Regulation of genes involved in hepatic drug elimination by female hormones

说明(申请人提供)：在怀孕期间，孕妇最好避免使用药物来保护发育中的胎儿免受任何潜在的不良影响。但孕妇先前存在的疾病或疾病要求继续使用药物。这种不可避免的药物治疗需要准确的药代动力学信息，因为接触不足或过量可能导致有害的临床结果，不仅对母亲，而且对胎儿也是如此。对孕妇的临床研究表明，许多药物的药代动力学曲线在怀孕期间会发生改变。一般情况下，药物的口服吸收延迟，药物的分布和肾脏排泄增加。另一方面，肝脏药物代谢被认为在妊娠期间以代谢途径依赖的方式改变。然而，除了其他因素的变化外，目前尚不清楚这些数据是否反映了肝脏药物代谢酶表达或功能的真实变化，例如药物的蛋白质结合或流向肝脏的血液的变化。这就需要一个体外系统，在这个系统中，可以专门检查肝脏药物代谢的变化。妊娠期间的生理变化被认为是导致妊娠期间药物代谢改变的原因。孕妇最显著的变化是雌激素和黄体酮等女性荷尔蒙的产生急剧增加。激素升高可能是孕期肝脏药物代谢改变的原因。在怀孕期间达到较高浓度时，女性荷尔蒙的功能与其作为性腺激素的传统作用不同。孕酮结合并激活转录调节因子孕烷异源受体(PXR)，诱导主要药物代谢酶的表达，包括细胞色素P450酶和UDP-葡萄糖醛酸基转移酶(UGTS)。此外，雌激素通过雌激素受体？？的作用，促进Hep G2细胞UGT1A4和-1A9的表达。(呃？)基于这些证据，我们建议检验一种假设，即雌激素和孕酮在怀孕期间升高，全局调节肝脏药物代谢酶的表达，导致改变肝脏药物的消除。据此设计了以下具体目标。(1)确定雌激素和孕酮对肝脏药物代谢的酶特异性影响。用雌性激素处理人肝细胞后，通过检测不同探针药物对肝细胞的清除率的变化，来探讨激素调节的功能意义。(2)明确雌激素调节UGT1A4和-1A9表达的机制。将进行电泳迁移率改变分析、DNase I保护和突变分析。(3)确定肝脏中雌二醇或孕酮的反应基因。基因芯片实验(Affymetrix Human Genome U133 Plus 2.0 GeneChip阵列)将使用激素处理后的人肝细胞制备的RNA样本进行。公共卫生相关性：孕妇使用药物是很常见的，但这一人群的药物行为通常不同于非孕妇或男性。了解这些变化对于确定最佳给药方案很重要。我们建议研究这种药物行为变化的机制。从这项研究中获得的知识可以扩大到优化其他妇女群体的药物治疗，例如口服避孕药使用者，从而使一般妇女受益。

DESCRIPTION (provided by applicant): During pregnancy, it is ideal for pregnant women to avoid use of medications to protect the developing fetus from any potential adverse effects. But pre-existing medical conditions or disorders to which pregnant women are susceptible mandate continued use of medicines. This unavoidable drug treatment requires accurate pharmacokinetic information because under- or over-exposure may result in detrimental clinical outcomes not only in the mother but also in the fetus. Clinical studies in pregnant women have suggested that pharmacokinetic profiles of many drugs are altered during pregnancy. Generally, oral absorption of drugs is delayed, and distribution and renal excretion of drugs increase. Hepatic drug metabolism, on the other hand, has been suggested to change in a metabolic pathway-dependent manner in pregnancy. However, it is currently unclear whether the data reflect true changes in expression or function of hepatic drug metabolizing enzymes, apart from changes in other factors, such as changes in protein binding of a drug or blood flow to the liver. This necessitates an in vitro system where changes in hepatic drug metabolism can be exclusively examined. Physiological changes accompanying pregnancy are expected to be responsible for altered drug metabolism in pregnancy. The most pronounced change in pregnant women is a dramatic increase in the production of female hormones such as estrogen and progesterone. The elevated hormones may be responsible for altered hepatic drug metabolism in pregnancy. At the high concentrations attained in pregnancy, female hormones have functions different from their conventional role as gonadal hormones. Progesterone binds to and activates a transcriptional regulator pregnane xenobiotic receptor (PXR), which induces expression of major drug metabolizing enzymes including cytochrome P450 enzymes and UDP- glucuronosyltransferases (UGTs). Furthermore, estrogen promotes the expression of UGT1A4 and -1A9 in HepG2 cells, mediated via the action of estrogen receptor-? (ER?). Based on the evidence, we propose to test the hypothesis that the hormones elevated during pregnancy, estrogen and progesterone, globally modulate expression of hepatic drug metabolizing enzymes, leading to altered hepatic elimination of drugs. The following specific aims are designed accordingly. (1) Determine the enzyme-specific effects of estrogen and progesterone on hepatic drug metabolism. The functional significance of hormonal regulation will be investigated by examining how the hepatic elimination rates of various probe drugs change in human hepatocytes after treatment of the hepatocytes with female hormones. (2) Define the mechanisms by which estradiol regulates the expression of UGT1A4 and -1A9. Electrophoretic mobility shift assay, DNase I protection, and mutation assays will be performed. (3) Identify estradiol- or progesterone-responsive genes in the liver. Microarray experiments (Affymetrix Human Genome U133 plus 2.0 GeneChip array) will be performed using RNA samples prepared from human hepatocytes after hormone treatment. PUBLIC HEALTH RELEVANCE: Medication use by pregnant women is common, but drug behaviors in this population are generally different from those in non-pregnant women or men. Understanding of these changes is important in determination of optimal dosing regimen. We propose to investigate mechanisms of such changes in drug behaviors. The knowledge obtained from this study can be expanded to optimize drug therapy in other groups of women, such as oral contraceptive users, thus benefiting women in general.