Regulation of genes involved in hepatic drug elimination by female hormones

描述（由申请人提供）：在怀孕期间，孕妇最好避免使用药物，以保护发育中的胎儿免受任何潜在的不良影响。但孕妇易患的既往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，因为暴露不足或过度暴露不仅会对母亲而且会对胎儿造成有害的临床结果。孕妇的临床研究表明，许多药物的药代动力学特征在怀孕期间发生改变。一般来说，药物的口服吸收延迟，药物的分布和肾排泄增加。另一方面，肝脏药物代谢已被认为在妊娠期间以代谢途径依赖的方式发生变化。然而，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，除了其他因素的变化，如药物蛋白结合或肝脏血流量的变化。这需要一个体外系统，其中肝脏药物代谢的变化可以专门检查。妊娠期伴随的生理变化可能是导致妊娠期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雌性激素的分泌急剧增加。激素升高可能是妊娠期肝脏药物代谢改变的原因。在怀孕期间达到高浓度时，雌性激素的功能不同于其传统的性腺激素作用。孕酮结合并激活转录调节因子妊娠异种受体（PXR），诱导主要药物代谢酶的表达，包括细胞色素P450酶和UDP-葡萄糖醛酸转移酶（UGTs）。此外，雌激素通过雌激素受体-?介导UGT1A4和- 1a9在HepG2细胞中的表达。(嗯?)在此基础上，我们提出验证妊娠期间雌激素和孕激素水平升高，从而全面调节肝脏药物代谢酶的表达，导致肝脏药物消除改变的假设。据此设计了以下具体目标。(1)确定雌激素和孕激素对肝脏药物代谢的酶特异性作用。通过观察女性激素对人肝细胞处理后，各种探针药物在人肝细胞中的肝脏消除率的变化，探讨激素调节的功能意义。(2)明确雌二醇调控UGT1A4和-1A9表达的机制。电泳迁移转移测定，dna酶I保护和突变测定将被执行。(3)鉴定肝脏中雌二醇或黄体酮应答基因。微阵列实验（Affymetrix Human Genome U133 + 2.0 GeneChip阵列）将使用激素治疗后的人肝细胞制备的RNA样品进行。公共卫生相关性：孕妇使用药物是常见的，但这一人群的药物行为通常与非孕妇或男性不同。了解这些变化对确定最佳给药方案很重要。我们建议研究这种药物行为改变的机制。从这项研究中获得的知识可以扩展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.