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Cytochrome P450 2D6 (CYP2D6) is a major drug‐metabolizing enzyme that exhibits large interindividual variability. Recent studies suggest that differential transcriptional regulation of CYP2D6 in part may be responsible for the variability. In this study, we characterized potential determinants of CYP 2D6 transcript levels in healthy human liver tissue samples (n = 115), including genetic polymorphisms in CYP2D6 and the genes encoding transcription regulators for CYP2D6 expression; mRNA expression of the transcription factors and their known target genes; and hepatic levels of bile acids and retinoids, agents that modulate the expression/activity of the transcription factors. Their associations with CYP2D6 mRNA levels in the tissues were examined. Results from multivariable linear regression analysis revealed CYP8B1 mRNA level and rs3892097, the single‐ nucleotide polymorphism defining the nonfunctional CYP2D6*4 allele, as the two most significant predictors of CYP2D6 mRNA levels in the liver tissue samples, explaining 30% of the variability.

CYP2D6-mediated drug metabolism exhibits large interindividual variability. Although genetic variations in the CYP2D6 gene are well known contributors to the variability, the sources of CYP2D6 variability in individuals of the same genotype remain unexplained. Accumulating data indicate that transcriptional regulation of CYP2D6 may account for part of CYP2D6 variability. Yet, our understanding of factors governing transcriptional regulation of CYP2D6 is limited. Recently, mechanistic studies of increased CYP2D6-mediated drug metabolism in pregnancy revealed two transcription factors, small heterodimer partner (SHP) and Krüppel-like factor 9, as a transcriptional repressor and an activator, respectively, of CYP2D6. Chemicals that increase SHP expression (e.g., retinoids and activators of farnesoid X receptor) were shown to downregulate CYP2D6 expression in the humanized mice as well as in human hepatocytes. This review summarizes the series of studies on the transcriptional regulation of CYP2D6 expression, potentially providing a basis to better understand the large interindividual variability in CYP2D6-mediated drug metabolism.

UDP-glucuronosyltransferase 1A9 (UGT1A9) is a major phase II enzyme responsible for elimination of drugs and endogenous molecules. Clinical data have shown increased elimination of UGT1A9 substrates in pregnant women or oral contraceptive users, but the role of estrogen in the regulation of UGT1A9 expression remains unknown. In this study, we investigated the effect of 17β-estradiol (E2) on UGT1A9 expression and the role of ERα in the transcriptional regulation of UGT1A9. E2 significantly increased UGT1A9 promoter activity in HepG2 cells in the presence of ERα. UGT1A9 induction by E2 was abrogated by antiestrogen ICI182,780 in HepG2 cells that constitutively express ERα. Results from transient transfection of ERα mutants into HepG2 cells demonstrated that mutation at DNA-binding domain of ERα abrogates increased UGT1A9 promoter activity by E2. Deletion and mutation assays of UGT1A9 promoter revealed a putative ERE located within −2262/−1987 region. Examination of healthy human liver tissues revealed significantly higher UGT1A9 expression in women as compared to men. Together, these findings provide a mechanistic basis for the previous clinical reports and may shed a light on identifying sources for inter-individual variability in UGT1A9-mediated drug metabolism., Graphical abstract 17β-Estradiol up-regulates UDP-glucuronosyltransferases 1A9 (UGT1A9) expression via estrogen receptor α (ERα). This potentially contributes to sex difference in hepatic UGT1A9 expression. fx1

CYP2D6 genetic polymorphisms are considered a major contributor to the large interindividual variability in CYP2D6-mediated drug metabolism, but fail to explain a significant portion of the variability. The aim of this study was to assess the ability of the CYP2D6 activity score (AS) estimated from CYP2D6 genotype to predict CYP2D6 expression and enzyme activity. The CYP2D6 gene region was sequenced in 115 healthy human liver tissue samples to determine their CYP2D6 AS. Additionally, CYP2D6 enzyme activity, protein, and mRNA levels were estimated. CYP2D6 AS explained 23% of the interindividual variability in CYP2D6 activity, but only 7.5% in tissues assigned AS 1-2. The CYP2D6 protein level was found to be the major determinant of CYP2D6 activity, explaining 59% of variability. These findings suggest that while CYP2D6 AS is a good predictor of poor metabolizer phenotype, additional nongenetic factors may govern the rate of CYP2D6-mediated metabolism in those without the poor metabolizer phenotype.

Medical conditions accompanying obesity often require drug therapy, but whether and how obesity alters the expression of drug-metabolizing enzymes and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high-fat diet (HFD) feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 CYP2D6, including hepatocyte nuclear factor 4α (HNF4α, a transcriptional activator of CYP2D6) and small heterodimer partner (SHP, a transcriptional repressor of CYP2D6). The objective of this study was to examine whether diet-induced obesity alters CYP2D6 expression by modulating HNF4α and SHP expression. Male CYP2D6-humanized transgenic (Tg-CYP2D6) mice were fed with HFD or matching control diet for 18 weeks. Hepatic mRNA expression of CYP2D6 decreased to a small extent in the HFD group (by 31%), but the differences in CYP2D6 protein and activity levels in hepatic S9 fractions were found insignificant between the groups. Although hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased by ∼30% in the HFD group. Among major mouse endogenous cytochrome P450 genes, Cyp1a2 and Cyp2c37 showed significant decreases in the HFD group, whereas Cyp2e1 expression did not differ between groups. Cyp2b10 and Cyp3a11 expression was higher in the HFD group, with corresponding 2.9-fold increases in hepatic CYP3A activities in HFD-fed mice. Together, these results suggest that obesity has minimal effects on CYP2D6-mediated drug metabolism, although it modulates the expression of mouse endogenous P450s in a gene-specific manner.

TM208 and TM209, dithiocarbamate derivatives with potential anti-cancer effects, were evaluated in reversible and time-dependent cytochrome P450 (CYP) 3A inhibition assays in rat liver microsomes using testosterone as probe substrate. Both compounds were found to be weak reversible inhibitors and moderate mechanism-based inhibitors of rat CYP3A. For reversible inhibition on rat CYP3A, the K i values of competitive inhibition model were 12.10±1.75 and 13.94±1.31 μM, respectively. For time-dependent inhibition, the inactivation constants ( K l ) were 31.93±12.64 and 32.91±15.58 μM, respectively, and the maximum inactivation rates ( k inact ) were 0.03497±0.0069 and 0.07259±0.0172 min −1 respectively. These findings would provide useful in vitro information for future in vivo DDI studies on TM208 or TM209.

To develop a pharmacokinetic/pharmacodynamic (PK/PD) model describing the receptor/gene-mediated induction of CYP3A1/2 by dexamethasone (DEX) in rats.A group of male Sprague-Dawley rats receiving DEX (100 mg/kg, ip) were sacrificed at various time points up to 60 h post-treatment. Their blood sample and liver were collected. The plasma concentration of DEX was determined with a reverse phase HPLC method. CYP3A1/2 mRNA, protein levels and enzyme activity were measured using RT-PCR, ELISA and the testosterone substrate assay, respectively. Data analyses were performed using a first-order conditional estimate (FOCE) with INTERACTION method in NONMEM version 7.1.2.A two-compartment model with zero-order absorption was applied to describe the pharmacokinetic characteristics of DEX. Systemic clearance, the apparent volume of distribution and the duration of zero-order absorption were calculated to be 172.7 mL·kg(-1)·h(-1), 657.4 mL/kg and 10.47 h, respectively. An indirect response model with a series of transit compartments was developed to describe the induction of CYP3A1/2 via PXR transactivation by DEX. The maximum induction of CYP3A1 and CYP3A2 mRNA levels was achieved, showing nearly 21.29- and 8.67-fold increases relative to the basal levels, respectively. The CYP3A1 and CYP3A2 protein levels were increased by 8.02-fold and 2.49-fold, respectively. The total enzyme activities of CYP3A1/2 were shown to increase by up to 2.79-fold, with a lag time of 40 h from the Tmax of the DEX plasma concentration. The final PK/PD model was able to recapitulate the delayed induction of CYP3A1/2 mRNA, protein and enzyme activity by DEX.A mechanism-based PK/PD model was developed to characterize the complex concentration-induction response relationship between DEX and CYP3A1/2 and to resolve the drug- and system-specific PK/PD parameters for the course of induction.

We have recently reported that transactivation of cytochrome P450 (CYP) 2D6 promoter by hepatocyte nuclear factor (HNF) 4α is enhanced during pregnancy, and this is triggered in part by altered expression of small heterodimer partner (SHP) and Kruppel-like factor 9 (KLF9). The objective of this study is to determine whether this is conserved for mouse endogenous Cyp2d gene(s). Among the eight Cyp2d homologs of mouse we examined, only Cyp2d40 expression was found induced (by 6-fold) at term pregnancy as compared to pre-pregnancy level. In mice where hepatic Hnf4α was knocked-down, the pregnancy-mediated increase in Cyp2d40 expression was abrogated. Results from transient transfection, promoter reporter assays, and electrophoretic mobility shift assays indicated that HNF4α transactivates Cyp2d40 promoter via direct binding to −117/−105 of the gene. Chromatin immunoprecipitation assay showed a 2.3-fold increase in HNF4α recruitment to Cyp2d40 promoter during pregnancy. Results from mice treated with an SHP inducer (i.e., GW4064) and HepG2 cells co-transfected with KLF9 suggest that neither SHP nor KLF9 is involved in the increased HNF4α transactivation of Cyp2d40 promoter during pregnancy. Together, our results indicate that while the underlying molecular mechanism is different from that for CYP2D6, Cyp2d40 is induced during pregnancy through enhanced transactivation by HNF4α.

Sulfotransferase (SULT) 2A1 catalyzes sulfonation of drugs and endogenous compounds and plays an important role in xenobiotic metabolism as well as in the maintenance of steroid and lipid homeostasis. A recent study showed that 17β-estradiol (E2) increases the mRNA levels of SULT2A1 in human hepatocytes. Here we report the underlying molecular mechanisms. E2 enhanced SULT2A1 expression in human hepatocytes and HepG2-ER cells (HepG2 stably expressing ERα). SULT2A1 induction by E2 was abrogated by antiestrogen ICI 182,780, indicating a key role of ERα in the induction. Results from deletion and mutation assays of SULT2A1 promoter revealed three cis-elements located within –257/+140 region of SULT2A1 that are potentially responsible for the induction. Chromatin immunoprecipitation assay verified the recruitment of ERα to the promoter region. Electrophoretic mobility shift assays revealed that AP-1 proteins bind to one of the cis-elements. Interestingly, SULT2A1 promoter assays using ERα mutants revealed that the DNA-binding domain of ERα is indispensable for SULT2A1 induction by E2, suggesting that direct ERα binding to the SULT2A1 promoter is also necessary for the induction. Taken together, our results indicate that E2 enhances SULT2A1 expression by both the classical and nonclassical mechanisms of ERα action.