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Designing robust assays to study non-CYP mediated metabolism

In vitro drug metabolism studies are critical in:

  • Estimating the extent of parent compound drug metabolism as well as determining the rate of formation and identity of any metabolites formed.
  • Identifying which enzymes are responsible for drug metabolism
  • Determining potential drug-drug interactions (DDI)
  • Understanding potential interspecies differences

The cytochrome P450 (CYP) superfamily of enzymes play a key role in phase I oxidative drug metabolism and, when performing reaction phenotyping studies to understand which enzymes are involved in metabolism, CYPs are typically assumed to be the main culprits. However, through structural based changes, medicinal chemists often focus on designing out potential CYP liability, and this can lead to an increase in alternative routes of metabolism including non-CYP mediated metabolism. Aldehyde oxidase (AO) and xanthine oxidase (XO), which are major cytosolic molybdenum hydroxylases, are two such non-CYP enzymes. Despite being structurally similar, aldehyde oxidase and xanthine oxidase exhibit distinct differences in substrate and inhibitor specificities.

As the regulatory authorities require knowledge of the enzymes involved when significant drug metabolism occurs, robust in vitro assays are required for this purpose. One aspect to consider when evaluating non-CYP metabolism and specifically xanthine oxidase is the source of the cytosol. Allopurinol, a selective inhibitor of xanthine oxidase, is often used in the perfusion solution when harvesting the liver, and as a result can contaminate the cytosol. This may, therefore, inhibit cytosolic activity of xanthine oxidase and so influence any in vitro drug metabolism studies. As a consequence, it is important to source allopurinol-free liver cytosol when evaluating non-CYP metabolism by xanthine oxidase. Another important consideration when studying cytosolic metabolism is the choice of selective inhibitors of aldehyde oxidase and xanthine oxidase for judicious identification of the role of these enzymes in the metabolism of drugs.

Research presented by Cyprotex at the 11th International ISSX meeting in Busan, South Korea evaluates these factors in the design of aldehyde oxidase and xanthine oxidase in vitro drug metabolism studies.

Download the poster.