The early detection of up and/or down regulation of genes involved in metabolic and transport processes caused by a lead compound is an increasingly integral part of being able to help assess potential future development issues and can assist in de-risking a molecule from these. This publication highlights some of our early validation data using cultured human hepatocytes which were exposed a selection of both small molecule hepatotoxins and large molecule (cytokine) controls.
Small molecule controls used: Omeprazole (25 µM), Rifampicin (20 µM), Phenobarbital (500 µM), α-Napthaflavone (50 µM), Diclofenac (50 µM), Na Butyrate (100 µM) and CITCO (0.5 µM).
Large molecule (cytokine) controls used – Interleukin-2 (IL-2), Interleukin-2 (IL-6), Interleukin-2 (IL-10), Interferon-λ (IFN- λ) and Tumour necrosis factor-α (TNF- α). Cytokines were tested as individual components (125 pg/mL) and as part of a cocktail.
Target CYP genes – CYP1A1, 1A2, 2A6, 2B4, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5
Target UGT genes – UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15
Target Transporter genes – P-GP, BCRP, BSEP, MRP1, MRP2, OATP1B1, OATP1B3, OCT1, OCT2, OAT1, OAT2, OAT3, MATE1, MATE2-K
Cryopreserved human hepatocytes (pool, n = 10 donors) were cultured using matrigel overlay for 72 hours. During this period, hepatocytes were exposed to the control compounds with a fresh media change/dose at 0.5% (v/v) every 24 hours . At the end of this cycle, hepatocytes were lysed and gene activity determined via flow cytometry using branched DNA analysis.
Data is presented as (X) fold increase vs solvent control response and normalised to housekeeping gene GAPDH in Table 1. Activation/up-regulation was indicated by a >2 fold increase in gene—specific bDNA compared to solvent controls. Suppression was indicated by a >50% decrease in gene-specific bDNA.
Small molecules – As anticipated, the prototypical CYP inducing agents Omeprazole, Phenobarbital, Rifampicin, α-Napthaflavone and CITCO effected up-regulation of CYP genes. In line with their typical human induction profiles, maximum up-regulation was observed as follows – Omeprazole CYP1A1 (14) and CYP1A2 (31), Phenobarbital (6), Rifampicin (5), α-Napthaflavone CYP1A1 (16) and CYP1A2 (40) and CITCO CYP2B6 (5). More minor cross-talk with other CYP activities were also observed with each compound. UGT1A enzymes were also up-regulated by each compound, with UGT1A1 being most sensitive (5-7). Down-regulation of CYP genes was not observed with small molecules apart from with Rifampicin and CYP2E1 (0.26). Down-regulation of transporter genes was not observed with any compound.
The hepatotoxin diclofenac and general proliferation agent sodium butyrate had no effect on any gene activity investigated.
Cytokines - As anticipated, the major effect mediated by exposure to cytokines was a down-regulatory of all CYP, UGT and transporter genes in a concentration-related manner.
At lower concentrations of 125 pg/mL (Cytokine cocktail CCA) the only effect was down-regulation of UGT2B7 (0.2). Down-regulation increased with increase in concentration such that at the highest concentrations of cytokines (500 – 20,000 pg/mL, CCC) most CYP, UGT and Transporter genes were markedly down-regulated (0.03 – 0.25) indicating a classic inflammatory response.