Different substrate elimination rates of model drugs pH-dependently mediated by flavin-containing monooxygenases and cytochromes P450 in human liver microsomes.

The predicted contributions of flavin-containing monooxygenase 3 (FMO3) to drug candidate N-oxygenations can be estimated using classic base dissociation constants of the N-containing moiety. In this study, metabolic clearance values in human liver microsomes were experimentally determined for available model drugs. Typical metabolic clearance values (34-96 µL/min/mg protein) at pH 8.4 of trimethylamine, benzydamine, and itopride were two-to fourfold higher than those at pH 7.4. In contrast, the metabolic clearance of control drug midazolam at pH 8.4 was half that at pH 7.4. The ratios of clearance values at pH 8.4 to those at pH 7.4 and the substrate pKa (base) values of reported metabolic N-oxygenation sites of trimethylamine, benzydamine, clomipramine, chlorpromazine, tamoxifen, itopride, loxapine, xanomeline, tozasertib, dasatinib, and clozapine were significantly correlated (r = 0.60, p < 0.05, n = 11). These results suggested that the simple comparison of metabolic clearance values at pH 8.4 and at pH 7.4 could be useful for predicting the contributions of FMO3 to the N-oxygenations of new drug candidates. This method, along with in silico pKa (base) values > 8.4, could prove useful for predicting the contributions of FMO3 to N-oxygenations as part of drug development.

Predicted Contributions of Flavin-containing Monooxygenases to the N-oxygenation of Drug Candidates Based on their Estimated Base Dissociation Constants.

AIMS: Base dissociation constants of 30 model chemicals were investigated to constitute potential determinant factors predicting the contributions of flavin-containing monooxygenases (FMOs). BACKGROUND: The contributions of FMOs to the metabolic elimination of new drug candidates could be underestimated under certain experimental conditions during drug development. OBJECTIVE: A method for predicting metabolic sites and the contributions of FMOs to N-oxygenations is proposed using a molecular descriptor, the base dissociation constant (pKa base), which can be estimated in silico using commonly available chemoinformatic prediction systems. METHODS: Model drugs and their oxidative pathways were surveyed in the literature to investigate the roles of FMOs in their N-oxygenations. The acid and base dissociation constants of the nitrogen moieties of 30 model substrates were estimated using well-established chemoinformatic software. RESULTS: The base dissociation constants of 30 model chemicals were classified into two groups based on the reported optimal in vitro pH of 8.4 for FMO enzymes as a key determinant factor. Among 18 substrates (e.g., trimethylamine, benzydamine, and itopride) with pKa (base) values in the range of 8.4-9.8, all N-oxygenated metabolites were reported to be predominantly catalyzed by FMOs. Except for three cases (xanomeline; L-775,606; and tozasertib), the nine substrates with pKa (base) values in the range 2.7-7.9 were only moderately or minorly N-oxygenated by FMOs in addition to their major metabolic pathway of oxidation mediated by cytochrome P450s. N-Oxygenation of T-1032 (with a pKa of 4.8) is mediated predominantly by P450 3A5, but not by FMO1/3. CONCLUSION: The predicted contributions of FMOs to the N-oxygenation of drug candidates can be simply estimated using classic base dissociation constants.

Benzydamine N-oxygenation as an index for flavin-containing monooxygenase activity and benzydamine N-demethylation by cytochrome P450 enzymes in liver microsomes from rats, dogs, monkeys, and humans.

Benzydamine is an anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to benzydamine N-oxide; however, benzydamine N-demethylation is also catalyzed by liver microsomes. In this study, benzydamine N-oxygenation and N-demethylation mediated by liver microsomes from rats, dogs, monkeys, and humans were characterized comprehensively. Values of the maximum velocity/Michaelis constant ratio for benzydamine N-oxygenation by liver microsomes from dogs and rats were higher than those from monkeys and humans, despite roughly similar rates of N-demethylation in the four species. Benzydamine N-oxygenation by liver microsomes was extensively suppressed by preheating liver microsomes at 45 °C for 5 min or at 37 °C for 5-10 min without NADPH, and benzydamine N-demethylation was strongly inhibited by 1-aminbobenztriazole. Liver microsomal benzydamine N-oxygenation was inhibited by dimethyl sulfoxide and methimazole, whereas N-demethylation was inhibited by quinidine. High benzydamine N-oxygenation activities of recombinant human FMO1 and FMO3 and human kidney microsomes were observed at pH 8.4, whereas N-demethylation by cytochrome P450 2D6 was faster at pH 7.4. These results suggest that benzydamine N-oxygenation and N-demethylation are mediated by FMO1/3 and P450s, respectively, and that the contribution of FMO to metabolic eliminations of new drug candidates might be underestimated under certain experimental conditions suitable for P450 enzymes.