In Vitro Hepatic Oxidative Biotransformation of Trimethoprim

Abstract

Trimethoprim (TMP) has been widely used since the 1960s, both alone and in combination with sulfamethoxazole. Unfortunately, information regarding the role that cytochrome P450 enzymes (P450s) play in the formation of TMP primary metabolites is scarce. Hence, we undertook in vitro studies to identify and more fully characterize the P450s that catalyze formation of six TMP primary metabolites: TMP 1-N-oxide (1-NO-TMP) and 3-N-oxide (3-NO-TMP), 3'- and 4'-desmethyl-TMP, a benzylic alcohol (Cα-OH-TMP), and an N-acetyl cysteine (NAC) adduct of TMP (Cα-NAC-TMP). Formation kinetics for each TMP metabolite in human liver microsomes (HLMs) were consistent with single-enzyme Michaelis-Menten kinetics, and Km values were markedly above (≥10-fold) the therapeutic concentrations of TMP (50 µM). The combined results from correlation studies between rates of metabolite formation and marker P450 activities in a panel of HLMs along with inhibition studies utilizing selective P450 inhibitors incubated with pooled HLMs suggested that 1-NO-TMP, Cα-NAC-TMP, and Cα-OH-TMP were predominantly formed by CYP3A4. In contrast, 3-NO-TMP was formed predominantly by CYP1A2 in HLMs and inhibited by α-naphthoflavone. 4'-Desmethyl-TMP, which is believed to be a reactive TMP metabolite precursor, was formed by several P450s, including CYP3A4, correlated with multiple P450 activities, but was inhibited primarily by ketoconazole (up to 50%), suggesting that CYP3A4 makes a major contribution to TMP 4'-demethylation. TMP 3'-demethylation was catalyzed by multiple P450s, including CYP2C9, correlated with CYP2C9 activity, and was inhibited by sulfaphenazole (up to 40%). Overall, CYP2C9 and CYP3A4 appear to be the most significant contributors to TMP primary metabolism.

Fig. 1.

Metabolic scheme for the conversion of TMP to its primary oxidative metabolites.

Fig. 2.

Variability in the formation rates of trimethoprim primary metabolites. TMP (5 µM) was incubated with HLMs prepared from individual donors (n = 16) as described under Materials and Methods. Microsomes from each donor were incubated in triplicate. Box and whisker plots were constructed for the rate of formation for each metabolite. The box extends from the 25^th to the 75^th percentile with the line in the middle at the median. The whiskers extend to the minimum and maximum values, respectively. (A) TMP N-oxidation (1- and 3-NO-TMP). (B) TMP demethylation (3′- and 4′-desmethyl-TMP). (C) TMP Cα-oxidation (Cα-OH-TMP and Cα-NAC-TMP).

Fig. 3.

N-oxidation of TMP to primary metabolites. (A) 1- and 3-NO-TMP formation by human cDNA-expressed P450 enzymes. TMP (5 µM) was incubated with cDNA-expressed P450 enzymes as described under Materials and Methods. Rates shown are corrected for rates from microsomes containing vectors alone. Each bar represents the mean of the duplicate incubations. (B) Sample-to-sample variation in the rates of TMP 1- and 3-N-oxidation by HLMs at a substrate concentration of 5 µM. Each bar represents the mean of the triplicate experiments ± S.D. (C) Effects of various P450 isoform–selective inhibitors on the formation of 1- and 3-NO-TMP by pooled HLMs at a substrate concentration of 50 µM, as described under Materials and Methods. Each bar represents the mean of the triplicate incubations. Uninhibited mean rates of 1- and 3-NO-TMP formation were 8.2 and 1.6 pmol/mg protein/min, respectively.

Fig. 4.

Demethylation of TMP to primary metabolites. (A) 3′- and 4′-desmethyl-TMP formation by human cDNA-expressed P450 enzymes. TMP (5 µM) was incubated with cDNA-expressed P450 enzymes as described under Materials and Methods. Rates shown are corrected for rates from microsomes containing vectors alone. Each bar represents the mean of the duplicate incubations. (B) Sample-to-sample variation in the rates of TMP 3′- and 4′-desmethyl-TMP by HLMs at a substrate concentration of 5 µM. Each bar represents the mean of the triplicate experiments ± S.D. (C) Effects of various P450 isoform–selective inhibitors on the formation of 3′- and 4′-desmethyl-TMP by pooled HLMs at a substrate concentration of 50 µM, as described under Materials and Methods. Each bar represents the mean of the triplicate incubations. Uninhibited rates of 3′- and 4′-desmethyl-TMP formation were 48.1 and 29.2 pmol/mg protein/min, respectively.

Fig. 5.

Cα-oxidation of TMP to primary metabolites. (A) Cα-OH-TMP and Cα-NAC-TMP formation by human cDNA-expressed P450 enzymes. TMP (5 µM) was incubated with cDNA-expressed P450 enzymes as described under Materials and Methods. Rates shown are corrected for rates from microsomes containing vectors alone. Each bar represents the mean of the duplicate incubations. (B) Sample-to-sample variation in the rates of Cα-OH-TMP and Cα-NAC-TMP formation by HLMs at a substrate concentration of 5 µM. Each bar represents the mean of the triplicate experiments ± S.D. (C) Effects of various P450 isoform–selective inhibitors on the formation of Cα-OH-TMP and Cα-NAC-TMP by pooled HLMs at a substrate concentration of 50 µM, as described under Materials and Methods. Each bar represents the mean of the triplicate incubations. Uninhibited rates of Cα-OH-TMP and Cα-NAC-TMP formation were 4.6 and 2.1 pmol/mg protein/min, respectively.