Hyunyoung Jeong

Up-Regulation of UDP-Glucuronosyltransferase (UGT) 1A4 by 17β-Estradiol: A Potential Mechanism of Increased Lamotrigine Elimination in Pregnancy

Oral clearance of lamotrigine, an antiepileptic drug commonly used in pregnant women, is increased in pregnancy by unknown mechanisms. In this study, we show that 17β-estradiol (E2) up-regulates expression of UDP glucuronosyltransferase (UGT) 1A4, the major enzyme responsible for elimination of lamotrigine. Endogenous mRNA expression levels of UGT1A4 in estrogen receptor (ER) α-negative HepG2 cells were induced 2.3-fold by E2 treatment in the presence of ERα expression. E2 enhanced transcriptional activity of UGT1A4 in a concentration-dependent manner in HepG2 cells when ERα was cotransfected. Induction of UGT1A4 transcriptional activity by E2 was also observed in ERα-positive MCF7 cells, which was abrogated by pretreatment with the antiestrogen fulvestrant (ICI 182,780). Analysis of UGT1A4 upstream regions using luciferase reporter assays identified a putative specificity protein-1 (Sp1) binding site (–1906 to –1901 base pairs) that is critical for the induction of UGT1A4 transcriptional activity by E2. Deletion of the Sp1 binding sequence abolished the UGT1A4 up-regulation by E2, and Sp1 bound to the putative Sp1 binding site as determined by a electrophoretic mobility shift assay. Analysis of ERα domains using ERα mutants revealed that the activation function (AF) 1 and AF2 domains but not the DNA binding domain of ERα are required for UGT1A4 induction by E2 in HepG2 cells. Finally, E2 treatment increased lamotrigine glucuronidation in ERα-transfected HepG2 cells. Together, our data indicate that up-regulation of UGT1A4 expression by E2 is mediated by both ERα and Sp1 and is a potential mechanism contributing to the enhanced elimination of lamotrigine in pregnancy.

Altered drug metabolism during pregnancy: hormonal regulation of drug-metabolizing enzymes.

Importance of the field: Medication use during pregnancy is prevalent, but pharmacokinetic information of most drugs used during pregnancy is lacking in spite of known effects of pregnancy on drug disposition. Accurate pharmacokinetic information is essential for optimal drug therapy in mother and fetus. Thus, understanding how pregnancy influences drug disposition is important for better prediction of pharmacokinetic changes of drugs in pregnant women. Areas covered in this review: Pregnancy is known to affect hepatic drug metabolism, but the underlying mechanisms remain unknown. Physiological changes accompanying pregnancy are probably responsible for the reported alteration in drug metabolism during pregnancy. These include elevated concentrations of various hormones such as estrogen, progesterone, placental growth hormones and prolactin. This review covers how these hormones influence expression of drug-metabolizing enzymes (DMEs), thus potentially responsible for altered drug metabolism during pregnancy. What the reader will gain: The reader will gain a greater understanding of the altered drug metabolism in pregnant women and the regulatory effects of pregnancy hormones on expression of DMEs. Take home message: In-depth studies in hormonal regulatory mechanisms as well as confirmatory studies in pregnant women are warranted for systematic understanding and prediction of the changes in hepatic drug metabolism during pregnancy.

Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells

The objective of this study was to characterize Huh7 cells’ baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

Decreased Warfarin Clearance Associated With the CYP2C9 R150H (*8) Polymorphism

The cytochrome P450 (CYP) 2C9 R150H (*8) allele occurs commonly in African Americans and is associated with lower warfarin dose requirements. We conducted a pharmacokinetic study to examine whether the CYP2C9*8 allele impacts warfarin clearance in African‐American patients. We also conducted an in vitro kinetic study of S‐warfarin 7‐hydroxylation using complementary DNA (cDNA)‐expressed CYP2C9 enzymes. We observed a 30% reduction in the unbound oral clearance of S‐warfarin and a 25% lower R‐ to S‐warfarin plasma concentration ratio in patients with the CYP2C9*8 allele (n = 12) as compared to CYP2C9*1 homozygotes (n = 26). Consistent with these findings, the in vitro intrinsic clearance of S‐warfarin was 30% lower with the cDNA‐expressed R150H protein as compared to the wild‐type protein. These data show that the R150H variant protein expressed by the CYP2C9*8 allele is associated with lower S‐warfarin clearance. This finding provides clinical and experimental evidence to explain the lower warfarin dose requirements in patients with the CYP2C9*8 allele.

Isoform-Specific Regulation of Cytochromes P450 Expression by Estradiol and Progesterone

Results from clinical studies suggest that pregnancy alters hepatic drug metabolism in a cytochrome P450 (P450) isoform-specific manner, and rising concentrations of female hormones are potentially responsible for the changes. The objective of this study was to comprehensively characterize the effects of estrogen and progesterone on the expression and activity of major drug-metabolizing P450s. To this end, primary human hepatocytes were treated with estradiol and progesterone, and mRNA expression and activity levels of 10 different P450 isoforms were determined. The results showed that estradiol enhances CYP2A6, CYP2B6, and CYP3A4 expression, whereas progesterone induces CYP2A6, CYP2B6, CYP2C8, CYP3A4, and CYP3A5 expression. The induction was mainly observed when the average hormone concentrations were at the levels reached during pregnancy, suggesting that these effects are likely pregnancy-specific. Estradiol also increased enzyme activities of CYP2C9 and CYP2E1 without affecting the mRNA expression levels by unknown mechanisms. Taken together, our results show differential effects of estrogen and progesterone on P450 expression, suggesting involvement of different regulatory mechanisms in female hormone-mediated P450 regulation. Our findings potentially provide a basis in mechanistic understanding for altered drug metabolism during pregnancy.

Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination

The authors recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, it was hypothesized that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol. Labetalol glucuronidation, which is the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyse labetalol glucuronidation. The effect of female hormones (progesterone, oestradiol, oestriol, or oestrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of oestrogen receptor α (ERα) and pregnane X receptor (PXR) was examined by co-transfecting ERα- or PXR-constructs. UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone, but not by oestrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time polymerase chain reaction (PCR) assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration dependent. Promoter activities of UGT2B7 were not affected by either oestrogens or progesterone. The results suggest a potential role for progesterone in regulating labetalol elimination by modulating the expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.

Estradiol induces cytochrome P450 2B6 expression at high concentrations: Implication in estrogen-mediated gene regulation in pregnancy

Pregnancy alters the rate and extent of drug metabolism, but little is known about the underlying molecular mechanism. We have found that 17β-estradiol (E2) upregulates expression of the major drug-metabolizing enzyme CYP2B6 in primary human hepatocytes. Results from promoter reporter assays in HepG2 cells revealed that E2 activates constitutive androstane receptor (CAR) and enhances promoter activity of CYP2B6, for which high concentrations of E2 reached during pregnancy were required. E2 triggered nuclear translocation of CAR in primary rat hepatocytes that were transiently transfected with human CAR as well as in primary human hepatocytes, further confirming transactivation of CAR by E2. E2-activated estrogen receptor (ER) also enhanced CYP2B6 promoter activity. The DNA-binding domain of ER was not required for the induction of CYP2B6 promoter activity by E2, suggesting involvement of a non-classical mechanism of ER action. Results from deletion and mutation assays as well as electrophorectic mobility shift and supershift assays revealed that two AP-1 binding sites (-1782/-1776 and -1664/-1658 of CYP2B6) are critical for ER-mediated activation of the CYP2B6 promoter by E2. Concurrent activation of both ER and CAR by E2 enhanced CYP2B6 expression in a synergistic manner. Our data demonstrate that at high concentrations reached during pregnancy, E2 activates both CAR and ER that synergistically induce CYP2B6 expression. These results illustrate pharmacological activity of E2 that would likely become prominent during pregnancy.

Altered expression of small heterodimer partner governs cytochrome P450 (CYP) 2D6 induction during pregnancy in CYP2D6-humanized mice

Background: CYP2D6-mediated drug metabolism is enhanced during pregnancy, but the underlying mechanisms remain unknown. Results: In CYP2D6-humanized mice, CYP2D6 induction during pregnancy was linked to decreased expression of SHP, a repressor of CYP2D6 expression. Conclusion: Decreased SHP expression may account for CYP2D6 induction during pregnancy. Significance: This may provide a mechanistic basis in designing optimal dosage regimens in pregnant women. Substrates of a major drug-metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6-humanized (Tg-CYP2D6) mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, expression of a known positive regulator of CYP2D6, hepatocyte nuclear factor 4α (HNF4α), did not change during pregnancy. However, HNF4α recruitment to CYP2D6 promoter increased at term pregnancy, accompanied by repressed expression of small heterodimer partner (SHP). In HepG2 cells, SHP repressed HNF4α transactivation of CYP2D6 promoter. In transgenic (Tg)-CYP2D6 mice, SHP knockdown led to a significant increase in CYP2D6 expression. Retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy in Tg-CYP2D6 mice. Administration of all-trans-retinoic acid led to a significant decrease in the expression and activity of hepatic CYP2D6 in Tg-CYP2D6 mice. This study provides key insights into mechanisms underlying altered CYP2D6-mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.

Altered cytochrome P450 expression in mice during pregnancy.

Human pregnancy is known to influence hepatic drug metabolism in a cytochrome (P450)-specific manner. However, the underlying mechanisms remain unknown, in part due to a lack of experimental models to study altered drug metabolism during pregnancy. In this study, we examined how pregnancy influences expression of major P450 isoforms in mice. Liver tissues were isolated from female FVB/N-mice at different gestational time points: prepregnancy, 7, 14, and 21 days of pregnancy, and 7 days postpartum. mRNA expression levels of major P450 isoforms (Cyp1a2, Cyp2a5, Cyp2b10, Cyp2c37, Cyp2d22, Cyp2e1, Cyp3a11, and Cyp3a41) in the liver tissues were determined by quantitative real-time polymerase chain reaction. Whereas Cyp2a5 expression was unchanged, Cyp3a41 expression was significantly increased during pregnancy. In contrast, expression of Cyp1a2, Cyp2c37, Cyp2d22, Cyp2e1, and Cyp3a11 was decreased. Expression of Cyp2d22 and Cyp2e1 isoforms correlated with that of peroxisome proliferator-activated receptor (PPAR)α in the mouse livers, suggesting potential involvement of PPARα in down-regulation of the P450 expression during pregnancy. Effects of pregnancy on expression of other P450 mouse isoforms as well as on in vivo drug disposition remain to be characterized. These results provide a guide for future studies on P450 regulation during pregnancy.

Role of P-glycoprotein in the hepatic metabolism of tacrolimus

The main objective was to determine the potential effect of P-glycoprotein (P-gp) modulation on hepatic metabolism of tacrolimus, a P-gp and cytochrome P450(CYP)3A4 substrate, and to investigate various potential factors that may contribute to the interaction between P-gp and CYP. An isolated perfused rat liver system was used to study the hepatic disposition of tacrolimus in the presence of a P-gp inhibitor, GF120918, and a comprehensive pharmacokinetic analysis was conducted. GF120918 significantly decreased mean intrinsic metabolic clearance (by 86 and 41% based on the well-stirred and tube models, respectively) as well as hepatic clearance (from 47.3 to 44.2 ml min(-1)). Potential factors that might contribute to these observations, such as the effects of GF120918 on hepatic metabolism or on distribution of tacrolimus, were investigated and found to be negligible. In conclusion, it was shown that P-gp inhibition by GF120918 reduces hepatic clearances of its substrate drugs although the mechanism is yet to be determined.