Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience |
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Authors: | Hoflack J-C Mueller L Fowler S Braendli-Baiocco A Flint N Kuhlmann O Singer T Roth A |
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Institution: | F. Hoffmann-La Roche Ltd., Pharma Research and Early Development, Non-Clinical Safety, Grenzacherstrasse 124, 4070 Basel, Switzerland |
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Abstract: | IntroductionDalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay.MethodsLiver samples were obtained from ~ 1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored.ResultsCyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250 mg/kg/day) to 160-fold (750 mg/kg/day), which declined slightly at 2000 mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750 mg/kg/day).DiscussionThese data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay. |
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Keywords: | ABCC3 ATP-binding cassette sub-family C (CFTR/MRP) member 3 ABCB1a ATP-binding cassette sub-family B (MDR/TAP) member 1A ALAS1 aminolevulinic acid synthase 1 BCA bicinchoninic acid BROD benzyloxyresorufin O-debenzylase BSA bovine serum albumin BrdU bromodeoxyuridine CAR constitutive androstane receptor Ct cycle threshold Cyp cytochrome P450 DMSO dimethyl sulfoxide EROD ethoxyresorufin O-deethylase GAPDH glyceraldehyde-3-phosphate dehydrogenase LC-MS/MS liquid chromatography-mass spectrometry/mass spectrometry PROD pentoxyresorufin O-dealkylase NADPH reduced nicotinamide adenine dinucleotide phosphate qRT-PCR quantitative real time polymerase chain reaction TCPOBOP 1 4-bis[2-(3 5-dichloropyridyloxy)]benzene SD standard deviation |
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