Hydralazine As a Selective Probe Inactivator of Aldehyde Oxidase in Human Hepatocytes: Estimation of the Contribution of Aldehyde Oxidase to Metabolic Clearance

Strelevitz, Timothy J.; Orozco, Christine C.; Obach, R. Scott

doi:10.1124/dmd.112.045195

Cited by 71 publications

(68 citation statements)

References 32 publications

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“…However, work performed by Brian Ogilvie's group at Xenotech, LLC indicated hydralazine is a more potent inhibitor of CYP2D6 than first thought, with an IC 50 of 18 mM in hepatocytes using dextromethorphan as a probe substrate and monitoring dextrorphan formation (B. Ogilvie, personal communication). Since the IC 50 of hydralazine for CYP2D6 is similar to the IC 90 obtained for AO by Strelevitz et al (2012), f m(AO) experiments with CYP2D6 substrates should not use hydralazine.…”

Section: In Vitro Considerations To Assess Aldehyde Oxidase Metabolismmentioning

confidence: 69%

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

2014

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During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase. IntroductionIn an era where combination drug therapy to treat several conditions simultaneously is common, drug companies emphasize the need for optimal absorption, distribution, metabolism, and excretion (ADME) properties, with the purpose of optimization of efficacy and minimization of the risk of adverse events. This includes a proper assignment and an extensive understanding of the routes of metabolism to aid in the prediction of human pharmacokinetics, and to help avoid a potential "object drug" scenario when coadministration is likely. The attributes of the coadministered drug and/or the patient has the potential to increase the probability of a drug-drug interaction (DDI), i.e., enzyme inhibitor, inducer, polymorphic genotype. Hence, the greater the percentage attributed to a single metabolic route, the greater the potential for a DDI and possible "black box warning" being issued as part of the drug package insert. Furthermore, the pharmacokinetic implications of a single polymorphic enzyme being responsible for a majority of the metabolism of a drug may have either an efficacy (extensive metabolizers) or toxicological (poor metabolizers) impact on exposure. To address these concerns, early drug discovery teams strive for balanced metabolism across multiple enzymes and clearance mechanisms (hepatic, renal, biliary). These discovery efforts center on in vitro reaction phenotyping to support enhanced chemical design.There is a general agreement among the major regulatory agencies that pharmaceutical companies should provide a characterization of the metabolic profile of a new chemical entity (NCE) and understand the enzymology of the major clearance mechanisms ...

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Section: In Vitro Considerations To Assess Aldehyde Oxidase Metabolismmentioning

confidence: 69%

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

2014

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“…1) (Hutzler et al, 2012). The role of aldehyde oxidase in the production of BIBU1476 was confirmed by a decrease in the observed in vitro clearance in cryopreserved human hepatocytes when the AO-selective inhibitor hydralazine was coincubated (Hutzler et al, 2012), an in vitro phenotyping methodology also reported by Strelevitz et al (2012).…”

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confidence: 70%

Cynomolgus Monkey as a Surrogate for Human Aldehyde Oxidase Metabolism of the EGFR Inhibitor BIBX1382

et al. 2014

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BIBX1382 was an epidermal growth factor receptor inhibitor under clinical investigation for treatment of cancer. This candidate possessed an attractive preclinical absorption, distribution, metabolism, and excretion profile, yet failed in clinical studies due in part to poor oral exposure, resulting from extensive metabolism by aldehyde oxidase (AO). In vitro metabolism studies were performed in liver cytosol and cryopreserved hepatocytes from multiple species. In addition, a pharmacokinetic study was performed in cynomolgus monkey for comparison with the reported human pharmacokinetics of BIBX1382. Estimated hepatic clearance of BIBX1382 in rhesus (42 ml/min per kg) and cynomolgus monkey (43 ml/min per kg) liver cytosol was comparable to human ( ‡93% of liver blood flow). Metabolite identification after incubation of BIBX1382 in liver cytosol fortified with the AO inhibitor raloxifene confirmed that AO is involved in the formation of the predominant metabolite (BIBU1476, M1) in cynomolgus monkey. After intravenous and oral administration of BIBX1382 to cynomolgus monkeys, high plasma clearance (118 ml/min per kg) and low oral exposure (C max = 12.7 nM and 6% oral bioavailability) was observed, with the exposure of M1 exceeding BIBX1382 after oral dosing. This pharmacokinetic profile compared favorably with the human clinical data of BIBX1382 (plasma clearance 25-55 ml/min per kg and 5% oral bioavailability). Thus, it appears that cynomolgus monkey represents a suitable surrogate for the observed human AO metabolism of BIBX1382. To circumvent clinical failures due to uncharacterized metabolism by AO, in vitro studies in the appropriate subcellular fraction, followed by pharmacokinetic and toxicokinetic studies in the appropriately characterized surrogate species should be conducted for substrates of AO.

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“…Allopurinol has been characterized as a highly selective inhibitor for XO (Panoutsopoulos et al, 2004), and thus, was used to determine whether XO had a significant effect on the oxidation of DACA. In addition, to ensure that the DACA metabolism was mediated exclusively by AO, hydralazine was used as a highly specific inhibitor for AO activity (Hutzler et al, 2011;Strelevitz et al, 2012). Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

Evidence for Substrate-Dependent Inhibition Profiles for Human Liver Aldehyde Oxidase

Barr

Jones

2012

Drug Metab Dispos

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The goal of this study was to provide a reasonable assessment of how probe substrate selection may impact the results of in vitro aldehyde oxidase (AO) inhibition experiments. Here, we used a previously studied set of seven known AO inhibitors to probe the inhibition profile of a pharmacologically relevant substrate N-[(2-dimethylamino)ethyl]acridine-4-carboxamide (DACA). DACA oxidation in human liver cytosol was characterized with a measured V max of 2.3 6 0.08 nmol product · min 21 · mg 21 and a K m of 6.3 6 0.8 mM.The K ii and K is values describing the inhibition of DACA oxidation by the panel of seven inhibitors were tabulated and compared with previous findings with phthalazine as the substrate. In every case, the inhibition profile shifted to a much less uncompetitive mode of inhibition for DACA relative to phthalazine. With the exception of one inhibitor, raloxifene, this change in inhibition profile seems to be a result of a decrease in the uncompetitive mode of inhibition (an affected K ii value), whereas the competitive mode (K is ) seems to be relatively consistent between substrates. Raloxifene was found to inhibit competitively when using DACA as a probe, and a previous report showed that raloxifene inhibited uncompetitively with other substrates. The relevance of these data to the mechanistic understanding of aldehyde oxidase inhibition and potential implications on drug-drug interactions is discussed. Overall, it appears that the choice in substrate may be critical when conducting mechanistic inhibition or in vitro drug-drug interactions prediction studies with AO IntroductionAldehyde oxidase (AO) is a member of the molybdo-flavin family of enzymes, a group that is characterized by containing molybopterin and FAD groups that are essential for catalytic activity. Perhaps the most widely studied molybdo-flavin enzyme, xanthine oxidase (XO), is quite similar in structure and sequence to AO; however, the two enzymes have a marked difference in substrate affinity and specificity (Kitamura et al., 2006;Torres et al., 2007). AO has broad substrate specificity, with the ability to perform redox chemistry on a variety of functional groups (e.g., azaheterocycles, aldehydes, and iminium ions) over a wide range of substrate sizes (Pryde et al., 2010;Garattini and Terao, 2012). As a consequence, AO has a significant role in the metabolism of many different xenobiotic compounds.In recent years, AO has garnered more interest from researchers in the field of drug metabolism. This is primarily due to the azaheterocycleoxidase activity that the enzyme exhibits. More and more, azaheterocycle groups are found in new drugs, and the number of azaheterocyclecontaining drugs has been predicted to increase (Pryde et al., 2010). Azaheterocyle moieties are typically introduced into lead compounds for a variety of reasons, including increased solubility, lower lipophilicity, and optimization of binding with the drug target. In general, these groups also lead to an increased stability of a compound to cytochrome P450...

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Hydralazine As a Selective Probe Inactivator of Aldehyde Oxidase in Human Hepatocytes: Estimation of the Contribution of Aldehyde Oxidase to Metabolic Clearance

Cited by 71 publications

References 32 publications

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

Cynomolgus Monkey as a Surrogate for Human Aldehyde Oxidase Metabolism of the EGFR Inhibitor BIBX1382

Evidence for Substrate-Dependent Inhibition Profiles for Human Liver Aldehyde Oxidase

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