CYP2C19 and CYP2C9 : new aspects of pharmacogenetics and transcriptional regulation
Author: Mwinyi, Jessica
Date: 2010-10-15
Location: föreläsningssal Petrén, Nobelsväg 12b, Karolinska Institutet
Time: 09.00
Department: Institutionen för fysiologi och farmakologi / Department of Physiology and Pharmacology
Abstract
Cytochrome P450s (CYPs) are responsible for approximately 75% of the
phase I-dependent 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 co-transfection 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 . The estrogen mediated regulation may explain the clinically observed inhibitory effects of oral contraceptives on CYP2C19 and CYP2C9 activity.
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 co-transfection 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 . The estrogen mediated regulation may explain the clinically observed inhibitory effects of oral contraceptives on CYP2C19 and CYP2C9 activity.
List of papers:
I. Baldwin RM, Ohlsson S, Pedersen RS, Mwinyi J, Ingelman-Sundberg M, Eliasson E, Bertilsson L. (2008). "Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers." Br J Clin Pharmacol 65: 767-774.
Pubmed
View record in Web of Science®
II. Ohlsson Rosenborg S, Mwinyi J, Andersson M, Baldwin RM, Pedersen RS, Sim SC, Bertilsson L, Ingelman-Sundberg M, Eliasson E. (2008). "Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects." Eur J Clin Pharmacol 64: 1175-1179.
Pubmed
View record in Web of Science®
III. Mwinyi J, Hofmann Y, Pedersen RS, Nekvindová J, Cavaco I, Mkrtchian S, Ingelman-Sundberg M (2009). "The transcription factor GATA-4 regulates cytochrome P4502C19 gene expression" Life Sci 86: 699-706.
Pubmed
View record in Web of Science®
IV. Mwinyi J, Nekvindová J, Cavaco I, Hofmann Y, Pedersen RS, Landman E, Mkrtchian S, Ingelman-Sundberg M. "New insights into the regulation of CYP2C9 gene expression: the role of the transcription factor GATA-4." Drug Metab Dispos 38: 415-421.
Pubmed
View record in Web of Science®
V. Mwinyi J, Cavaco I, Pedersen RS, Persson A, Burkhardt S, Mkrtchian S and Ingelman-Sundberg M (2010). "Regulation of CYP2C19 expression by estrogen receptor alpha. Implications for estrogen dependent inhibition of drug metabolism." Mol Pharmacy. [Accepted]
Pubmed
View record in Web of Science®
VI. Mwinyi J, Cavaco I, Yurdakok B, Mkrtchian S and Ingelman-Sundberg M (2010). "Cytochrome P4502C9 (CYP2C9) regulation by the ligands of estrogen receptor α". [Manuscript]
I. Baldwin RM, Ohlsson S, Pedersen RS, Mwinyi J, Ingelman-Sundberg M, Eliasson E, Bertilsson L. (2008). "Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers." Br J Clin Pharmacol 65: 767-774.
Pubmed
View record in Web of Science®
II. Ohlsson Rosenborg S, Mwinyi J, Andersson M, Baldwin RM, Pedersen RS, Sim SC, Bertilsson L, Ingelman-Sundberg M, Eliasson E. (2008). "Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects." Eur J Clin Pharmacol 64: 1175-1179.
Pubmed
View record in Web of Science®
III. Mwinyi J, Hofmann Y, Pedersen RS, Nekvindová J, Cavaco I, Mkrtchian S, Ingelman-Sundberg M (2009). "The transcription factor GATA-4 regulates cytochrome P4502C19 gene expression" Life Sci 86: 699-706.
Pubmed
View record in Web of Science®
IV. Mwinyi J, Nekvindová J, Cavaco I, Hofmann Y, Pedersen RS, Landman E, Mkrtchian S, Ingelman-Sundberg M. "New insights into the regulation of CYP2C9 gene expression: the role of the transcription factor GATA-4." Drug Metab Dispos 38: 415-421.
Pubmed
View record in Web of Science®
V. Mwinyi J, Cavaco I, Pedersen RS, Persson A, Burkhardt S, Mkrtchian S and Ingelman-Sundberg M (2010). "Regulation of CYP2C19 expression by estrogen receptor alpha. Implications for estrogen dependent inhibition of drug metabolism." Mol Pharmacy. [Accepted]
Pubmed
View record in Web of Science®
VI. Mwinyi J, Cavaco I, Yurdakok B, Mkrtchian S and Ingelman-Sundberg M (2010). "Cytochrome P4502C9 (CYP2C9) regulation by the ligands of estrogen receptor α". [Manuscript]
Issue date: 2010-09-24
Publication year: 2010
ISBN: 978-91-7457-049-6
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