CYP2C19 and CYP2C9: New aspects of pharmacogenetics and transcriptional regulation

Cytochrome P450s (CYPs) are responsible for approximately 75% of the phase Idependent drug metabolism. Several important polymorphisms in these enzymes are known to significantly affect the individual drug response. CYP2C9 and CYP2C19 are polymorphically expressed CYP family members which are responsible for the metabolism of many different clinically important drugs, e.g. anticoagulants, antidepressants and antiulcer drugs. This thesis focuses on novel aspects with regard to the regulation of CYP2C9 and CYP2C19 gene expression. The influence of the recently found common allele variant CYP2C19*17 on CYP2C19 enzyme activity towards two CYP2C19 substrates was investigated in the frame of two pharmacokinetic in vivo studies. The studies compared the single-dose pharmacokinetics of omeprazole and the steady-state kinetics of escitalopram in healthy CYP2C19*17/*17 carriers with the pharmacokinetic outcome obtained for CYP2C19 wild-type subjects. While no significant differences in the metabolic efficacy were observed for escitalopram, CYP2C19*17/*17 subjects showed significantly lower plasma levels of omeprazole compared to CYP2C19 wild type carriers. This observation suggests that CYP2C19*17/*17 carriers might be at a higher risk for therapy failure during treatment with proton pump inhibitors. The transcriptional regulation of CYP2C9 and CYP2C19 gene expression by GATA transcription factors and by estrogen receptor α (ERα) was investigated in vitro. In the proximal promoter regions of both the CYP2C9 and the CYP2C19 gene, two adjacent putative GATA-binding sites with an ER-binding half-site in their vicinity were predicted in silico and initially studied by luciferase gene reporter assay. HepG2 and Huh-7 hepatoma cells were transfected with CYP2C9 or CYP2C19 promoter fragment carrying pGL3basic-constructs along with expression vectors for the transcription factors GATA-2, GATA-4, or ERα. Luciferase activities driven by wild-type CYP2C19 or CYP2C9 promoter were highly increased by GATA-4 and GATA-2 in both cell lines, whereas mutations introduced into the GATA binding sites or the cotransfection of the GATA-4 antagonist FOG-2 caused a significant loss of luciferase activity. In contrast, treatment with estradiol derivatives of ERα-transfected cells caused a significant inhibition of CYP2C19 and CYP2C9 promoter activity that was antagonized by site-directed mutagenesis of the putative ERα-binding half-sites. Additionally, estradiol derivatives significantly suppressed both CYP2C9 and CYP2C19 mRNA expression in human hepatocytes, as measured by real time PCR. Electrophoretic mobility shift assays revealed sequence-specific binding of GATA-4, GATA-6, and ERα to the two adjacent GATA binding sites and to the predicted ER binding half sites, respectively. ChIP assay in the cultured cells furthermore confirmed the association of both GATA-4 and ERα with CYP2C9 and CYP2C19 gene promoter. In conclusion, we have established novel mechanisms of CYP2C9 and CYP2C19 transcriptional regulation that involve transcription factors from the GATA family and estrogen receptor α. The estrogen mediated regulation may explain the clinically observed inhibitory effects of oral contraceptives on CYP2C19 and CYP2C9 activity. LIST OF PUBLICATIONS I. Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers. Baldwin RM, Ohlsson S, Pedersen RS, Mwinyi J, IngelmanSundberg M, Eliasson E, Bertilsson L. Br J Clin Pharmacol. 2008;65:767-74. II. Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects. Ohlsson Rosenborg S, Mwinyi J, Andersson M, Baldwin RM, Pedersen RS, Sim SC, Bertilsson L, Ingelman-Sundberg M, Eliasson E. Eur J Clin Pharmacol. 2008;64:1175-9. III. The transcription factor GATA-4 regulates cytochrome P4502C19 gene expression. Mwinyi J, Hofmann Y, Pedersen RS, Nekvindová J, Cavaco I, Mkrtchian S, Ingelman-Sundberg M. Life Sci. 2010;86:699-706. IV. New insights into the regulation of CYP2C9 gene expression: the role of the transcription factor GATA-4. Mwinyi J*, Nekvindová J*, Cavaco I, Hofmann Y, Pedersen RS, Landman E, Mkrtchian S**, Ingelman-Sundberg M**. Drug Metab Dispos. 2010;38:415-21. V. Regulation of CYP2C19 expression by estrogen receptor alpha. Implications for estrogen dependent inhibition of drug metabolism. Mwinyi J, Cavaco I, Pedersen RS, Persson A, Burkhardt S, Mkrtchian S and Ingelman-Sundberg M. Mol Pharmacol. 2010 July 30 [Epub ahead of print]. VI. Cytochrome P4502C9 (CYP2C9) regulation by the ligands of estrogen receptor α Mwinyi J*, Cavaco I*, Yurdakok B, Mkrtchian S** and Ingelman-Sundberg M**. Submitted Manuscript * Equal contribution ** Shared last authorship