Inhibitor Of Aldehyde Oxidase Research Articles

Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) that is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km=2.7 μM and Vmax=794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in lungs of control mice with even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.

Investigating the potential of 6-substituted 3-formyl chromone derivatives as anti-diabetic agents using in silico methods

In exploring nature's potential in addressing diabetes-related conditions, this study investigates the therapeutic capabilities of 3-formyl chromone derivatives. Utilizing in silico methodologies, we focus on 6-substituted 3-formyl chromone derivatives (1–16) to assess their therapeutic potential in treating diabetes. The research examined the formyl group at the chromone’s C-3 position. ADMET, biological activities, were conducted along with B3LYP calculations using 3 different basis sets. The analogues were analyzed based on their parent structure obtained from PubChem. The HOMO–LUMO gap confirmed the bioactive nature of the derivatives, NBO analysis was performed to understand the charge transfer. PASS prediction revealed that 3-formyl chromone derivatives are potent aldehyde oxidase inhibitors, insulin inhibitors, HIF1A expression inhibitors, and histidine kinase. Molecular docking studies indicated that the compounds had a strong binding affinity with proteins, including CAD, BHK, IDE, HIF-α, p53, COX, and Mpro of SARS-CoV2. 6-isopropyl-3-formyl chromone (4) displayed the highest affinity for IDE, with a binding energy of − 8.5 kcal mol−1. This result outperformed the affinity of the reference standard dapagliflozin (− 7.9 kcal mol−1) as well as two other compounds that target human IDE, namely vitexin (− 8.3 kcal mol−1) and myricetin (− 8.4 kcal mol−1). MD simulations were revealed RMSD value between 0.2 and 0.5 nm, indicating the strength of the protein–ligand complex at the active site.

Contribution of aldehyde oxidase to methotrexate-induced hepatotoxicity: in vitro and pharmacoepidemiological approaches

ABSTRACT Background Methotrexate (MTX) is partially metabolized by aldehyde oxidase (AOX) in the liver and its clinical impact remains unclear. In this study, we aimed to demonstrate how AOX contributes to MTX-induced hepatotoxicity in vitro and clarify the relationship between concomitant AOX inhibitor use and MTX-associated liver injury development using the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS). Methods We assessed intracellular MTX accumulation and cytotoxicity using HepG2 cells. We used the FAERS database to detect reporting odds ratio (ROR)-based MTX-related hepatotoxicity event signals. Results AOX inhibition by AOX inhibitor raloxifene and siRNA increased the MTX accumulation in HepG2 cells and enhanced the MTX-induced cell viability reduction. In the FAERS analysis, the ROR for MTX-related hepatotoxicity increased with non-overlap of 95% confidence interval when co-administered with drugs with higher Imax, u (maximum unbound plasma concentration)/IC50 (half-maximal inhibitory concentration for inhibition of AOX) calculated based on reported pharmacokinetic data. Conclusion AOX inhibition contributed to MTX accumulation in the liver, resulting in increased hepatotoxicity. Our study raises concerns regarding MTX-related hepatotoxicity when co-administered with drugs that possibly inhibit AOX activity at clinical concentrations.

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.

Abstract 701: Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines

Abstract Radiation therapy (RT) is a cornerstone of oncologic therapy for the majority of solid malignancies, and unrepaired DNA double-strand breaks are responsible for radiation-induced cytotoxicity. RT-induced DNA lesions can be repaired by multiple mechanisms including homologous recombination (HR) and non-homologous end joining (NHEJ), and disruption of either of these pathways can enhance cytotoxicity. A key component of the NHEJ pathway is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and loss of kinase activity can significantly increase radiosensitivity. Herein we describe the results of our medicinal chemistry campaign to develop a potent and selective DNA-PKcs inhibitor, WNC0901. WNC0901 inhibits DNA-PKcs kinase activity in a cell free system with an IC50 of 0.071 nM and demonstrated at least 30-fold higher sensitivity than other family members (ATM, ATR, mTOR, PI3K). WNC0901 has limited aldehyde oxidase (AO) liability with a T1/2 >2000 min in liver cytosol and is stable for over 120 minutes in the presence and absence of an AO inhibitor. A preliminary pharmacokinetic analysis in wista han rat (IV=2mg/kg, PO=20mg/kg) demonstrated 116% apparent absolute oral bioavailability, 2.6 hour terminal half-life, 33.0 mL/min/kg clearance, and a 2.6% unbound brain-to-plasma partition coefficient (Kpuu). WNC0901 also had favorable pharmacokinetic properties in beagle dog (IV=2mg/kg, PO=20mg/kg) with moderate clearance (8.5 mL/min/kg), high apparent absolute oral bioavailability (131%) with good exposure (AUC=36092 h*ng/mL), moderate half-life (4.28 h), and low protein binding (74.3% fraction unbound). The volume of distribution was moderate in both species (Vss = 1.32 L/kg in wistar han rat and 1.87 L/kg in beagle dog). In cell culture, WNC0901 inhibited autophosphorylation of DNA-PKcs in HT29 cells irradiated with 10 Gy with an IC50 of 32.7 nM and robustly inhibited autophosphorylation in both U251 glioma and A549 lung cancer cell lines at 300 nM in combination with 5Gy. In a clonogenic assay, 5Gy irradiation (10% survival) combined with 100nM WNC0901 demonstrated modestly enhanced cell killing (1.5% survival), and maximal effects were seen at 300nM (0.04% survival, p<0.01). Similar radiosensitizing effects with 300 nM WNC0901 were seen in the A549 cell line (0.2% survival with combination compared to 19% with 5Gy alone, p<0.01). In summary, WNC0901 inhibits DNA-PK kinase activity and provides potent radiosensitization in a GBM and lung cancer cell line. Future studies will assess the combination of WNC0901 and RT in GBM patient-derived xenograft models in vivo. Citation Format: Ann C. Mladek, Danielle L. Burgenske, William F. Elmquist, Wei Zhong, Jann N. Sarkaria. Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 701.

Non-cytochrome P450 enzyme aldehyde oxidase is involved in the oxidative metabolic pathway of diquat and its detoxification effect

Diquat (DQ) poisoning has garnered attention in recent years, primarily due to the rising incidence of cases worldwide, coupled with the absence of a viable antidote for its treatment. Despite the fact that diquat monopyridone (DQ-M) has been identified as a significant metabolite of DQ, the enzyme responsible for its formation remains unknown. In this study, we have identified aldehyde oxidase (AOX) as a vital enzyme involved in DQ oxidative metabolism. The metabolism of DQ to DQ-M was significantly inhibited by AOX inhibitors including raloxifene and hydralazine. The source of oxygen incorporated into DQ-M was proved to be from water through a H218O incubation experiment which further corroborated DQ-M formation via AOX metabolism. The product of DQ-M in vitro generated by fresh rat tissues co-incubation was consistent with its AOX expression. The result of the molecular docking analysis of DQ and AOX protein showed that DQ is capable of binding to AOX. Furthermore, the cytotoxicity of DQ was significantly higher than DQ-M at the same concentration tested in six cell types. This work is the first to uncover the involvement of aldehyde oxidase, a non-cytochrome P450 enzyme, in the oxidative metabolic pathway of diquat, thus providing a potential target for the development of detoxification treatment.

Comparative study on the occurrence of adverse effects in the concomitant use of azathioprine and aldehyde oxidase inhibitors

ABSTRACT Objectives Aldehyde oxidase (AO) is a molybdenum-containing redox enzyme similar to xanthine oxidase that is involved in the thiopurine metabolism. This study investigated the effects of drug-drug interactions (DDIs) between azathioprine (AZA) and AO inhibitors on hematologic and hepatic disorders using the U.S. Food and Drug Administration Adverse Event Reporting System and the Japanese Adverse Drug Event Report database. Methods The presence of DDI was assessed using the interaction signal scores (ISSs) calculated via the reporting odds ratios and 95% confidence intervals. The study used reports of ‘azathioprine’ as a suspect drug for adverse effects. AO inhibitors were selected based on previous in vitro reports. Results Some drugs tested positive for ISSs in each database and type of adverse effect (hematologic or hepatic disorder) analysis. Among these drugs, chlorpromazine, clozapine, hydralazine, and quetiapine could inhibit AZA metabolism via AO, given the previously reported clinical blood concentration and inhibitory effects of each drug. Conclusion Concomitant use of AO inhibitors increased the signals for AZA-induced adverse effects. To date, no studies have evaluated the clinical importance of AO as a drug-metabolizing enzyme, and further in vitro and clinical research is needed to clarify the contribution of AO to the pharmacokinetics of thiopurines.

SGX523 causes renal toxicity through aldehyde oxidase-mediated less-soluble metabolite formation in chimeric mice with humanized livers

SGX523 is a c-Met tyrosine kinase inhibitor that failed in clinical trials because of renal toxicity caused by crystal deposits in renal tubules. SGX523 is metabolized by aldehyde oxidase (AOX) in a species-dependent manner to the considerably less soluble 2-quinolinone-SGX523, which is likely involved in the clinically observed obstructive nephropathy. This study investigated the metabolism and renal toxicity of SGX523 in chimeric mice with humanized livers (humanized-liver mice). The 2-quinolinone-SGX523 formation activity was higher in humanized-liver mouse and human hepatocytes than in mouse hepatocytes. Additionally, this activity in the liver cytosolic fraction from humanized-liver mice was inhibited by the AOX inhibitors raloxifene and hydralazine. After oral SGX523 administration, higher maximum concentrations, larger areas under the plasma concentration versus time curves, and higher urinary concentrations of 2-quinolinone-SGX523 were observed in humanized-liver mice than in non-humanized mice. Serum creatinine and blood urea nitrogen levels were elevated in humanized-liver mice following repeated oral SGX523 administration. The accumulation of amorphous material in the tubules and infiltration of inflammatory cells around tubules were observed in the kidneys of humanized-liver mice after repeated oral SGX523 administration. These findings demonstrate that humanized-liver mice are useful for understanding the metabolism and toxicity of SGX523.

Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine.

The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide, O6 -benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate. O6 -Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with O6 -benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.

Weak Inhibitory Effects of Anthocyanins on Human Aldehyde Oxidase Activity: An In Vitro Study.

Aldehyde oxidase (AO) plays an important role in the metabolism of antitumor and antiviral drugs, including methotrexate, favipiravir, and acyclovir. The consumption of blueberry fruits or their extracts, which contain large amounts of anthocyanins, has recently increased. The intake of large amounts of anthocyanins occurs through the frequent consumption of blueberries or their functional foods, which may result in unwanted interactions between anthocyanins and medicinal drugs. Therefore, the present study examined the inhibition of AO by anthocyanins, anthocyanidins, and blueberry extracts in human liver cytosol using a HPLC assay. A comparison of the 50% inhibitory concentration (IC50) values of the test compounds showed that anthocyanidins slightly suppressed AO activity, whereas the inhibitory effects of anthocyanins and blueberry extracts were negligible. The inhibitory activities of the anthocyanins tested were approximately 60- to 130-fold weaker than that of the positive control menadione and were almost negligible. Furthermore, they were approximately 2,000-fold less potent than that of raloxifene, a typical AO inhibitor, and, thus, unlikely to interfere with drug metabolism by AO. In addition, since the plasma concentrations of anthocyanins after their administration were generally lower than the IC50 level, the inhibition of AO substrate metabolism by anthocyanins does not appear to be severe.

Drug-Drug Interaction of Aldehyde Oxidase Inhibitor and Xanthine Oxidase Inhibitor with Favipiravir

Aim: Favipiravir is an effective antiviral used in the treatment of COVID-19. It is metabolized by aldehyde oxidase (AO) and xanthine oxidase (XO). This study investigated drug-drug interactions between favipiravir with both AO substrate and XO enzyme inhibitor, allopurinol, and an XO inhibitor, verapamil. 
 Material and Methods: 25 Sprague-Dawley female rats, 250-300 g, were divided into five equal groups. Blood samples were taken from the jugular vein at the end of 0, 15, 30, and 45 minutes, and at the end of the 1st, 2nd, 4th, 6th, and 8th hours after the drugs were administered. The drug-blood concentration was determined in the HPLC-UV device using plasma. The ELISA method measured AO and XO enzyme activities in rat liver tissue.
 Results: Allopurinol prolonged the time taken for favipiravir to reach Cmax (Tmax), decreased maximum serum concentration (Cmax), elimination half-life (T1/2), area under the curve (AUC), and mean residence time (MRT). Allopurinol significantly reduced clearance per unit time (Cl/f) when co-administered with favipiravir. Verapamil accelerated the elimination of favipiravir, significantly reducing T1/2, MRT, and AUC. On the other hand, Favipiravir decreased the absorption of verapamil and slowed its elimination. Cmax, AUC, and Cl values of verapamil decreased. In addition, T1/2, MRT, and volume of distribution (Vd) increased. 
 Conclusion: In conclusion, the concomitant use of favipiravir with other drugs that affect AO and/or XO enzyme activities may cause changes in the pharmacokinetic profiles of drugs and the levels of enzymes that metabolize drugs.

Role of Human Aldehyde Oxidase in the Generation of Reactive Oxygen Species during the Metabolism of Nicotine

Nicotine is the major addictive constituent of tobacco and alternative tobacco products including electronic cigarettes (e‐cigs). While e‐cig vaping has been proposed as safer than traditional tobacco smoking, recent studies has demonstrated that these products can also trigger oxidant stress and inflammation with resultant pulmonary and cardiovascular disease. E‐cig liquids contain high levels of nicotine typically of 12 to 24 mg/ml or more and with the use of high power delivery devices high, poorly controlled levels of nicotine can be delivered. When inhaling tobacco cigarette smoke or e‐cig vape, nicotine is rapidly absorbed through the lungs. The major pathway of nicotine metabolism in humans consists of the C‐oxidation of nicotine to produce cotinine involving a two‐step process: the first step is CYP2A6‐catalyzed 5′‐oxidation to nicotine‐Δ1′(5′)‐iminium ion (NI), which is in equilibrium with 5′‐hydroxynicotine; and the second step is aldehyde oxidase (AO) catalyzed oxidation of NI to cotinine.Using both cytochrome‐c reduction and EPR spin trapping techniques, we show for the first time that the oxidation of NI by human AO produces superoxide radical anion (O2• −) with high flux rates. Purified recombinant human AO (hAO) was prepared and characterized by SDS gel electrophoresis and optical absorption spectroscopy. hAO, 40 nM was incubated in the presence of NI concentrations from 1‒50 µM. The kinetic parameters, Km and Vmax values, for superoxide generation from NI were determined to be 2.7 ± 0.4 μM and 694 ± 24 nmol min−1 mg−1 of protein, respectively. From quantitation of the measured superoxide and the amount of NI consumed, we estimate that ~60% of the oxygen consumed in this process is converted to superoxide with ~40% hydrogen peroxide. Superoxide generation was almost completely quenched by either 0.5 μM raloxifene, a potent inhibitor of AO, or 0.1 μM Copper‐Zinc‐superoxide dismutase (SOD1) with no superoxide generation detected either in the absence of either AO or nicotine‐Δ1′(5′)‐iminium ion.Therefore, human AO efficiently catalyzes the oxidation of NI to cotinine with concomitant production of reactive oxygen species. Due to the high expression levels of AO in lung tissue as well as its rapid rate of NI metabolism with low Km, AO rapidly and efficiently converts NI to cotinine with the formation of superoxide and hydrogen peroxide. Thus, nicotine metabolism will be associated with the formation of superoxide, hydrogen peroxide and secondary reactive oxygen species that can in turn cause lung oxidative stress and inflammation.

Progesterone Metabolism by Human and Rat Hepatic and Intestinal Tissue.

Following oral administration, the bioavailability of progesterone is low and highly variable. As a result, no clinically relevant, natural progesterone oral formulation is available. After oral delivery, first-pass metabolism initially occurs in the intestines; however, very little information on progesterone metabolism in this organ currently exists. The aim of this study is to investigate the contributions of liver and intestine to progesterone clearance. In the presence of NADPH, a rapid clearance of progesterone was observed in human and rat liver samples (t1/2 2.7 and 2.72 min, respectively). The rate of progesterone depletion in intestine was statistically similar between rat and human (t1/2 197.6 min in rat and 157.2 min in human). However, in the absence of NADPH, progesterone was depleted at a significantly lower rate in rat intestine compared to human. The roles of aldo keto reductases (AKR), xanthine oxidase (XAO) and aldehyde oxidase (AOX) in progesterone metabolism were also investigated. The rate of progesterone depletion was found to be significantly reduced by AKR1C, 1D1 and 1B1 in human liver and by AKR1B1 in human intestine. The inhibition of AOX also caused a significant reduction in progesterone degradation in human liver, whereas no change was observed in the presence of an XAO inhibitor. Understanding the kinetics of intestinal as well as liver metabolism is important for the future development of progesterone oral formulations. This novel information can inform decisions on the development of targeted formulations and help predict dosage regimens.

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.

Methyl-hydroxylation and subsequent oxidation to produce carboxylic acid is the major metabolic pathway of tolbutamide in chimeric TK-NOG mice transplanted with human hepatocytes

Tolbutamide is an oral anti-hyperglycaemic agent used to treat non-insulin–dependent diabetes mellitus with species-dependent metabolic profiles. In this study, we investigated tolbutamide metabolism in chimeric TK-NOG mice transplanted with human hepatocytes (humanised-liver mice). Substantial 4-hydroxytolbutamide and 4-carboxytolbutamide production was observed in hepatocytes from humanised-liver mice (Hu-Liver cells) and humans, whereas 4-carboxytolbutamide production was not detected in mouse hepatocytes. In Hu-Liver cells, 4-hydroxytolbutamide formation was inhibited by sulfaphenazole (CYP2C9 inhibitor), whereas 4-carboxytolbutamide formation was inhibited by raloxifene/ethinyloestradiol (aldehyde oxidase inhibitor) and disulfiram (aldehyde dehydrogenase inhibitor). After a single oral dose of tolbutamide (10 mg/kg), the plasma levels of 4-carboxytolbutamide and p-tolylsulfonylurea were higher in humanised-liver mice than in TK-NOG mice. Urinary excretion was the predominant route (>99% of unchanged drug and metabolites detected in excreta) of elimination in both groups. 4-Carboxytolbutamide was the most abundant metabolite in humanised-liver mouse urine, as similarly reported for humans, whereas 4-hydroxytolbutamide was predominantly excreted in TK-NOG mouse urine. These results suggest that humanised-liver mice might represent a suitable animal model for studying the successive oxidative metabolism of tolbutamide by multiple drug-metabolising enzymes. Future work is warranted to study the general nature of primary alcohol metabolism using humanised-liver mice.

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.

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.

Case Study 11: Considerations for Enzyme Mapping Experiments-Interaction Between the Aldehyde Oxidase Inhibitor Hydralazine and Glutathione.

Often it may be convenient and efficient to address multiple research questions with a single experiment. In many instances, however, the best approach is to design the experiment to address one question at a time. The design of enzyme mapping experiments is discussed in this chapter, focusing on considerations pertinent to the study of aldehyde oxidase (AO) vs. cytochrome P450 metabolism. Specifically, a case is presented in which reduced glutathione (GSH) was included in an experiment with human liver S9 fraction to trap reactive metabolites generated from cytochrome P450-mediated metabolism of lapatinib and its O-dealkylated metabolite, M1 (question 1). The AO inhibitor hydralazine was included in this experiment to investigate the involvement of AO-mediated metabolism of M1 (question 2). The presence of GSH was found to interfere with the inhibitory activity of hydralazine. Consideration of the time-dependent nature of hydralazine inhibitory activity toward AO when designing this experiment could have predicted the potential for GSH to interfere with hydralazine. This case underscores the importance of clearly identifying the research question, tailoring the experimental protocol to answer that question, and then meticulously considering how the experimental conditions could influence the results, particularly if attempting to address multiple questions with a single experiment.

Aldehyde Oxidase Contributes to All-Trans-Retinoic Acid Biosynthesis in Human Liver.

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

P104 - Evaluation of cytochrome P450 selectivity for hydralazine as an aldehyde oxidase inhibitor for reaction phenotyping

P104 - Evaluation of cytochrome P450 selectivity for hydralazine as an aldehyde oxidase inhibitor for reaction phenotyping