Metabolism of Xenobiotics by Aldehyde Oxidase

Deepak Dalvie1, Michael Zientek1

1 Pfizer Global Research and Development, LaJolla Laboratories, San Diego
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.41
DOI:  10.1002/0471140856.tx0441s63
Online Posting Date:  February, 2015
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Abstract

Aldehyde oxidase (AO) is a cytosolic molybdoflavoprotein whose contribution to the metabolism and clearance of xenobiotics‐containing heterocyclic rings has attracted increased interest in recent years. This unit details methods for identification and confirmation of AO as a metabolic pathway as well as a method for estimating clearance of compounds that are AO substrates. © 2015 by John Wiley & Sons, Inc.

Keywords: aldehyde oxidase (AO); heteroaromatic rings; liver cytosol; liver S9 fraction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of AO as an Enzyme in Metabolism of Zoniporide
  • Basic Protocol 2: Confirmation of AO Metabolism
  • Basic Protocol 3: Estimating Hepatic Intrinsic Clearance of Compounds Metabolized by AO
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification of AO as an Enzyme in Metabolism of Zoniporide

  Materials
  • Human liver S9 (pooled, mixed gender, ten donors; 23 mg/ml protein concentration; CelsisIVT)
  • 100 mM potassium phosphate buffer, pH 7.4 (see recipe)
  • 1000 mM MgCl 2 (see recipe)
  • ∼3 mM zoniporide stock (see recipe)
  • HPLC‐grade acetonitrile (Sigma‐Aldrich)
  • Nitrogen source
  • HPLC‐grade water with formic acid (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 37°C shaking water bath
  • Vortex
  • Tabletop centrifuge
  • Pyrex disposable centrifuge tube, capacity 15‐ml × 126‐mm (Sigma‐Aldrich)
  • TurboVap evaporator
  • 1‐ml HPLC vials
  • Agilent HP‐1100 HPLC system (Agilent Technologies)
  • ThermoFinnigan LCQ Deca XP ion trap mass spectrometer (ThermoScientific)
  • Xcalibur v1.4 software (ThermoScientific)

Basic Protocol 2: Confirmation of AO Metabolism

  Materials
  • 1 mM raloxifene stock (Sigma)
  • DMSO
  • 12 × 75–mm borosilicate glass tubes
  • For additional reagents and equipment, see protocol 1.

Basic Protocol 3: Estimating Hepatic Intrinsic Clearance of Compounds Metabolized by AO

  Materials
  • Human liver S9 (pooled, mixed gender of ten donors; protein concentration, 23 mg/ml; CelsisIVT)
  • 0.1 M potassium phosphate buffer, pH 7.4 (see recipe)
  • 1 mM MgCl 2 (see recipe)
  • 50 μM zoniporide stock (see recipe)
  • Buspirone powder (see recipe)
  • HPLC‐grade (Milli‐Q) water (Sigma Aldrich)
  • 12 ml × 75 mm borosilicate tube
  • 96‐well polypropylene PCR plates (e.g., Axygen)
  • 37°C heating block
  • 96‐well caps (Nalge Nunc)
  • Vortexer
  • Benchtop centrifuge
  • Synergi Polar RP (2 × 30–mm, 4‐μm, Phenomenex)
  • CTC PAL autoinjector (Leap Technologies)
  • Shimadzu HPLC pumps (Shimadzu Scientific Instruments)
  • API 4000‐triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex)
  • MDS Sciex Analyst Software v.1.4.1
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Figures

Videos

Literature Cited

Literature Cited
  Akabane, T., Gerst, N., Masters, J.N., and Tamura, K. 2012a. A quantitative approach to hepatic clearance prediction of metabolism by aldehyde oxidase using custom pooled hepatocytes. Xenobiotica 42:863‐871.
  Akabane, T., Gerst, N., Naritomi, Y., Masters, J.N., and Tamura, K. 2012b. A practical and direct comparison of intrinsic metabolic clearance of several non‐CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes. Drug Metab. Pharmacokinet. 27:181‐191.
  Barr, J.T., Choughule, K., and Jones, J.P. 2014. Enzyme kinetics, inhibition, and regioselectivity of aldehyde oxidase. Methods Mol. Biol. 1113:167‐186.
  Choughule, K.V., Barr, J.T., and Jones, J.P. 2013. Evaluation of rhesus monkey and guinea pig hepatic cytosol fractions as models for human aldehyde oxidase. Drug Metab. Dispos. 41:1852‐1858.
  Dalvie, D., Zhang, C., Chen, W., Smolarek, T., Obach, R.S., and Loi, C.M. 2010. Cross‐species comparison of the metabolism and excretion of zoniporide: Contribution of aldehyde oxidase to interspecies differences. Drug Metab. Dispos. 38:641‐654.
  Garattini, E. and Terao, M. 2011. Increasing recognition of the importance of aldehyde oxidase in drug development and discovery. Drug. Metab. Rev. 43:374‐386.
  Garattini, E. and Terao, M. 2012. The role of aldehyde oxidase in drug metabolism. Expert Opin. Drug Metab. Toxicol.
  Garattini, E., Fratelli, M., and Terao, M. 2008. Mammalian aldehyde oxidases: Genetics, evolution and biochemistry. Cell Mol. Life Sci. 65:1019‐1048.
  Garattini, E., Fratelli, M., and Terao, M. 2009. The mammalian aldehyde oxidase gene family. Hum. Genomics 4:119‐130.
  Garattini, E., Mendel, R., Romao, M.J., Wright, R., and Terao, M. 2003. Mammalian molybdo‐flavoenzymes, an expanding family of proteins: Structure, genetics, regulation, function and pathophysiology. Biochem. J. 372:15‐32.
  Houston, J.B. and Galetin, A. 2008. Methods for predicting in vivo pharmacokinetics using data from in vitro assays. Curr. Drug Metab. 9:940‐951.
  Hutzler, J.M., Obach, R.S., Dalvie, D., and Zientek, M.A. 2013. Strategies for a comprehensive understanding of metabolism by aldehyde oxidase. Expert Opin. Drug Metab. Toxicol. 9:153‐168.
  Hutzler, J.M., Yang, Y.S., Brown, C., Heyward, S., and Moeller, T. 2014. Aldehyde oxidase activity in donor‐matched fresh and cryopreserved human hepatocytes and assessment of variability in 75 donors. Drug Metab. Dispos. 42:1090‐1097.
  Hutzler, J.M., Yang, Y.S., Albaugh, D., Fullenwider, C.L., Schmenk, J., and Fisher, M.B. 2012. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes. Drug Metab. Dispos. 40:267‐275.
  Johnson, C., Stubley‐Beedham, C., and Stell, J.G. 1985. Hydralazine: A potent inhibitor of aldehyde oxidase activity in vitro and in vivo. Biochem. Pharmacol. 34:4251‐4256.
  Kitamura, S., Sugihara, K., and Ohta, S. 2006. Drug‐metabolizing ability of molybdenum hydroxylases. Metab. Pharmacokinet. 21:83‐98.
  Mendel, R.R. 2009. Cell biology of molybdenum. BioFactors 35:429‐434.
  Obach, R.S. 1999. Prediction of human clearance of twenty‐nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half‐life approach and nonspecific binding to microsomes. Drug Metab. Dispos. 27:1350‐1359.
  Obach, R.S. 2004. Potent inhibition of human liver aldehyde oxidase by raloxifene. Drug Metab. Dispos. 32:89‐97.
  Obach, R.S., Huynh, P., Allen, M.C., and Beedham, C. 2004. Human liver aldehyde oxidase: Inhibition by 239 drugs. Br. J. Clin Pharmacol. 44:7‐19.
  Pryde, D.C., Dalvie, D., Hu, Q., Jones, P., Obach, R.S., and Tran, T.D. 2010. Aldehyde oxidase: An enzyme of emerging importance in drug discovery. J. Med. Chem. 53:8441‐8460.
  Strelevitz, T.J., Orozco, C.C., and Obach, R.S. 2012. Hydralazine as a selective probe inactivator of aldehyde oxidase in human hepatocytes: Estimation of the contribution of aldehyde oxidase to metabolic clearance. Drug Metab. Dispos. 40:1441‐1448.
  Zientek, M., Jiang, Y., Youdim, K., and Obach, R.S. 2010. In vitro‐in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. Drug Metab. Dispos. 38:1322‐1327.
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