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    Induction of Drug Metabolism and Transport Genes and Activation of Human Pregnane X Receptor by Tamoxifen

    Scientific Abstract:
    Title: Induction of drug metabolism and transport genes and activation of human Pregnane X Receptor by Tamoxifen Background: Tamoxifen is an important therapeutic agent in the treatment and chemoprevention of breast cancers. Since it acts as an anti-estrogen in breast cancer tissue but as an estrogen receptor agonist in uterine tissue it is considered to be a selective estrogen receptor modulator (SERM). While tamoxifen is clinically very effective, its use is associated with several serious problems. These include drug-drug interactions, increased risk of endometrial cancer and development of drug resistance. Tamoxifen metabolism may be a contributive factor in these events. Tamoxifen metabolites a-hydroxytamoxifen and 4-hydroxytamoxifen upon further activation can form DNA adducts: a causative factor implicated in endometrial carcinogenesis. Cytochrome P450 (CYP) 3A4 isozyme is an enzyme of central importance to tamoxifen metabolism and is highly inducible. CYP3A4 induction has significant implications for tamoxifen-mediated drug-drug interactions and adverse effects. It is often co-expressed with MDR1 gene that encodes P-glycoprotein, an efflux transporter associated with multi drug resistance as well as drug interactions. The molecular mechanisms underlying pharmacokinetic drug interactions of tamoxifen with other drugs (e.g. aromatase inhibitors letrozole and anastrazole) are yet unknown. In recently completed studies we made two novel findings. First, tamoxifen and 4-hydroxytamoxifen markedly induced CYP3A4 in primary human hepatocytes but not in colon cancer cell line LS174T, a model system useful in assessing intestinal induction. Second, these two agents also activated human Pregnane X Receptor (hPXR), a ligand dependent transcriptional regulator of CYP3A4, in cell-based reporter assays hinting that CYP3A4 induction may be a result of hPXR activation by tamoxifen. However, mechanisms underlying tissue specific induction still remain poorly understood. Further, it is unclear if these agents also induce other hPXR target genes like MDR1 and UDP glucuronysyl transferases (UGTs) which may also contribute to drug-drug interactions. This knowledge gap represents a significant barrier to optimizing the use of tamoxifen and to the development of the next generation of SERMS. Our long-term goal is to gain a comprehensive understanding of the molecular mechanisms that underlie the modulation of drug metabolizing enzymes and drug transport proteins by tamoxifen and related compounds. Hypothesis: The objective of this proposal is to test the hypothesis that tamoxifen and 4-hydroxytamoxifen serve as ligands for hPXR. Further, we postulate that these anti-estrogens induce MDR1 and UGTs. Specific aims: 1) To identify the role of nuclear receptors in the induction of CYP3A4 and MDR1 by tamoxifen and 4-hydroxytamoxifen and 2) To determine the extent to which these agents induce MDR1 and UGT genes in primary human hepatocytes and LS174T human colon carcinoma cells. Study design: Nuclear receptor activation will be examined employing cell based reporter gene assays. Transgenic humanized PXR mice will be treated with anti-estrogens and liver and intestinal tissue will be processed to assess induction. MDR1 and UGT activation will be evaluated by examining enzymatic activity, immunoreactive protein and mRNA levels. Potential benefits: We anticipate that these studies will advance our understanding of the mechanisms that underlie adverse drug-drug interactions and side effects associated with tamoxifen use. This will ultimately be useful in optimizing tamoxifen-containing dosing regimens in order to enhance their clinical efficacy while reducing the toxicities. Additionally, the knowledge acquired may help in the development of next generation of SERMs.

    Lay Abstract:
    Title: Induction of drug metabolism and transport genes and activation of human Pregnane X Receptor by Tamoxifen Breast cancer affects thousands of women each year and is the second most common cause of death in women. Tamoxifen is regarded as one of the most useful drugs in treatment of breast cancer and is an essential part of adjuvant therapy. Furthermore, tamoxifen is now also used for prevention of this disease in women who are considered to be at high risk of developing breast cancer. However, there are several problems associated with the use of tamoxifen. Firstly, when used with other drugs (including other anti-breast cancer drugs like letrozole and anastrazole), tamoxifen has the potential to reduce their effectiveness. When co-administered with letrozole, which belongs to the class of aromatase inhibitors, tamoxifen reduces its plasma levels by over 33%. Secondly, in women who take tamoxifen for a long period of time (which is usually the case) there is an increased risk of developing endometrial cancer. Thirdly, over time tamoxifen loses its effectiveness in those individuals who initially respond to the drug (development of resistance). The exact mechanisms underlying these events are unknown. The major hypothesis of our work is that tamoxifen and related compounds induce (increase) the levels of key metabolism and transporter enzymes in the liver and the small intestine called cytochrome P4503A4 (CYP3A4), MDR1 which encodes for multi-drug resistance protein P-glycoprotein and phase II metabolizing enzymes UDP glucuronyl transferases (UGTs). CYP3A4 is of central importance as it is responsible for metabolism of tamoxifen as well as over 50% of other clinically used drugs. The levels of these enzymes are regulated by a nuclear receptor called Pregnane X Receptor. Induction of CYP3A4, MDR1 and UGTs may contribute to the above-mentioned problems associated with tamoxifen use. This induction may lead to increased elimination of co-administered drugs. Also, increased levels of CYP3A4 may lead to increased formation of certain tamoxifen metabolites, which are linked to endometrial cancers associated with long term tamoxifen use. The goal of our research is to gain a deeper understanding of the events at molecular level that finally result in the increased production of these enzymes. We will employ a battery of tests to assess the increase in enzyme levels using freshly cultured human liver cells (to assess increase in enzymes in liver) and colon cancer cells (to assess increase in enzymes in intestine). Liver and intestine are the tissues of interest since these are the two most important sites of metabolism. The potential of tamoxifen to activate PXR will be determined. We will also employ “humanized” mice in which the mouse homolog of receptor PXR, (which regulates the formation of CYP3A4, MDR1 and UGTs) is replaced by human PXR, to mimic the events occurring in humans. This model is important and necessary to determine the in vivo relevance of this study because mouse PXR and human PXR do not recognize tamoxifen in identical manner. The livers and intestines of these mice will be processed to test the role of hPXR. We believe that this understanding will ultimately help us in refining the use of tamoxifen so that we can eliminate/reduce the adverse drug-drug interactions and the adverse side effects associated with the use of tamoxifen. Also, the knowledge acquired during this project can be employed to improve the clinical effectiveness while reducing toxicity of the next generation of compounds being developed for use in breast cancer therapy.