Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.
Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.
Boehringer-Ingelheim Pharmaceuticals Inc., Translational Research (Drug Metabolism and Pharmacokinetics), 175 Briar Ridge Road, R&D 10578, Ridgefield, CT 06877, USA. Matt.Hutzler@Boehringer-Ingelheim.com
J Matthew
JM
Hutzler
Young-Sun
YS
Yang
Daniel
D
Albaugh
Cody L
CL
Fullenwider
Jennifer
J
Schmenk
Michael B
MB
Fisher
D042931:Aldehyde Oxidase / Q000009:adverse effects / Q000378:metabolism*; D000465:Algorithms; D023401:Animal Use Alternatives; D002478:Cells, Cultured; D015925:Cryopreservation; D003600:Cytosol / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D006830:Hydralazine / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D004364:Pharmaceutical Preparations; D006830:Hydralazine; C526436:AOX1 protein, human; D042931:Aldehyde Oxidase