Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers

Abstract

Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 μM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 μM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0–50 μM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3.

Introduction

The flavin-containing monooxygenases (FMOs, EC 1.14.13.8) are a family of NADPH-dependent enzymes that catalyze the oxygenation of a wide variety of nucleophilic compounds containing a nitrogen, sulfur, phosphorous, or selenium atom [1], [2]; such compounds include the anti-inflammatory drug benzydamine, antithyroid drug methimazole, gastroprokinetic agent itopride, hereditary polyposis drug sulindac sulfide, anti-cancer agent tozasertib, and diet-derived trimethylamine (Fig. 1) [3], [4], [5]. Histamine H₂-receptor antagonist ranitidine [6], dipeptidyl peptidase IV inhibitor teneligliptin [7], α7 neuronal nicotinic receptor agonist AZD0328 [8], and peroxisome proliferator-activated receptor dual agonist MK-0767 [9] are listed as medicinal and new drug candidate substrates of FMO.

FMO3 is considered the prominent functional FMO form expressed in adult human liver [10], [11]. Human protein-coding gene FMO3 and its mRNA expression levels are given in http://www.ncbi.nlm.nih.gov/UniGene/under [UniGene 240387 – Hs.445350]. Genetic polymorphism of FMO3 [12], [13], [14] and/or post-translational modification by environmental factors such as nitric oxide could cause interindividual differences in FMO3 levels or FMO3 catalytic function [15], [16]. Loss-of-function mutations, nonsense mutations, and missense mutations of FMO3 [17], [18], [19] produce phenotypes associated with the inherited disorder trimethylaminuria (also known as fish odor syndrome). In the literature, reported mutations in the FMO3 gene are given using systematic and trivial names [18]. In the course of identification of novel mutations of FMO3 and functional analysis in Japanese individuals suffering from trimethylaminuria, we found common and unique FMO3 variants [19]. We previously reported that Val257Met FMO3 protein had almost the same activity as wild-type FMO3, but [Glu158Lys; Glu308Gly] and Arg205Cys FMO3 proteins exhibited slightly and moderately decreased activities, respectively, with respect to N-oxygenation of trimethylamine [19].

FMOs form a complementary enzyme system to the cytochrome P450 enzymes and have been found to oxygenate several soft, highly polarizable nucleophilic heteroatom-containing chemicals and several drugs analyzed [20]. It has previously been suggested that the products of FMO3-mediated metabolism are generally benign, highly polar, and readily excreted [20]. The absence of induction of FMO3 by xenobiotics is strikingly different to the case for cytochrome P450 enzymes, which are induced by a number of xenobiotics [1]. FMO has been shown to exhibit a stable 4a-flavin hydroperoxide intermediate capable of oxygenating both nucleophiles and electrophiles in its catalytic cycle, even in the absence of oxygenatable substrate [21]. In this context, little information regarding apparent drug interactions has been reported so far for FMO3 [22]. Some of the preclinical research and development areas related to FMOs are not yet fully mature. Therefore, in the present study, we investigated the effects of a variety of N- and S-containing chemicals on benzydamine N-oxygenation to test its suitability as an index reaction for the enzymatic activity of recombinant human FMO3. Further investigations were carried out on FMO3-mediated oxidations of trimethylamine, methimazole, itopride, and tozasertib (selected as strong suppressors of benzydamine N-oxygenation) and sulindac sulfide (selected as typical S-containing clinical substrate). Strong suppression by S-containing methimazole on sulindac sulfide S-oxygenation by polymorphic FMO3 was observed. The present results suggest that a battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities would be useful as probe substrate reactions to clarify drug interactions mediated via polymorphic human FMO3.