Evaluation of Icotinib as a Potent and Selective Inhibitor of Aldehyde Oxidase for Reaction Phenotyping in Human Hepatocytes.

Aldehyde oxidase (AO) is a molybdenum cofactor-containing cytosolic enzyme that has gained prominence due to its involvement in the developmental failure of several drug candidates in first-in-human trials. Unlike cytochrome P450s (P450) and glucuronosyltransferase, AO substrates have been plagued by poor in vitro to in vivo extrapolation, leading to low systemic exposures and underprediction of human dose. However, apart from measuring a drug's AO clearance rates, it is also important to determine the relative contribution to metabolism by this enzyme (fm,AO). Although hydralazine is the most well-studied time-dependent inhibitor (TDI) of AO and is frequently employed for AO reaction phenotyping in human hepatocytes to derive fm,AO, multiple studies have expressed concerns pertaining to its utility in providing accurate estimates of fm,AO values due to its propensity to significantly inhibit P450s at the concentrations typically used for reaction phenotyping. In this study, we characterized icotinib, a cyclized analog of erlotinib, as a potent TDI of AO-inactivating human liver cytosolic zoniporide 2-oxidation equipotently with erlotinib with a maximal inactivate rate/inactivator concentration at half maximal inactivation rate (K I) ratio of 463 and 501 minute-1mM-1 , respectively. Moreover, icotinib also exhibits selectivity against P450 and elicits significantly weaker inhibition against human liver microsomal UGT1A1/3 as compared with erlotinib. Finally, we evaluated icotinib as an inhibitor of AO for reaction phenotyping in cryopreserved human hepatocytes and demonstrated that it can yield more accurate prediction of fm,AO compared with hydralazine and induce sustained suppression of AO activity at higher cell densities, which will be important for reaction phenotyping endeavors of low clearance drugs SIGNIFICANCE STATEMENT: In this study, we characterized icotinib as a potent time-dependent inhibitor of AO with ample selectivity margins against the P450s and UGT1A1/3 and demonstrated its utility for reaction phenotyping in human hepatocytes to obtain accurate estimates of fm,AO for victim DDI risk predictions. We envisage the adoption of icotinib in place of hydralazine in AO reaction phenotyping.

Involvement of aldehyde oxidase in the metabolism of aromatic and aliphatic aldehyde-odorants in the mouse olfactory epithelium.

Xenobiotic-metabolizing enzymes (XMEs) expressed in the olfactory epithelium (OE) are known to metabolize odorants. Aldehyde oxidase (AOX) recognizes a wide range of substrates among which are substrates with aldehyde groups. Some of these AOX substrates are odorants, such as benzaldehyde and n-octanal. One of the mouse AOX isoforms, namely AOX2 (mAOX2), was shown to be specifically expressed in mouse OE but its role to metabolize odorants in this tissue remains unexplored. In this study, we investigated the involvement of mouse AOX isoforms in the oxidative metabolism of aldehyde-odorants in the OE. Mouse OE extracts effectively metabolized aromatic and aliphatic aldehyde-odorants. Gene expression analysis revealed that not only mAOX2 but also the mAOX3 isoform is expressed in the OE. Furthermore, evaluation of inhibitory effects using the purified recombinant enzymes led us to identify specific inhibitors of each isoform, namely chlorpromazine, 17ß-estradiol, menadione, norharmane, and raloxifene. Using these specific inhibitors, we defined the contribution of mAOX2 and mAOX3 to the metabolism of aldehyde-odorants in the mouse OE. Taken together, these findings demonstrate that mAOX2 and mAOX3 are responsible for the oxidation of aromatic and aliphatic aldehyde-odorants in the mouse OE, implying their involvement in odor perception.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

Enzyme Kinetics, Pharmacokinetics, and Inhibition of Aldehyde Oxidase.

Aldehyde oxidase (AO) has emerged as an important drug metabolizing enzyme over the last decade. Several compounds have failed in the clinic because the clearance or toxicity was underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. Metabolic products from AO-catalyzed oxidation are generally nonreactive and often they have much lower solubility. AO metabolism is not limited to oxidation as AO can also catalyze reduction of oxygen and nitrite. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. AO is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite, and hydroxamic acids. These reductive transformations may cause toxicity due to the formation of reactive metabolites. Moreover, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening.

Evaluation of vanillin as a probe drug for aldehyde oxidase and phenotyping for its activity in a Western Indian Cohort.

BACKGROUND: Aldehyde oxidase (AO), a molybdoflavoenzyme, is emerging as a key player in drug discovery and metabolism. Despite having several known substrates, there are no validated probes reported for studying the activity of AO in vivo. Vanillin (4-hydroxy 3-methoxy benzaldehyde) is an excellent substrate of AO, in vitro. In the present study, vanillin has been validated as an in vivo probe for AO. Subsequently, a phenotyping study was carried out using vanillin in a subset of Indian population with 100 human volunteers. METHODS: For the purposes of in vitro probe validation, initially the metabolism of vanillin was characterized in partially purified guinea pig AO fraction. Further, vanillin was incubated with partially purified xanthine oxidase fraction and AO fractions, and liver microsomes obtained from different species (in presence and absence of specific inhibitors). For the phenotyping study, an oral dose of 500 mg of vanillin was administered to the participants in the study and cumulative urine samples were obtained up to 8 h after giving the dose. The samples were analyzed by high-performance liquid chromatography and metabolic ratios were calculated as peak area ratio of vanillic acid/vanillin. RESULTS: (a) The results of the in vitro validation studies clearly indicated that vanillin is preferentially metabolized by AO. (b) Normal distribution tests and probit analysis revealed that AO activity was not normally distributed and that 73.72% of the participants were fast metabolizers, 24.28% intermediate metabolizers, and 2% were slow metabolizers. CONCLUSIONS: Data of the phenotyping study suggest the existence of AO polymorphism, in a Western Indian cohort.

A comprehensive review of in silico approaches for the prediction and modulation of aldehyde oxidase-mediated drug metabolism: The current features, challenges and future perspectives.

The importance of aldehyde oxidase (AOX) in drug metabolism necessitates the development and application of the in silico rational drug design methods as an integral part of drug discovery projects for the early prediction and modulation of AOX-mediated metabolism. The current study represents an up-to-date and thorough review of in silico studies of AOX-mediated metabolism and modulation methods. In addition, the challenges and the knowledge gap that should be covered have been discussed. The importance of aldehyde oxidase (AOX) in drug metabolism is a hot topic in drug discovery. Different strategies are available for the modulation of the AOX-mediated metabolism of drugs. Application of the rational drug design methods as an integral part of drug discovery projects is necessary for the early prediction of AOX-mediated metabolism. The current study represents a comprehensive review of AOX molecular structure, AOX-mediated reactions, AOX substrates, AOX inhibition, approaches to modify AOX-mediated metabolism, prediction of AOX metabolism/substrates/inhibitors, and the AOX related structure-activity relationship (SAR) studies. Furthermore, an up-to-date and thorough review of in silico studies of AOX metabolism has been carried out. In addition, the challenges and the knowledge gap that should be covered in the scientific literature have been discussed in the current review.

Reduction of fatty liver in rats by nicotinamide via the regeneration of the methionine cycle and the inhibition of aldehyde oxidase.

Nonalcoholic fatty liver disease, which has been rapidly increasing in the world in recent years, is roughly classified into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis. This study was based on our previous reports that stated that the combination treatment of N1-methylnicotinamide (MNA) and hydralazine (HYD) improves fatty liver in NAFL model rats. This finding was attributed to the MNA metabolism inhibition by HYD, which is a strong inhibitor of aldehyde oxidase (AO); this results in an increase in hepatic MNA and improved fatty liver. We hypothesized that orally administered nicotinamide (NAM), which is the precursor of MNA and is a form of niacin, would be efficiently metabolized by nicotinamide N-methyltransferase in the presence of exogenous S-adenosylmethionine (SAM) in NAFL rats. To address this issue, NAFL model rats were orally administered with NAM, SAM, and/or HYD. As a result, liver triglyceride (TG) and lipid droplet levels were barely altered by the administration of NAM, SAM, NAM+SAM, or NAM+HYD. By contrast, the triple combination of NAM+SAM+HYD significantly reduced hepatic TG and lipid droplet levels and significantly increased hepatic MNA levels. These findings indicated that the combination of exogenous SAM with AO inhibitors, such as HYD, has beneficial effects for improving fatty liver with NAM.

In Vitro Inhibition of Human Aldehyde Oxidase Activity by Clinically Relevant Concentrations of Gefitinib and Erlotinib: Comparison with Select Metabolites, Molecular Docking Analysis, and Impact on Hepatic Metabolism of Zaleplon and Methotrexate.

Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) with activity against metastatic non-small cell lung cancer. Aldehyde oxidase-1 (AOX1) is a cytosolic drug-metabolizing enzyme. We conducted an experimental and molecular docking study on the effect of gefitinib, erlotinib, and select metabolites on the in vitro catalytic activity of AOX1, as assessed by carbazeran 4-oxidation, and determined the impact of AOX1 inhibition on hepatic metabolism of zaleplon and methotrexate. Gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib inhibited human hepatic cytosolic carbazeran 4-oxidation by a competitive mode, with inhibition constants in submicromolar or low micromolar concentrations. Desmethylgefitinib did not affect AOX1 catalytic activity. A similar pattern was obtained when investigated with human kidney cytosol or recombinant AOX1. The differential effect of gefitinib on human, rat, and mouse hepatic AOX1 catalytic activity suggests species-dependent chemical inhibition of AOX1. Erlotinib was considerably more potent than gefitinib in decreasing hepatic cytosolic zaleplon 5-oxidation and methotrexate 7-oxidation. Molecular docking analyses provided structural insights into the interaction between EGFR-TKIs and AOX1, with key residues and bonds identified, which provided favorable comparison and ranking of potential inhibitors. Based on the US Food and Drug Administration guidance to assess the risk of drug-drug interactions, the calculated R1 values indicate that further investigations are warranted to determine whether gefitinib and erlotinib impact AOX1-mediated drug metabolism in vivo. Overall, erlotinib desmethylerlotinib, didesmethylerlotinib, gefitinib, and desmorpholinopropylgefitinib are potent inhibitors of human AOX1 catalytic function and hepatic metabolism of zaleplon and methotrexate, potentially affecting drug efficacy or toxicity. SIGNIFICANCE STATEMENT: As epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib are first-line pharmacotherapy for metastatic non-small cell lung cancer. Our experimental findings indicate that clinically relevant concentrations of gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib, but not desmethylgefitinib, inhibit human aldehyde oxidase (AOX1) catalytic activity and hepatic cytosolic metabolism of zaleplon and methotrexate. Molecular docking analysis provide structural insights into the key AOX1 interactions with these EGFR-TKIs. Our findings may trigger improved strategies for new EGFR-TKI design and development.

Inhibition of vertebrate aldehyde oxidase as a therapeutic treatment for cancer, obesity, aging and amyotrophic lateral sclerosis.

The aldehyde oxidases (AOXs) are a small sub-family of cytosolic molybdo-flavoenzymes, which are structurally conserved proteins and broadly distributed from plants to animals. AOXs play multiple roles in both physiological and pathological processes and AOX inhibition is of increasing significance in the development of novel drugs and therapeutic strategies. This review provides an overview of the evolution and the action mechanism of AOX and the role of each domain. The review provides an update of the polymorphisms in the human AOX. This review also summarises the physiology of AOX in different organs and its role in drug metabolism. The inhibition of AOX is a promising therapeutic treatment for cancer, obesity, aging and amyotrophic lateral sclerosis.

Aldehyde oxidase at the crossroad of metabolism and preclinical screening.

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.

In Vitro and In Silico Analyses of the Inhibition of Human Aldehyde Oxidase by Bazedoxifene, Lasofoxifene, and Structural Analogues.

Tamoxifen, raloxifene, and nafoxidine are selective estrogen receptor modulators (SERMs) reported to inhibit the catalytic activity of human aldehyde oxidase 1 (AOX1). How these drugs interact with AOX1 and whether other SERMs inhibit this drug-metabolizing enzyme are not known. Therefore, a detailed in vitro and in silico study involving parent drugs and their analogs was conducted to investigate the effect of specific SERMs, particularly acolbifene, bazedoxifene, and lasofoxifene on AOX1 catalytic activity, as assessed by carbazeran 4-oxidation, an AOX1-selective catalytic marker. The rank order in the potency (based on IC50 values) of AOX1 inhibition by SERMs was raloxifene > bazedoxifene ∼ lasofoxifene > tamoxifen > acolbifene. Inhibition of liver cytosolic AOX1 by bazedoxifene, lasofoxifene, and tamoxifen was competitive, whereas that by raloxifene was noncompetitive. Loss of 1-azepanylethyl group increased the inhibitory potency of bazedoxifene, whereas the N-oxide group decreased it. The 7-hydroxy group and the substituted pyrrolidine ring attached to the tetrahydronaphthalene structure contributed to AOX1 inhibition by lasofoxifene. These results are supported by molecular-docking simulations in terms of predicted binding modes, encompassing binding orientation and efficiency, and analysis of key interactions, particularly hydrogen bonds. The extent of AOX1 inhibition by bazedoxifene was increased by estrone sulfate and estrone. In summary, SERMs differentially inhibited human AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contributed to AOX1 inhibition, whereas those of acolbifene rendered it considerably less susceptible to AOX1 inhibition. Overall, our novel biochemical findings and molecular-docking analyses provide new insights into the interaction between SERMs and AOX1. SIGNIFICANCE STATEMENT: Aldehyde oxidase (AOX1) is a molybdo-flavoprotein and has emerged as a drug-metabolizing enzyme of potential therapeutic importance because drugs have been identified as AOX1 substrates. Selective estrogen receptor modulators (SERM), which are drugs used to treat and prevent various conditions, differentially inhibit AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contribute to AOX1 inhibition, whereas those of acolbifene render it considerably less susceptible to AOX1 inhibition. Our novel biochemical findings, together with molecular- docking analyses, provide new insights into the differential inhibitory effect of SERMs on the catalytic activity of human AOX1, how SERMs bind to AOX1, and increase our understanding of the AOX1 pharmacophore in the inhibition of AOX1 by drugs and other chemicals.

Role of Molybdenum-Containing Enzymes in the Biotransformation of the Novel Ghrelin Receptor Inverse Agonist PF-5190457: A Reverse Translational Bed-to-Bench Approach.

(R)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT: PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies.

Evaluation of Cytochrome P450 Selectivity for Hydralazine as an Aldehyde Oxidase Inhibitor for Reaction Phenotyping.

Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metabolized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay conditions, at concentrations that chemically knocked out most of the AO activities (≥50 µM). Furthermore, hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases.

Effects of Phenothiazines on Aldehyde Oxidase Activity Towards Aldehydes and N-Heterocycles: an In Vitro and In Silico Study.

BACKGROUND: Aldehyde oxidase (AOX) is an important molybdenum-containing enzyme with high similarity with xanthine oxidase (XO). AOX involved in the metabolism of a large array of aldehydes and N-heterocyclic compounds and its activity is highly substrate-dependent. OBJECTIVES: The aim of this work was to study the effect of five important phenothiazine drugs on AOX activity using benzaldehyde and phenanthridine as aldehyde and N-heterocyclic substrates, respectively. METHODS: The effect of trifluperazine, chlorpromazine, perphenazine, thioridazine and promethazine on rat liver AOX was measured spectrophotometrically. To predict the mode of interactions between the studied compounds and AOX, a combination of homology modeling and a molecular docking study was performed. RESULTS: All phenothiazines could inhibit AOX activity measured either by phenanthridine or benzaldehyde with almost no effect on XO activity. In the case of benzaldehyde oxidation, the lowest and highest half-maximal inhibitory concentration (IC50) values were obtained for promethazine (IC50 = 0.9 µM), and trifluoperazine (IC50 = 3.9 µM), respectively; whereas perphenazine (IC50 = 4.3 µM), and trifluoperazine (IC50 = 49.6 µM) showed the strongest and weakest inhibitory activity against AOX-catalyzed phenanthridine oxidation, respectively. The in silico findings revealed that the binding site of thioridazine is near the dimer interference, and that hydrophobic interactions are of great importance in all the tested phenothiazines. CONCLUSION: The five studied phenothiazine drugs showed dual inhibitory effects on AOX activity towards aldehydes and N-heterocycles as two major classes of enzyme substrates. Most of the interactions between the phenothiazine-related drugs and AOX in the binding pocket showed a hydrophobic nature.

Alleviation of fatty liver in a rat model by enhancing N1-methylnicotinamide bioavailability through aldehyde oxidase inhibition.

Nonalcoholic fatty liver disease (NAFLD) has increased worldwide in recent years. NAFLD is classified into two types, nonalcoholic fatty liver (NAFL), with few complications, and nonalcoholic steatohepatitis (NASH), which leads to liver cirrhosis or cancer. This study was based on previous reports that N1-methylnicotinamide (MNA) can stabilise sirtuin 1 protein, leading to decreased lipid levels in the liver. We hypothesised that fatty liver improvement by MNA would be further enhanced by suppressing its rapid metabolism by aldehyde oxidase in the liver. To test this, hydralazine (HYD), a potent aldehyde oxidase inhibitor, was administered orally to NAFL model rats. Liver triglyceride (TG) levels in the model were nearly unchanged by administration of MNA alone. In contrast, TG levels were marked decreased in NAFL rats treated with a combination of MNA and HYD. In addition, TG levels were decreased even in NAFL rats treated with only HYD. These findings supported our hypothesis that maintaining MNA concentrations in the liver, by suppressing MNA metabolism, would at least partially ameliorate fatty liver.

Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O6-benzylguanine as substrates. 17ß-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O6-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O6-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.

The metabolism of imidacloprid by aldehyde oxidase contributes to its clastogenic effect in New Zealand rabbits.

Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified in Regulation N° 1272/2008 of the European Commision as "harmful if swallowed and very toxic to aquatic life, with long-lasting effects". IMI is metabolized in vitro both by aldehyde oxidase (AOX) (reduction) and by cytochrome P450s enzymes (CYPs). In the present study, the AOX inhibitor sodium tungstate dihydrate (ST) was used to elucidate the relative contribution of CYP 450 and AOX metabolic pathways on IMI metabolism, in male rabbits exposed to IMI for two months. To evaluate the inhibition effectiveness, various metabolite concentrations in the IMI and IMI + ST exposed groups were monitored. DNA damage was also evaluated in micronucleus (MN) and single cell electrophoresis (SCGC) assays in both groups, along with oxidative stress (OS) with the inflammatory status of the exposed animals, in order to clarify which metabolic pathway is more detrimental in this experimental setting. A significant increase in the frequency of binucleated cells with MN (BNMN, 105%) and micronuclei (MN, 142%) was observed after exposure to IMI (p < 0.001). The increase in the ST co-exposed animals was less pronounced (BNMN 75%, MN 95%). The Cytokinesis Block Proliferation Index (CBPI) showed no significant difference between controls and exposed animals at any time of exposure (p > 0.05), which indicates no cytotoxic effect. Similarly, comet results show that the IMI group exhibited the highest achieved tail intensity, which reached 70.7% over the control groups, whereas in the IMI + ST groups the increase remained at 48.5%. No differences were observed between all groups for oxidative-stress biomarkers. The results indicate that the AOX metabolic pathway plays a more important role in the systemic toxicity of IMI.

An iminium ion metabolite hampers the production of the pharmacologically active metabolite of a multikinase inhibitor KW-2449 in primates: irreversible inhibition of aldehyde oxidase and covalent binding with endogenous proteins.

We previously reported that KW-2449, (E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine, a novel multikinase inhibitor developed for the treatment of leukemia patients, was oxidized to an iminium ion intermediate by monoamine oxidase B (MAO-B) and then converted to its oxo-piperazine form (M1) by aldehyde oxidase (AO). However, it was found that the significant decrease in the pharmacologically active metabolite M1 following repeated administration of KW-2449 in primates might hamper the effectiveness of the drug. The mechanism underlying this phenomenon was investigated and it was found that the AO activity was inhibited in a time-dependent manner in vitro under the co-incubation of KW-2449 and MAO-B, while neither KW-2449 nor M1 strongly inhibited MAO-B or AO activity. These results clearly suggest that MAO-B catalysed iminium ion metabolite inhibited AO, prompting us to investigate whether or not the iminium ion metabolite covalently binds to endogenous proteins, as has been reported with other reactive metabolites as a cause for idiosyncratic toxicity. The association of the radioactivity derived from 14 C-KW-2449 with endogenous proteins both in vivo and in vitro was confirmed and it was verified that this covalent binding was inhibited by the addition of sodium cyanide, an iminium ion-trapping reagent, and pargyline, a MAO-B inhibitor. These findings strongly suggest that the iminium ion metabolite of KW-2449 is highly reactive in inhibiting AO irreversibly and binding to endogenous macromolecules covalently.

A rapid and sensitive fluorometric method for determination of aldehyde oxidase activity.

Previous research has characterized the important role of aldehyde oxidases (AOX) in biotransformation of N-heterocyclic therapeutic drugs and environmental contaminants in mammals. Research pertaining to AOX activity in non-mammalian vertebrates, however, is scarce, despite its biological role as a potentially important metabolic pathway for xenobiotics. One of the limiting factors of research on AOX is that available photometric methods are relatively insensitive, limited in throughput, and prone to cross-reactivity from other enzymes. Therefore, this study aimed to develop a novel and improved fluorometric AOX assay. This assay is based on the conversion of the exogenous aldehyde substrate 4-(dimethyl)amino cinnamaldehyde to its corresponding fluorescent acid by AOX, and was evaluated using partially purified hepatic cytosol from rat, human, and rainbow trout. Purification of native cytosol by heat treatment and ammonium sulfate precipitation resulted in increased specific activity of AOX. Michaelis-Menten kinetic parameters (Kmand Vmax) were comparable to values previously generated by photometric methods. Furthermore, effects of the inhibitor hydralazine on AOX activity revealed half maximal inhibitory concentrations comparable to those generated using conventional methods. Product identity was confirmed by liquid chromatography and mass spectrometry. In summary, this study successfully developed a rapid and sensitive assay for determination of AOX activity in across different vertebrate species that is 4- to 10-fold more sensitive compared to conventional absorbance-based methods. It can be applied in environmental, toxicological, and pharmacological studies relating to identification of AOX substrates, as well as the induction of AOX expression through drugs and environmental contaminants.

Species-Specific Involvement of Aldehyde Oxidase and Xanthine Oxidase in the Metabolism of the Pyrimidine-Containing mGlu5-Negative Allosteric Modulator VU0424238 (Auglurant).

Aldehyde oxidase (AO) and xanthine oxidase (XO) are molybdo-flavoenzymes that catalyze oxidation of aromatic azaheterocycles. Differences in AO activity have been reported among various species, including rats, humans, and monkeys. Herein we report a species difference in the enzymes responsible for the metabolism of the negative allosteric modulator of metabotropic glutamate receptor subtype 5 (mGlu5 NAM) VU0424238 (VU238, auglurant). Hepatic S9 incubations with AO and XO specific inhibitors hydralazine and allopurinol indicated that rats and cynomolgus monkeys both oxidized VU238 to the 6-oxopyrimidine metabolite M1 via an AO-mediated pathway, whereas secondary oxidation to the 2,6-dioxopyrimidine metabolite M2 was mediated predominantly by AO in monkeys and XO in rats. Despite differences in enzymatic pathways, intrinsic clearance (CLint) of M1 was similar between species (cynomolgus and rat CLint = 2.00 ± 0.040 and 2.19 ± 0.201 µl/min per milligram of protein, respectively). Inhibitor studies in the S9 of multiple species indicated that oxidation of VU238 to M1 was mediated predominantly by AO in humans, cynomolgus and rhesus monkeys, rats, mice, guinea pigs, and minipigs. Oxidation of M1 to M2 was mediated predominantly by XO in rats and mice and by AO in monkeys and guinea pigs, whereas low turnover prevented enzyme phenotyping in humans and minipigs. Additionally, inhibitor experiments indicated that oxidation at the 2-position of the pyrimidine ring of the known AO substrate, BIBX1382, was mediated by AO in all species, although production of this metabolite was comparatively low in rats and mice. These data may suggest low reactivity of rat AO toward 2-oxidation of pyrimidine-containing compounds and highlight the importance of thoroughly characterizing AO-metabolized drug candidates in multiple preclinical species.