SCREENING ASSAY FOR THE INDUCTION OF HUMAN CYP3A4, P-GLYCOPROTEIN AND RAT CYP3A1 BY AEDS IN VITRO: INSIGHTS INTO POTENTIAL PHARMACOKINETIC RESISTANCE
Abstract number :
1.200
Submission category :
7. Antiepileptic Drugs
Year :
2009
Submission ID :
9583
Source :
www.aesnet.org
Presentation date :
12/4/2009 12:00:00 AM
Published date :
Aug 26, 2009, 08:12 AM
Authors :
John Lamb, J. Raucy, M. Franklin and H. White
Rationale: Induction of cytochrome P450s (CYPs) and p-glycoprotein via the pregnane X receptor (PXR) can lead to clinically relevant adverse drug interactions and contribute to pharmaco-resistance in the treatment of epilepsy. We have utilized a hepatic cell line that contains the human CYP3A4 gene promoter, an intestinal cell line that contains the human p-glycoprotein gene promoter, both of which contain the human PXR, and a separate hepatic cell line containing the rat PXR. In all three cell lines PXR induces gene expression through the PXRE (PXR DNA response element) which has been linked to luciferase. These constructs are stably expressed and can be utilized to access the potential for antiepileptic drugs (AEDs) to induce human CYP3A4, p-glycoprotein and rat CYP3A1. Methods: DPX2 (CYP3A4 PXR), RPXR (rat CYP3A1), and MDR1.C (human p-glycoprotein) cells were exposed to increasing concentrations of several AEDs. Viability was determined by MultiTox (Promega) and induction was determined by luminesent activity using BrightGlo (Promega). Results on induction of CYP3A4 were compared to guidelines laid out by the FDA(2004-2006) on the induction potential compared to the induction seen with 10uM rifiampicin. ED50s were determined using Sigma Plot software. Results: Moderate to high human PXR (CYP3A4) activation was seen for carbamazepine, clonazepam, diazepam, flunarizine, oxazepam, phenobarbital, phenytoin, and topiramate. For lamotrigine only a moderate activation of human PXR (CYP3A4) was seen. There was negligible activation seen for ethosuxamide, felbamate, levetiracetam, phenacemide, phensuximide, primidone, valproic acid, and zonisamide. In the human p-glycoprotein (MDR) cell line a greater than two fold induction was observed for carbamazepine, clonazepam, diazepam, flunarizine, lamotrigine, oxazepam, phenobarbital, phenytoin, and topiramate. For rat PXR (CYP3A1), only valproic acid, clonazepam and diazepam caused a significant induction. Conclusions: These results give insights into the induction potential of AEDs for human CYP3A4, and p-glycoprotein, and the potential for adverse drug-drug interactions. They also give insights into differences in human vs. rat induction via a PXR mediated molecular mechanism. It is important to note the induction potential (as well as ED50s) seen for CYP3A4 occurred at physiologically relevant levels. Co-induction for CYP3A4 and p-glycoprotein underscore the importance of these enzymes in potential therapy resistance to AEDs. We have utilized these cell lines for accessing the liability of potential drug-drug interactions, we have found some compounds caused induction as expected, and therefore the assay is validated for the screening of novel compounds. The use of these cell lines will be invaluable in the discovery of new compounds for the treatment of epilepsy. Funded by N01-NS-4-2359
Antiepileptic Drugs