A novel flavin-containing monooxygenase from Pseudomonas guineae enables efficient biosynthesis of indigo from indole via both enzymatic and cell factory approaches.

Busulphan Metabolism Via Flavin-Containing Monooxygenase 3 (FMO3) Can Explain Several Interactions with Other Drugs

The antipsychotic olanzapine is reportedly metabolized by inducible human cytochrome P450 (CYP) 1A2 and variable copy-number CYP2D6 and polymorphic flavin-containing monooxygenase 3 (FMO3) in different pathways. We investigated individual differences in the metabolite formation and clearance of olanzapine in vitro and in vivo. Human liver microsomal olanzapine oxidation activities were evaluated, and plasma concentrations of olanzapine were determined in 21 Japanese patients (mean age: 50 years, range: 32-69 years, 14 male and 7 female, including 6 smokers) genotyped for CYP2D6 (*1, *5, and *10) and FMO3 (E158K, C197fsX, R205C, V257M, E308G, and R500X). Furafylline (a CYP1A2 inhibitor), quinidine (a CYP2D6 inhibitor), and heat treatment (inactivates FMO3) suppressed liver microsomal metabolic clearance of olanzapine by approximately 30%. Olanzapine N-demethylation and N-oxygenation were found to be catalyzed by CYP1A2 and CYP2D6 and by CYP2D6 and FMO3, respectively, in experiments using liver microsomes and recombinant enzymes. Plasma concentrations and clearance of olanzapine were not affected by CYP2D6 or FMO3 genotypes or smoking behavior. Olanzapine clearance was not affected by CYP2D6 or FMO3 genotypes or smoking behavior as a single factor under the present conditions because olanzapine clearance is mediated by multiple enzymes involved in two major and one minor pathways.

The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity. BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.

A variety of cytochrome P450 enzymes and flavin-containing monooxygenases in dogs and pigs commonly used as preclinical animal models

Overview of indigo biosynthesis by Flavin-containing Monooxygenases: History, industrialization challenges, and strategies

The 8-aminoquinoline drug primaquine (PQ) is currently the only drug in use against the persistent malaria caused by the hypnozoite-forming strains P. vivax and P. ovale. However, despite decades of research, its complete metabolic profile is still poorly understood. In the present study, the metabolism of PQ was evaluated by incubating the drug with pooled human hepatocytes cultured in vitro as well as with recombinant cytochrome P450 (CYP) iso- enzymes, monoamine oxidases (MAO), and flavin-containing monooxygenases (FMO). Targeted LC-MS/MS analysis of hepatocyte incubations using chemical inhibitors indicated that PQ was predominantly metabolized by CYPs 3A4, 1A2 and 2D6, MAO-A, -B and FMO-3. Confirmation of these results was sought by incubation of PQ with the corresponding recombinant enzymes. Small amounts of carboxyprimaquine (CPQ), the major observed PQ metabolite in vivo, were detected in recombinant MAO-A incubations along with another peak at m/z 261, and no significant formation of CPQ with any other recombinant enzymes was observed. Incubations with all recombinant enzymes identified as potentially active towards PQ from the hepatocyte-based assay resulted in significant parent loss over the course of 1 h. These results suggest that several enzymes, including CYPs in combination with FMOs and MAOs, play a role in the overall metabolism of PQ and indicate a major role for MAO-A. Future studies to elucidate the potential role in cytotoxicity and/or efficacy of the PQ metabolite observed at m/z 261, as observed in MAO-A isoenzyme studies, are needed.

Drug metabolism involves the biochemical modification of pharmaceutical substances or xenobiotics by living organisms, usually through specialized enzymes. This process normally converts lipophilic chemicals into less potent, and more hydrophilic products that facilitates their elimination from the body (Mittal et al., 2015). However, these processes may also convert the drug into more lipophilic, more potent or even toxic metabolites (Macherey and Dansette, 2015). In order to design effective and safe dosage regimens, the pharmacology, toxicology, and drug-drug interactions of the drug and its metabolites should be thoroughly understood (Tillement and Tremblay, 2007). As a result, the study of drug metabolism is vital to the pharmaceutical industry. Drug metabolism is generally divided into two distinct phases. Phase I reactions include oxidation, reduction and hydrolysis which are mediated by enzymes such as cytochrome P450 (CYPs), flavin-containing monooxygenases (FMOs), aldehyde oxidase (AO) and various hydrolases (Shehata, 2010). In phase II metabolism, enzymes such as uridine 5’-diphosphoglucuronosyltransferase (UGT), sulfotransferase, glutathione S-transferase (GST) and N-acetyl transferase (NAT) catalyze the conjugation of drugs with endogenous molecules (Testa and Clement, 2015). &#13;\nAlthough the liver is the primary site of drug metabolism, other organs including skin, lungs, kidneys, and intestine also possess considerable metabolic capacity (Krishna and Klotz, 1994). Furthermore, drug-metabolizing enzymes are known to be expressed in the lungs, albeit to a lesser extent than in liver (Somers et al., 2007). Recent reports have highlighted the metabolic role of lungs, a highly perfused organ that is in direct contact with inhaled xenobiotics and drugs (Borghardt et al., 2018). In addition, phase I enzymes such as CYP1A1 may play an important role in bioactivation of inhaled procarcinogens, such as those present in tobacco smoke (Anttila et al., 2011). From the perspective of pharmacotherapy, pulmonary drug metabolism may cause a first-pass effect for inhaled medicines, as well as contribute to overall clearance of systemically administered drugs (Winkler et al., 2004). Frequent exposure of the lungs to environmental xenobiotics may also lead to induction of drug-metabolizing enzymes via pregnane X receptors (PXR), constitutive androstane receptors (CAR), or aryl hydrocarbon receptors (AhR), a phenomenon that can significantly alter the rate of drug metabolism (Tolson and Wang, 2010). &#13;\nDespite the potential importance of lung metabolism for respiratory therapies, relatively little is known about the actual activity and protein abundance of drug-metabolizing enzymes in lung tissue (Hukkanen et al., 2002). The lack of robustness and consistency of existing experimental models of lung metabolism leads to considerable difficulties in the interpretation and prediction of drug clearance. This project was designed to address these challenges and establish a robust and predictive experimental model for rat and human pulmonary drug metabolism. Therefore the aim of this thesis was 1) to investigate the further development of a precision-cut lung slicing (PCLS) model to accurately estimate pulmonary drug clearance in rat, 2) to examine the pulmonary metabolic activity of rat and human phase I and phase II enzymes using this model, and 3) to compare the PCLS model with currently available in vitro and in vivo experimental models in order to better understand the contribution of pulmonary metabolism to drug elimination. &#13;\n1)\tEstablish a PCLS model to accurately estimate pulmonary drug clearance in rat &#13;\nPCLS technology is a 3D organotypic tissue model which reflects the natural and relevant microanatomy and the metabolic function of the lung (Neuhaus et al., 2017). Although the use of PCLS is becoming accepted as a research tool to investigate pulmonary drug metabolism, the protocols applied vary between laboratories and there is still opportunity to improve and standardize the methodology. Therefore, some of the key experimental factors used in the PCLS procedure were optimized with the aim of reducing variability and tissue damage and retaining lung metabolic activity. Due to the limited availability of fresh human lung tissue, method optimization was performed using rat lung tissue (under the assumption that rat is a suitable surrogate for human) and referring to a well-known CYP1A1 substrate, mavoglurant (AFQ056). The choice of mavoglurant as a test compound was based on two factors; 1) it is a CYP1A1 substrate and this enzyme is located primarily in extra-hepatic organs such as lung, kidney, brain and intestine (Drahushuk et al., 1998, Cheung et al., 1999, Paine et al., 1999, Smith et al., 2001) and 2) metabolism of mavoglurant by CYP1A1 produces a specific metabolite, CBJ474, that serves as a marker for CYP1A1 metabolic activity (Walles et al., 2013). During the optimization process of the PCLS model it was possible to achieve higher mavoglurant turnover by performing incubations on dynamic organ culture system. This investigation demonstrated the importance of optimization and standardization of PCLS conditions.&#13;\n2)\tInvestigate the activity of phase I and phase II metabolic enzymes in rat and human lungs using PCLS model&#13;\nPreclinical species such as rats and mice are commonly used for the optimization of pharmacokinetic (PK) properties and for testing in vivo efficacy of new chemical entities. The PK data from these preclinical species is also often used for the prediction of human PK, and therefore, it is desirable that drug metabolism in these species is representative of that in human. For the comparison of enzyme activity in rat and human lungs, a selection of phase I and phase II probe substrates (please refer to chapter 3.1, Table 3 for a list of the probe substrates tested) were incubated using the optimized PCLS model. The results showed that there are remarkable differences in pulmonary metabolic activity between rat and human, reflecting species dependent expression of drug-metabolizing enzymes. CYP-mediated metabolic activity was relatively low in both species, whereas phase II enzyme activities appeared to be more significant in rat than in human. Therefore, care should be taken when extrapolating metabolism data from animal models to humans.&#13;\n3)\tComparison of PCLS model with established in vitro and in vivo experimental models to be able to understand the contribution of pulmonary metabolism to drug elimination&#13;\nA variety of in vitro and ex vivo lung models such as cell culture, sub-cellular fractions, tissue slices and isolated perfused lung models have been used to investigate pulmonary metabolism. Each tool has advantages and disadvantages and can be used to answer specific scientific questions. However, due to the diversity of the cells in the lung it is difficult to obtain quantitative data. In this research project, mavoglurant and benzydamine (well-characterized FMO substrate) were incubated using rat lung microsomes (in vitro) and rat PCLS (ex vivo) and also administered intravenously (i.v.) and intra-arterially (i.a.) to rats (in vivo). Previous human in vivo studies had indicated that extra-hepatic metabolism contributes to the elimination of mavoglurant (Novartis internal data). The goal was to use these models to understand if the lung makes a significant contribution to total body clearance. Using the well-stirred organ model, lung clearance (CLlung) of mavoglurant was estimated from microsomal and PCLS data and compared to the in vivo data obtained from i.v. and i.a. dosing to rats. The data generated from these three experimental models were comparable and the data suggested that the contribution of pulmonary metabolism to the elimination of mavoglurant is negligible. The same experiments were also performed using benzydamine. Interestingly, calculated lung clearance from microsomal data were 8-fold higher than lung clearance estimates from the PCLS model. Hence, similar to the PCLS-derived predictions, in vivo data indicated very low pulmonary clearance. &#13;\n

Voriconazole is a potent second-generation triazole antifungal agent with broad-spectrum activity against clinically important fungi. It is cleared predominantly via metabolism in all species tested including humans. N-Oxidation of the fluoropyrimidine ring, its hydroxylation, and hydroxylation of the adjacent methyl group are the known pathways of voriconazole oxidative metabolism, with the N-oxide being the major circulating metabolite in human. In vitro studies have shown that CYP2C19, CYP3A4, and to a lesser extent CYP2C9 contribute to the oxidative metabolism of voriconazole. When cytochrome P450 (P450)-specific inhibitors and antibodies were used to evaluate the oxidative metabolism of voriconazole by human liver microsomes, the results suggested that P450-mediated metabolism accounted for approximately 75% of the total oxidative metabolism. The studies presented here provide evidence that the remaining approximately 25% of the metabolic transformations are catalyzed by flavin-containing monooxygenase (FMO). This conclusion was based on the evidence that the NADPH-dependent metabolism of voriconazole was sensitive to heat (45 degrees C for 5 min), a condition known to selectively inactivate FMO without affecting P450 activity. The role of FMO in the metabolic formation of voriconazole N-oxide was confirmed by the use of recombinant FMO enzymes. Kinetic analysis of voriconazole metabolism by FMO1 and FMO3 yielded K(m) values of 3.0 and 3.4 mM and V(max) values of 0.025 and 0.044 pmol/min/pmol, respectively. FMO5 did not metabolize voriconazole effectively. This is the first report of the role of FMO in the oxidative metabolism of voriconazole.

Benzydamine is a nonsteroidal anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to a stable N-oxide. This metabolite can be quantified with high specificity and sensitivity by using a simple reverse-phase high-performance liquid chromatography (HPLC) assay with fluorescence detection. Studies with recombinant FMO enzymes demonstrate that FMOI and FMO3 are the primary catalysts of benzydamine N-oxygenation, with minimal contributions from cytochrome P450 enzymes. Investigations conducted with human liver microsomes confirm that FMO3, in large part, is responsible for benzydamine N-oxide formation in this tissue. These features render benzydamine a useful in vitro probe for FMO activity in a wide range of tissues and cell types. In addition, benzydamine appears to be a suitable in vivo probe for human liver FMO3. This chapter provides a detailed account of the experimental protocol for determining rates of formation of benzydamine N-oxide by FMO-containing enzyme fractions.

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

NADPH regeneration for efficient biosynthesis of indigo by flavin-containing monooxygenase and formate dehydrogenase.

Role of cytochrome P450 enzymes in fimasartan metabolism in vitro

Nabumetone (NAB) is a non-steroidal anti-inflammatory drug used clinically, and its biotransformation includes the major active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). One of the key intermediates between NAB and 6-MNA may be 3-hydroxy nabumetone (3-OH-NAB). The aim of the present study was to investigate the role of flavin-containing monooxygenase (FMO) isoform 5 in the formation of 6-MNA from 3-OH-NAB. To elucidate the biotransformation of 3-OH-NAB to 6-MNA, an authentic standard of 3-OH-NAB was synthesised and used as a substrate in an incubation with human liver samples or recombinant enzymes. The formation of 3-OH-NAB was observed after the incubation of NAB with various cytochrome P450 (CYP) isoforms. However, 6-MNA itself was rarely detected from NAB and 3-OH-NAB. Further experiments revealed a 6-MNA peak derived from 3-OH-NAB in human hepatocytes. 6-MNA was also detected in the extract obtained from 3-OH-NAB by a combined incubation of recombinant human FMO5 and human liver S9. We herein demonstrated that the reaction involves carbon-carbon cleavage catalyzed by the Baeyer–Villiger oxidation (BVO) of a carbonyl compound, the BVO substrate, such as a ketol, by FMO5. Further in vitro inhibition experiments showed that multiple non-CYP enzymes are involved in the formation of 6-MNA from 3-OH-NAB.

A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli

Flavin-containing monooxygenases (FMOs) are a family of enzymes that are involved in the oxygenation of heteroatom-containing molecules. In humans, FMO3 is the major hepatic form, whereas FMO1 is predominant in the kidneys. FMO1 and FMO3 have also been identified in monkeys, dogs, and pigs. The predicted contribution of human FMO3 to drug candidate N-oxygenation could be estimated using the classic base dissociation constants of the N-containing moiety. A basic quinuclidine moiety was found in natural quinine and medicinal products. Consequently, N-oxygenation of quinuclidine was evaluated using liver and kidney microsomes from humans, monkeys, dogs, and pigs as well as recombinant FMO1, FMO3, and FMO5 enzymes. Experiments using simple reversed-phase liquid chromatography with fluorescence monitoring revealed that recombinant FMO1 mediated quinuclidine N-oxygenation with a high capacity in humans. Moreover, recombinant FMO1, FMO3, and/or FMO5 in monkeys, dogs, and pigs exhibited relatively broad substrate specificity toward quinuclidine N-oxygenation. Kinetic analysis showed that human FMO1 efficiently, and pig FMO1 moderately, mediated quinuclidine N-oxygenation with high capacity, which is consistent with the reported findings for larger substrates readily accepted by pig FMO1 but excluded by human FMO1. In contrast, human FMO3-mediated quinuclidine N-oxygenation was slower than that of the typical FMO3 substrate trimethylamine. These results suggest that some species differences exist in terms of FMO-mediated quinuclidine N-oxygenation in humans and some animal models (monkeys, dogs, and minipigs); however, the potential for quinuclidine, which has a simple chemical structure, to be inhibited clinically by co-administered drugs should be relatively low, especially in human livers. SIGNIFICANCE STATEMENT: The high capacity of human flavin-containing monooxygenase (FMO) 1 to mediate quinuclidine N-oxygenation, a basic moiety in natural products and medicines, was demonstrated by simple reversed-phase liquid chromatography using fluorescence monitoring. The substrate specificity of FMO1 and FMO3 toward quinuclidine N-oxygenation in monkeys, dogs, and pigs was suggested to be relatively broad. Human FMO3-mediated quinuclidine N-oxygenation was slower than trimethylamine N-oxygenation. The likelihood of quinuclidine, with its simple chemical structure, being clinically inhibited by co-administered drugs is relatively low.