P137 Profiling of NCEs in a PXR Reporter Cell Line and Human Hepatocytes to Adopt a Best-Practice Approach for Identifying CYP3A Inducers in Drug Discovery

Kevin J. Coe , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA
Judith Skaptason , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA
Robyn Buerger , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, Springhouse, PA
Chrissa Ferguson , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, Springhouse, PA
Zhengyin Yan , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, Springhouse, PA
Gary Caldwell , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, Springhouse, PA
Tatiana Koudriakova , Dmpk, Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA
Induction of CYP3A by drugs can result in undesirable ADME-properties, such as insufficient drug exposure due to autoinduction, increased risk of drug-drug interactions due to enhanced clearance, and potential for toxicity due to CYP3A-mediated bioactivation of drugs into reactive metabolites.  Thus, identification of new chemical entities (NCEs) that induce CYP3A expression is required to guide early chemistry efforts in order to avoid nomination of drugs that are inducers.  Drugs that induce CYP3A often activate the nuclear receptor pregnane X receptor (PXR) to increase gene expression of a battery of ADME-related genes, including CYP3A4.  The ligand binding domain of PXR is highly divergent across species, leading to potency differences that impede the use of pre-clinical species to extrapolate the risk of CYP3A induction in humans.  The study of CYP3A induction is therefore best explored in vitro in human models, such as engineered PXR reporter cell lines and human hepatocytes. 

Prototypical CYP3A inducers and a chemically diverse test set of NCEs were examined in the Puracyp™ PXR reporter cell line and human hepatocytes to measure transcriptional and activity response of CYP3A, respectively.  Although the PXR reporter cell line offers the throughput necessary for SAR building, follow-up work in human hepatocytes is paramount to confirm the success of the current chemistry strategy to address CYP3A induction.  In our traditional assay, hepatocytes are seeded in 48-well plates and incubated with a test compound for two days.  After two days, the hepatocyte media is replaced with fresh media containing a “CYP probe substrate cocktail” which includes midazolam (3 μM). The reaction is carried out for 30 min followed by LC-MS/MS analysis for the formation of the CYP-selective probe metabolites. In addition to a relatively modest throughput (~ 12 compounds) this detection method is sometimes susceptive to ion suppression and other bioanalytical problems (e.g. lack of sensitivity).  To overcome these issues, we examined the utility of a luminescent-based assay, using the CYP3A4-selective probe Luciferin-IPA (Promega).  Hepatocytes were seeded in 96-well plates, incubated with CYP inducers for two days and the CYP activity was measured using Luciferin-IPA substrate.   Comparison of the two methodologies for CYP3A activity reveals a good concordance and applicability of the luminescent-based assay to meet our throughput demands.  Findings from these bridging experiments reveal that PXR – hepatocyte relationships are often series-dependent and are especially timely if a series induces CYP3A in hepatocytes but not in the PXR reporter assay.  These observations, in turn, allow for a translation of the PXR reporter data to outcomes in hepatocytes, refine strategy for candidate selection, and reveal other possible ADMET liabilities such as CYP3A time dependent inhibition and hepatocyte toxicity.  In conclusion, the tandem profiling of compounds in PXR reporter and hepatocytes assays mitigate the risk in drug discovery of extensive characterizing and nominating drug candidates that harbor significant CYP3A induction.