Characterization of Aldehyde Oxidase Enzyme Activity in Cryopreserved Human Hepatocytes

Hutzler, J. Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L.; Schmenk, Jennifer; Fisher, Michael B.

doi:10.1124/dmd.111.042861

Cited by 73 publications

(69 citation statements)

References 30 publications

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“…In addition to the potent inhibition of GDC-0834 metabolism by AO inhibitors, GDC-0834 also inhibits the metabolism of several known AO substrates carbazeran, DACA, O 6 -benzylguanine, phthalazine, zaleplon, and zoniporide (Beedham et al, 1990;Beedham et al, 1995;Kawashima et al, 1999;Schofield et al, 2000;Obach et al, 2004;Dalvie et al, 2012;Hutzler et al, 2012) (Fig. 4, A-F).…”

Section: Discussionmentioning

confidence: 97%

A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

et al. 2015

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GDC-0834, a Bruton's tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834was rapidly hydrolyzed with a CL int of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC 50 values ranging from 0.86 to 1.87 mM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834.

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Section: Discussionmentioning

confidence: 97%

A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

et al. 2015

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“…In a recent report, our laboratory demonstrated that the predominant metabolite after incubation of BIBX1382 in cryopreserved human hepatocytes had a retention time and fragmentation pattern matching that of the authentic standard metabolite, BIBU1476 (4-[(3-chloro-4-fluorophenyl)amino]-6-[(1-methylpiperidin-4-yl)amino]pyrimido [5,4-day]pyrimidin-2(4aH)-one), with the position of oxidation occurring on the pyrimido-pyrimidine core ( Fig. 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: 83%

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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“…Based on analysis of chemical structure, a recent review suggested that a large number of drugs on the market (;13%) or candidate drugs (almost 45% of drugs in development) could be AO substrates (Pryde et al, 2010). With the increasing role of AO in drug metabolism, ongoing research activities have focused exclusively on the human liver (Obach et al, 2004;Hutzler et al, 2012Hutzler et al, , 2014, even if gene expression (Nishimura and Naito, 2006) and immunohistochemistry (Moriwaki et al, 2001) studies suggested a considerable extrahepatic presence, especially in kidneys and lungs. Moreover, efforts to predict AO clearance in vivo based on in vitro assays in hepatic experimental models generally resulted in underestimation (Zientek et al, 2010;Hutzler et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…With the increasing role of AO in drug metabolism, ongoing research activities have focused exclusively on the human liver (Obach et al, 2004;Hutzler et al, 2012Hutzler et al, , 2014, even if gene expression (Nishimura and Naito, 2006) and immunohistochemistry (Moriwaki et al, 2001) studies suggested a considerable extrahepatic presence, especially in kidneys and lungs. Moreover, efforts to predict AO clearance in vivo based on in vitro assays in hepatic experimental models generally resulted in underestimation (Zientek et al, 2010;Hutzler et al, 2012). The mRNA and protein expressions of AO were recently also detected in human skin van Eijl et al, 2012), but its activity to date remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Aldehyde Oxidase Activity in Fresh Human Skin

et al. 2014

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Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol×mg skin, resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 mM substrate. Hydroxylation activities for the two substrates were significantly correlated (r 2 = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 mM inhibitor. Reaction rates were linear up to 4 hours and well described by MichaelisMenten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin.

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Characterization of Aldehyde Oxidase Enzyme Activity in Cryopreserved Human Hepatocytes

Cited by 73 publications

References 30 publications

A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

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

Aldehyde Oxidase Activity in Fresh Human Skin

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