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 1A1, 1A2, 3A and 4A1. The assay uses sets of oligonucleotide probes end-labelled with [35S]-dATP to hybridize to mRNA in control- or drug-treated rat hepatocytes cultured on Cytostar-T 96-well scintillating microplates. 2. The rat hepatocyte induction potential (RHIP) assays for CYP3A, 1A1, 1A2 and 4A1 are sensitive and selective and have an excellent qualitative relationship with CYP induction data ex vivo. The robustness of the CYP3A assay was determined following a run of > 40 plates. The variation of the dexamethasone (DEX) response on each plate, calculated as %coefficient of variation, 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. In the CYP3A RHIP assay, pregnenalone 16alpha-carbonitrile (PCN), DEX, clotrimazole (CLOT) and miconazole (MIC) were all good inducers of CYP3A mRNA; beta-napthoflavone (BNF) and methylclofenapate (MCP), however, did not induce CYP3A mRNA, further defining the specificity of this methodology. Specificity was similarly confirmed for the other CYP isoforms. 4. Ind50, the concentration of inducer required to elicit a 50% induction of CYP-specific mRNA, was derived for prototypical CYP inducers: BNF 0.54 and 0.17 microM (CYP1A1 and 1A2 respectively), 3-methylcholanthrene (3MC) 0.11 and 0.04 microM (CYP1A1 and 1A2 respectively), PCN 0.03 microM, DEX 0.17 microM, CLOT 0.48 microM, MIC 3 microM, TAO 3 microM (CYP3A), MCP 1.8 microM, clofibrate (CLOF) 65 microM and ciprofibrate (CIP) 1.9 microM (CYP4A1). Ind50 for BNF and 3MC at CYP1A2 was 3-fold lower than that at CYP1A1 indicating a subfamily difference in inducer potency. 5. Reducing the numbers of animals and the amount of compound required to study CYP induction is an important advantage of the RHIP assays over conventional 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 be obtained from a single animal (< 2 x 10(8) viable cells) and CYP induction investigated using microg 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 preclinical data can contribute to improving the quality of compounds progressing into the expensive process of drug development.
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Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Harlow, UK. Dominic_Surry@Merck.com
Meneses-Lorente
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D000704:Analysis of Variance; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D002994:Clofibrate / Q000494:pharmacology; D002995:Clofibric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D058607:Fibric Acids; D005821:Genetic Techniques; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D020025:Peroxisome Proliferators / Q000494:pharmacology; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D058607:Fibric Acids; D005938:Glucocorticoids; D016376:Oligonucleotides, Antisense; D020025:Peroxisome Proliferators; D020033:Protein Isoforms; D012333:RNA, Messenger; D002995:Clofibric Acid; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D042926:Cytochrome P-450 CYP4A; D010089:Oxidoreductases, N-Demethylating; C019304:ciprofibrate; D002994:Clofibrate