Rapid determination of rat hepatocyte mRNA induction potential using oligonucleotide probes for CYP1A1, 1A2, 3A and 4A1

1. A new assay to quantify mRNA levels in small numbers of rat hepatocytes has been developed for cytochrome P450 (CYP) isoforms 1Al, 1A2, 3A and 4A1. The assay uses sets of oligonucleotide probes end-labelled with [S-35]-dAT P to hybridize to mRNA in controlor drug-treated rat hepatocytes cultured o n Cytostar-T(TM) 96-well scintillating microplates. 2. The rat hepatocyte induction potential (RHIP) assays for CYP3A, 1Al, 1A2 and 4A1 are sensitive and selective and have an excellent qualitative rela tionship with CYP induction data ex vivo. The robustness of the CYP3A assay was determined following a run of > 40 plates. The variation of the dexame thasone (DEX) response on each plate, calculated as %coefficient of variati on, showed that there was no significant difference between the variability of the response to DEX. 3. Assay specificity for each CYP isoform was achieved by designing probes (four per isoform) antisense to coding regions of each CYP gene sequence. I n the CYP3A RHIP assay, pregnenalone 16 alpha-carbonitrile (PCN), DEX, clot rimazole (CLOT) and miconazole (MIC) were all good inducers of CYP3A mRNA; beta-napthoflavone (BNF) and methyl-clofenapate (MCP), however, did not ind uce CYP3A mRNA, further defining the specificity of this methodology. Speci ficity was similarly confirmed for the other CYP isoforms. 4. Ind(50), the concentration of inducer required to elicit a 50%, inductio n of CYP-specific mRNA, was derived for prototypical CYP inducers: BNF 0.54 and 0.17 mu M (CYP1A1 and 1A2 respectively), 3-methylcholanthrene (3MC) 0. 11 and 0.04 mu M (CYP1A1 and 1A2 respectively), PCN 0.03 mu M, DEX 0.17 mu M, CLOT 0.48 mu M, MIC 3 mu M, TAO 3 mu M (CYP3A), MCP 1.8 mu M, clofibrate (CLOF) 65 mu M and ciprofibrate (CIP) 1.9 mu M (CYP4A1). Ind(50) for BNF a nd 3MC at CYP1A2 was 3-fold lower than that at CYP1A1 indicating a subfamil y difference in inducer potency. 5. Reducing the numbers of animals and the amount of compound required to s tudy CYP induction is an important advantage of the RHIP assays over conven tional evaluations in vivo. Typically four rats are dosed for 4 days using oral doses in the range 50-500 mg kg(-1) day(-1). In comparison, the amount of hepatocytes required to carry out all the studies reported herein may b e obtained from a single animal (< 2 x 10(8) viable cells) and CYP inductio n investigated using mu g rather than g quantities of drug substance. 6. With appropriately designed oligonucleotide probes, the RHIP technology can assess CYP induction in human hepatocytes, which together with preclini cal data can contribute to improving the quality of compounds progressing i nto the expensive process of drug development.