Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes

Abstract

Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.

Introduction

Tuberculosis (TB) continues to be a world health problem of staggering proportions. There are an estimated nine million new cases and two million deaths annually, making TB the most common infectious cause of death (Harries and Dye, 2006). Additionally, one-third the world's population is infected with Mycobacterium tuberculosis without exhibiting symptoms of the disease, although 5–10% will be symptomatic at some point in their lifetime (Harries and Dye, 2006). The World Health Organization declared TB a global emergency in 1993 (World Health Organization). Those individuals also infected with HIV are much more likely to develop active TB, further increasing incidence and mortality in areas of the world where HIV is rampant including sub-Saharan Africa (Harries and Dye, 2006, Kochi, 1994, Weyer and Kleeberg, 1992). Because of the emergence of multi-drug-resistant strains of M. tuberculosis, second-line drugs such as ethionamide (ETA) have become increasingly important as replacements for isoniazid and rifampicin (API Consensus Expert Committee, 2006, DeBarber et al., 2000, Kruijshaar et al., 2008, Lu et al., 2008, Shi et al., 2007, Umubyeyi et al., 2008). ETA, and other second-line anti-tuberculosis drugs, exhibit higher rates of adverse drug reactions, including hepatotoxicity (Donald et al., 1987, See et al., 1986, Wada, 1998).

Almost 20 years ago, it was recognized that S-oxygenation of the thiourea moiety of ETA was involved in bioactivation to a metabolite toxic to liver (Russ and Waring, 1991), in a manner similar to what had been shown earlier for thiocarbamides (Neal and Halpert, 1982, Krieter et al., 1984). This S-oxygenation also determines the therapeutic efficacy of ETA, i.e., ETA is a prodrug. In 2000, two separate laboratories identified the gene product Rv3855c (EtaA) of M. tuberculosis as the enzyme responsible for bioactivation of ETA (Baulard et al., 2000, DeBarber et al., 2000). This enzyme had properties consistent with that of a monooxygenase (Baulard et al., 2000). Subsequently, Vannelli et al. (2002) cloned, expressed and characterized the product of the M. tuberculosis gene Rv3854c as an FAD-containing enzyme capable of bioactivating ETA to two major products, the corresponding S-oxide (ETASO), and 2-ethyl-4-amidopyridine (ETAA). ETA and ETASO exhibit equivalent therapeutic potency and hepatotoxicity, whereas, ETAA is relatively non-toxic and without therapeutic efficacy. Further oxygenation of ETASO to the sulfinic acid (which spontaneously breaks down to ETAA and other products) is the postulated pathway for M. tuberculosis-dependent bioactivation of ETA or ETASO. To date, the sulfinic acid has not been isolated. Further studies by Qian and Ortiz de Montellano (2006) demonstrated that the activity toward activation of ETA by M. tuberculosis EtaA extended to other related compounds, such as thiacetoazone, prothioamide and isoxy (4,4′-diisoamyloxydiphenylthiourea) (Dover et al., 2007, Phetsuksiri et al., 2003, Wang et al., 2007).

Flavin-containing monooxygenases (FMOs) are a superfamily of flavoprotein monooxygenases active towards a wide range of xenobiotics. Humans express protein products of five genes (FMO1 to FMO5) in a developmental-, sex-, and tissue-specific manner (Cashman et al., 1995, Hernandez et al., 2004, Krueger and Williams, 2005, Ziegler, 2002). Several FMO pseudogenes have been described that do not appear to produce functional proteins (Hernandez et al., 2004, Hines et al., 2002). FMOs catalyze oxygenation of a wide variety of endogenous and xenobiotic compounds containing soft nucleophiles (nitrogen, sulfur, and phosphorus heteroatoms) (Cashman, 2003, Krueger et al., 2005, Ziegler, 2002). FMOs contain a single FAD and require NADPH and O₂ for catalytic activity (Ziegler, 2002). The highest concentrations are found in liver, but extrahepatic tissue, including lung can express high specific concentrations in a number of mammals (Cashman, 1995, Dolphin et al., 1998, Krueger et al., 2001, Shehin-Johnson et al., 1995, Ziegler, 1988). In human fetal liver, the major FMO is FMO1; shortly after birth, expression of FMO1 is shut off and FMO3 levels increase over time and become the major hepatic FMO in adults (Hines, 2006, Koukouritaki et al., 2002). FMO1 is the major form expressed in adult intestine and kidney (Yeung et al., 2000). FMO2 is typically expressed at high levels in lung, including those of non-human primates (Dolphin et al., 1998, Krueger et al., 2001). Humans exhibit an interesting genetic polymorphism in that both alleles of all Caucasians and Asians genotyped to date possess a C→T transition mutation resulting in a premature TAG stop codon 64 amino acids from the C-terminal (FMO2⁎2) (Dolphin et al., 1998, Whetstine et al., 2000). This protein (FMO 2.2) is not detectable on western blots with antibody that cross-reacts with the truncated protein (Dolphin et al., 1998, Krueger et al., 2002a), probably due to improper folding and rapid degradation. Conversely, 27% of individuals of African descent and 2–7% (depending if they are of Mexican or Puerto Rican descent) of Hispanics possess at least one FMO2⁎1 allele coding for the full length and enzymatically active enzyme (FMO 2.1) (Furnes et al., 2003, Krueger et al., 2002b, Krueger et al., 2004, Krueger et al., 2005, Whetstine et al., 2000).

M. tuberculosis EtaA appears to have properties in common with a Baeyer–Villiger monooxygenase (Fraaije et al., 2004). EtaA readily catalyzes the further oxygenation of ETASO (Vannelli et al., 2002); whereas, the human FMO 2.1 has been demonstrated to oxygenate certain thioureas only once (Henderson et al., 2004b). In this paper, we compare the activity of expressed mouse and human FMOs to ETA and demonstrate k_cats greater than EtaA. Furthermore, the second oxygenation to the sulfinic acid is slower than with M. tuberculosis EtaA and the sulfenic acid is long-lived enough to react with glutathione (GSH). Thus, the yield of ETAA through the sulfinic acid is much reduced compared to EtaA. These findings may explain the hepatotoxicity of ETA and also raise questions about ETA metabolism in human lung.