYPs (rCYPs), a panel of characterized HLM (n=16), or pooled HLM in the presence of selective CYP inhibitors were incubated with therapeutic concentrations of TMP (5 µM, 50 µM). RMs were trapped with NAC, and metabolite formation was quantified by UPLC/MS/MS. Correlation coefficients between the rates of metabolite formation and CYP marker reaction rates were determined using least-squares regression analysis and evaluated at α=0.05.
RESULTS: Formation of Cα-NAC-TMP was highly correlated with CYP3A4 activity (r=0.99) in HLM, inhibited by ketoconazole (CYP3A inhibitor) and catalyzed by rCYP3A4. TMP 3-demethylation was highly correlated with CYP2C9 activity (r=0.87), inhibited by sulphafenazole (CYP2C9 inhibitor), but was catalyzed by a variety of rCYPs. TMP 4-demethylation was highly correlated with CYP2C8 activity (r=0.93), but inhibited by ketoconazole and formed by several rCYPs, including rCYP3A4.
CONCLUSION: Cα-NAC-TMP and 4-desmethyl TMP appear to be primarily formed by CYP3A4, while 3-desmethyl TMP formation appears to be catalyzed by CYPs 2C9 and 3A4. Factors modulating activity of these enzymes may affect the risk of ADRs.