Gefitinib, at Its Clinically Relevant Concentrations, Inhibits Rifampicin‐Induced CYP3A4 Gene Expression in Human Hepatocytes

Abstract

Pregnane xenobiotic receptor (PXR) has been shown to induce chemoresistance during multidrug chemotherapy via upregulation of PXR target genes such as cytochrome P450 3A4 (CYP3A4). CYP3A4 contributes to the metabolism of over 50% of clinical drugs. Therefore, during multidrug chemotherapy, drug induction of hPXR‐mediated CYP3A4, can reduce the therapeutic concentration of coadministered drugs, leading to chemoresistance. It is possible to overcome a PXR agonist‐induced chemoresistance with a PXR antagonist. However, proposed antagonists lack appropriate pharmacological characteristics that allow them to be active in the clinic. We propose that a PXR antagonist would be a cancer drug itself that is part of a “cancer drug cocktail”, and effective as PXR antagonist at therapeutic concentrations. Gefitinib (GEF), a tyrosine kinase inhibitor approved for the treatment of advanced non‐small cell lung cancer, and shown to be effective in combinational chemotherapy treatment, is a promising candidate due to its PXR ligand‐like features. We therefore sought to determine whether GEF would behave as a PXR antagonist at its clinically relevant concentrations in human primary hepatocytes, human hepatocells, and HepG2 human hepatocellular carcinoma cells. Rifampicin, an agonist of PXR, was used to activate PXR‐mediated CYP3A expression. Reporter gene assays were conducted to determine PXR transactivation of CYP3A4 promoter activity. Cell viability and qRT‐PCR assays were performed to assess the cytotoxicity and CYP3A4 gene expression, respectively. PXR molecular docking studies were carried out to predict binding affinity of GEF. Finally, competitive ligand binding and steroid receptor coactivator‐1 (SRC‐1) recruitment assays were performed to examine GEF ability to bind to PXR and alter SRC‐1 recruitment. GEF, at its therapeutic concentrations, repressed rifampicin‐induced endogenous CYP3A4 gene expression. Additionally, GEF inhibited rifampicin induction of PXR‐mediated CYP3A4 promoter activity. These findings indicate that GEF could inhibit PXR agonists‐induced CYP3A4 gene expression. Molecular docking studies predicted that GEF can bind to multiple sites on PXR, including the ligand binding pocket, AF‐2 region and alpha‐8 pocket. Indeed, GEF bound to PXR and attenuated the PXR agonist‐induced SRC‐1 interaction, suggesting that GEF directly interacts with multiple sites on the PXR. Taken together, these results suggest that GEF, at its clinically relevant therapeutic concentration, can antagonize PXR agonists‐induced CYP3A4 gene expression. Thus, GEF could be a potential candidate for use in combinational chemotherapies to combat PXR‐mediated chemoresistance. Future studies are required to examine GEF selectivity towards PXR, and to determine GEF suppression of PXR agonists‐induced chemoresistance.Support or Funding InformationThe authors thank Drs. Coleman, Mansour, Schwartz & Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant and Animal Health and Disease Research Grant.