Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism.

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17ß-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Identification of Gut Bacterial Enzymes for Keto-Reductive Metabolism of Xenobiotics.

Human gastrointestinal microbiota are known for the keto-reductive metabolism of small-molecule pharmaceuticals; however, the responsible enzymes remain poorly understood. Through in vitro biochemical assays, we report the identification of enzymes encoded in the genome of Clostridium bolteae that can reduce the ketone groups of nabumetone, hydrocortisone, and tacrolimus. The homologues to a newly identified enzyme (i.e., DesE) are potentially widely distributed in the gut microbiome. The selected enzymes display different levels of activities against additional chemicals such as two dietary compounds (i.e., raspberry ketone and zingerone), chemotherapeutic drug doxorubicin, and its aglycone metabolite doxorubicinone. Thus, our results expand the repertoire of enzymes that can reduce the ketone groups in small molecules and could serve as the basis for future personalized medicine approaches.

Role of human flavin-containing monooxygenase (FMO) 5 in the metabolism of nabumetone: Baeyer-Villiger oxidation in the activation of the intermediate metabolite, 3-hydroxy nabumetone, to the active metabolite, 6-methoxy-2-naphthylacetic acid 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.

Presence or absence of microbiome modulates the response of mice organism to administered drug nabumetone.

The gut microbiota provides a wide range of beneficial functions for the host, and has an immense effect on the host's health status. The presence of microbiome in the gut may often influence the effect of an orally administered drug. Molecular mechanisms of this process are however mostly unclear. We investigated how the effect of a nonsteroidal drug nabumetone on expression of drug metabolizing enzymes (DMEs) in mice intestine and liver is changed by the presence of microbiota, here, using the germ free (GF) and specific pathogen free (SPF) BALB/c mice. First, we have found in a preliminary experiment that in the GF mice there is a tendency to increase bioavailability of the active form of nabumetone, which we have found now to be possibly influenced by differences in expression of DMEs in the GF and SPF mice. Indeed, we have observed that the expression of the most of selected cytochromes P450 (CYPs) was significantly changed in the small intestine of GF mice compared to the SPF ones. Moreover, orally administered nabumetone itself altered the expression of some CYPs and above all, in different ways in the GF and SPF mice. In the GF mice, the expression of the DMEs (CYP1A) responsible for the formation of active form of the drug are significantly increased in the small intestine and liver after nabumetone application. These results highlight the importance of gut microbiome in processes involved in drug metabolism in the both gastrointestinal tract and in the liver with possible clinical relevance.

A metabolic pathway for the prodrug nabumetone to the pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA) by non-cytochrome P450 enzymes.

The pathway for the transformation of the prodrug nabumetone, 4-(6-methoxynaphthalen-2-yl)butan-2-one, to the active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA), a potent cyclooxygenase-2 inhibitor, has not yet been clarified in humans.To confirm the activation pathway, authentic standards of the nabumetone intermediates, 2-(6-methoxynaphthalen-2-yl)ethyl acetate (6-MNEA), 2-(6-methoxynaphthalen-2-yl)ethan-1-ol (6-MNE-ol) and 2-(6-methoxynaphthalen-2-yl)acetaldehyde (6-MN-CHO) were synthesized. High performance liquid-chromatography and gas chromatography-mass spectrometry on nabumetone oxidation revealed the generation of three metabolites.The formation of 6-MNA after a 60-min incubation of nabumetone was detected and 6-MNE-ol, an alcohol-related intermediate, was also generated by in cryopreserved hepatocytes. However, 6-MNA was below detection limit, but 4-(6-methoxynaphthalen-2-yl)butan-2-ol (MNBO) and 4-(6-hydroxynaphthalen-2-yl)butan-2-one (M3) peak were found in both the microsomes and S9 extracts with any cofactors.Nabumetone has recently been proposed as a typical substrate of flavin-containing monooxygenase isoform 5 (FMO5) and was shown to be efficiently oxidized in vitro to 6-MNEA. 6-MNA was detected in the extract obtained from a combined incubation of recombinant FMO5 and S9 fractions.The specificity of FMO5 towards catalyzing this Baeyer-Villiger oxidation (BVO) was demonstrated by the inhibition of the BVO substrate, 4-methoxyphenylacetone. Further in vitro inhibition studies demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of 6-MNA.

Gut microbiota metabolizes nabumetone in vitro: Consequences for its bioavailability in vivo in the rodents with altered gut microbiome.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.

Determination of the Absolute Configuration of the Nabumetone Metabolite 4-(6-Methoxy-2-naphthyl)butan-2-ol Using the Chiral Derivatizing Agent, 1-Fluoroindan-1-carboxylic Acid.

The absolute configuration of (+)-4-(6-methoxy-2-naphthyl)butan-2-ol ((+)-MNBO), a nabumetone metabolite, was determined using 1-fluoroindan-1-carboxylic acid (FICA). Both enantiomers of the FICA methyl esters were derivatized to diastereomeric esters of (+)-MNBO by an ester exchange reaction. The results of 1H- and 19F-NMR spectroscopy of the diastereomeric FICA esters of (+)-MNBO confirmed the absolute configuration of (+)-MNBO was (S).