Why Do Most Human Liver Cytosol Preparations Lack Xanthine Oxidase Activity?

Abstract

When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions--an Industry Perspective

“…The involvement of AO or XO in oxidative metabolism of a NME can be determined directly by monitoring the metabolism of a NME in liver cytosol or S9 fraction in the absence of NADPH (Hutzler et al, 2013;Barr et al, 2014a). It is important that the source of liver tissue used in these studies was not derived from liver that had been perfused with solutions containing allopurinol, a common practice after full hepatectomy, because that tissue would be expected to have little or no XO activity due to residual allopurinol, an inhibitor of XO (Barr et al, 2014b).…”

“…Coadministration of famciclovir with a potent inhibitor of AO such as raloxifene could potentially lead to a reduction in antiviral efficacy (Obach, 2004), but studies to determine the magnitude of this interaction have not been conducted. It was recently demonstrated that inhibition of AO may be substrate dependent and occur by mixed modes of reversible and irreversible inhibition, so care should be exercised in predicting drug interactions based on in vitro data Jones, 2011, 2013a;Barr et al, 2014b).…”

Toward an Understanding of Structural Insights of Xanthine and Aldehyde Oxidases: An Overview of their Inhibitors and Role in Various Diseases

“…briefly discussed various substrates that involve both AOX and XO individually and in combination. Allopurinol is used to inhibit XO expression, while menadione hydralazine has been suggested as a potent and highly selective AOX inhibitors . Weidert et al.…”

In Vitro Oxidative Metabolism of 6-Mercaptopurine in Human Liver: Insights into the Role of the Molybdoflavoenzymes Aldehyde Oxidase, Xanthine Oxidase, and Xanthine Dehydrogenase

“…Pooled mixed-gender human liver samples (n = 8) devoid of allopurinol were obtained from St Jude's Children's Hospital human liver bank (Barr et al, 2014). The samples were prepared and stored as described in (Barr et al, 2014). Table 1 contains information on the demographics of the liver samples used to prepare cytosol.…”

“…Sequence grade trypsin was acquired from Promega (Madison, WI). Pooled mixed-gender human liver samples (n = 8) devoid of allopurinol were obtained from St Jude's Children's Hospital human liver bank (Barr et al, 2014). The samples were prepared and stored as described in (Barr et al, 2014).…”