Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.

Induction of cytochrome P450 can cause drug-drug interactions and efficacy failure. Induction risk in liver and gut is typically inferred from experiments with plated hepatocytes. Organoids are physiologically relevant, multicellular structures originating from stem cells. Intestinal stem cell-derived organoids retain traits of normal gut physiology, such as an epithelial barrier and cellular diversity. Matched human enteroid and colonoid lines, generated from ileal and colon biopsies from two donors, were cultured in extracellular matrix for 3 days, followed by a single 48-hour treatment with rifampin, omeprazole, CITCO, and phenytoin at concentrations that induce target genes in hepatocytes. After treatment, mRNA was analyzed for induction of target genes. Rifampin induced CYP3A4; estimated EC50 and maximal fold induction were 3.75 µM and 8.96-fold, respectively, for ileal organoids and 1.40 µM and 11.3-fold, respectively, for colon organoids. Ileal, but not colon, organoids exhibited nifedipine oxidase activity, which was induced by rifampin up to 14-fold. The test compounds did not increase mRNA expression of CYP1A2, CYP2B6, multidrug resistance transporter 1 (P-glycoprotein), breast cancer resistance protein, and UDP-glucuronosyltransferase 1A1 in ileal organoids. Whereas omeprazole induced CYP3A4 (up to 5.3-fold, geometric mean, n = 4 experiments), constitutive androstane receptor activators phenytoin and CITCO did not. Omeprazole failed to induce CYP1A2 mRNA but did induce CYP1A1 mRNA (up to 7.7-fold and 15-fold in ileal and colon organoids, respectively, n = 4 experiments). Despite relatively high intra- and interexperimental variability, data suggest that the model yields induction responses that are distinct from hepatocytes and holds promise to enable evaluation of CYP1A1 and CYP3A4 induction in gut. SIGNIFICANCE STATEMENT: An adult intestinal stem cell-derived organoid model to test P450 induction in gut was evaluated. Testing several prototypical inducers for mRNA induction of P450 isoforms, UDP-glucuronosyltransferase 1A1, P-glycoprotein, and breast cancer resistance protein with both human colon and ileal organoids resulted in a range of responses, often distinct from those found in hepatocytes, indicating the potential for further development of this model as a physiologically relevant gut induction test system.

Despite the availability of liquid chromatography (LC)–mass spectrometry (MS) methods for quantifying cytochrome P450 (P450) proteins, incorporation of P450 protein quantification into induction study workflows has not been widely adopted. To more readily enable P450 protein quantification in induction study workflows, DMPK research groups need a simple, robust, cost-effective, high-throughput method compatible with 96-well–plated human hepatocyte formats. Here, we provide such a methodology. Our method bypasses both microsomal enrichment and antibody-based enrichment to go directly from the plate to LC-MS/MS analysis. We use this "plate-to-peaks" approach for quantifying CYP3A4, CYP2B6, and CYP1A2, the major inducible hepatic P450s representative of pregnane X receptor–, constitutive androstane receptor–, and aryl hydrocarbon receptor–mediated induction, respectively. We leveraged our induction study-aligned assay format to assess induction across mRNA, protein, and enzyme activity using known induction control compounds. As expected, results from the three methods using model inducers were broadly concordant, but the magnitude of the induction response differed. Induction of CYP3A4 using 10 µM rifampicin was 12-fold for RNA, eightfold for protein, and threefold for activity; for CYP1A2 with 50 µM omeprazole, induction was 30-fold for RNA, 13-fold for protein, and 17-fold for activity; for CYP2B6 with 50 µM phenytoin, induction was 23-fold for RNA, twofold for protein, and fivefold for activity. Most importantly, we anticipate the relative ease of this method will enable researchers to routinely adopt P450 protein quantification as part of nonclinical evaluation of P450 induction. SIGNIFICANCE STATEMENT Current methodologies for quantifying P450 proteins by liquid chromatography (LC)–tandem mass spectrometry are either cumbersome, too costly, or both to be widely adopted into induction study workflows by the ADME research community. We present a simplified LC-MS/MS methodology for quantifying P450 proteins directly from human hepatocytes, without any form of enrichment, in 96-well induction assay plate format that should be readily adoptable by any ADME laboratory with LC–multiple-reaction monitoring capabilities.