• Metabolism-based Alterations of Constitutive Androstane Receptor (CAR) Activity and Downstream Effects

      Mackowiak, Bryan; Wang, Hongbing, Ph.D. (2019)
      The xenobiotic defense network in the liver has evolved so that many foreign compounds can activate xenobiotic receptors like the constitutive androstane receptor (CAR) and the pregnane x receptor (PXR), induce the expression of drug metabolizing enzymes, and enhance the clearance of drugs. Typically, autoinduction of a compound’s metabolism leads to its breakdown, disrupting the detoxification feedback loop. However, multiple lines of evidence suggest that metabolites of autoinducers can have diverse effects on xenobiotic receptors, including agonism and antagonism conversion, and cause unexpected consequences, including drug-drug interactions (DDIs) that can lead to liver toxicity. While the effect of xenobiotic receptor-mediated CYP induction on drug metabolism has been well-characterized, the effect of metabolism on the activity of xenobiotic receptors has received little attention. Although the “traditional” role of CAR revolves around inducing xenobiotic metabolism and detoxification, evidence has accumulated that CAR also plays important roles in energy metabolism, cellular proliferation, and liver homeostasis, making it a potential drug target for various liver disorders. In addition, the effects of CAR activation in human primary hepatocytes (HPH) are not well understood and need further study to determine whether or not CAR is a potential drug target for different types of liver dysfunction, including cancer. The overall objectives of this proposal are to investigate the effect of drug metabolism on CAR activity, identify FDA-approved drugs and metabolites that alter CAR activity, and determine whether CAR activation is beneficial for liver disorders such as cancer. Using CAR as a model xenobiotic receptor, my studies have shown that potent CAR antagonist PK11195 is metabolized to a CAR agonist in metabolically-competent systems. Therefore, I hypothesize that hepatic metabolism capacity and CAR activity can form a regulatory feedback loop, altering the PK/PD profiles of drug substrates. Successful completion of these studies has provided a model for metabolism-based changes in xenobiotic receptor activity, identified FDA-approved drugs that modulate CAR activity, and determined the clinically-relevant downstream effects of CAR activation.
    • Novel human Constitutive Androstane Receptor Activators and their Effects on Hepatic Energy Homeostasis

      Lynch, Caitlin; Wang, Hongbing, Ph.D. (2014)
      The constitutive androstane receptor (CAR, NR1I3) serves as a ligand-activated transcription factor transforming chemical stimuli into cellular responses by regulating the expression of its target genes. CAR is well-recognized as a key mediator affecting drug metabolism and transport in response to a broad range of xenobiotics. It is predominantly expressed in the liver and intestine; where it regulates the inductive expression of phase I and II drug metabolizing enzyme target genes such as cytochrome P450 (CYP) 2B6, CYP3A4, sulfotransferases, and glutathione s-transferases, and drug transporters such as multidrug resistance protein 1 and organic anion-transporting polypeptides. Recent animal studies revealed that CAR also influences gluconeogenesis, lipogenesis, and fatty acid synthesis, and potentially ameliorates diabetes and obesity. Thus, discovery of compounds activating or deactivating CAR offers a promising avenue for the prediction of induction-mediated drug-drug interactions and a potential therapeutic strategy for metabolic diseases. Strategies incorporating computational based virtual screening with biological approaches were employed to identify and validate novel human (h) CAR modulators. Luciferase reporter assays were performed by transfecting various hCAR expression and CYP2B6 reporter plasmids into HepG2 cells. A stable cell line expressing both CYP2B6 luciferase and hCAR vectors was generated to identify novel hCAR activators and deactivators in a high-throughput manner. Human primary hepatocytes were utilized to explore the effects of CAR activation on target gene expression and hepatic energy homeostasis. Overall, this project revealed that a combined computational and biological approach is effective for identifying hCAR activators. The novel double stable cell line generated here offers a unique cell model in identification of both hCAR agonists and antagonists at high-throughput levels. The most distinctive finding obtained through this research however, was the species difference between hCAR and mouse (m) CAR energy metabolism. Activation of mCAR represses the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis, while activation of hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. These findings warrant caution with respect to the interpretation and extrapolation of results obtained from animal models to humans.
    • The Role of Bile Salt Export Pump (BSEP; ABCB11) Gene Repression in Drug-Induced Cholestatic Liver Injury

      Garzel, Brandy N.; Wang, Hongbing, Ph.D. (2017)
      Drug-induced liver injury (DILI) is a persistent matter in the pharmaceutical field, with extensive research being dedicated to understanding the causes, risk factors, and potential biomarkers associated with it. While DILI manifests in a variety of ways and diseases, the most severe form is drug-induced cholestasis. Bile acids are amphipathic molecules responsible for the extraction of lipids from the diet, and while necessary for function, their amphipathic nature can cause toxicity if allowed to accumulate in tissue. Constant flow of bile acids through biliary tissue is facilitated by specific bile transporters, with the bile salt export pump (BSEP; ABCB11) dictating excretion of bile acids from the liver to the gall bladder. Disruption of BSEP function is a major contributor to both inherited and acquired cholestasis. The predominant mechanism of drug-induced cholestasis is direct inhibition of BSEP, however in the following work, the contribution of BSEP repression on BSEP function was investigated. In the following studies, human primary hepatocytes (HPH) were used predominantly as a physiologically relevant system for studying drug transporter function and expression upon drug treatment. The functional transport assay employed was modified from the prototypical inhibition assays primarily used in cholestatic studies, and represents a novel approach to assess the cholestatic potential of BSEP repressors. Additional BSEP repressors were predicted from reported cases of drug-induced cholestasis combined with Bayesian modeling. It was determined from our study, that compared to inhibition alone, BSEP inhibition combined with repression correlated with severe clinical manifestations of DILI. Mechanistically, activation of AMPK was found to repress basal BSEP expression, while known transcriptional regulators FXR and Nrf2 had no effect. Most importantly, under conditions which cause BSEP repression, bile acid efflux was also significantly reduced. Metformin was used as a model drug in our studies, however, a number of drugs associated with cholestasis do not inhibit BSEP directly. Employing a strategy to elucidate additional cholestatic BSEP repressors, tamoxifen was found to potently repress BSEP expression, indicating the potential discovery of additional repressors. Overall, these results confirm BSEP repression represents an understudied mechanism for drug-induced cholestasis.
    • The Role of CAR and Nrf2 Dual Activation in doxorubicin/cyclophosphamide-based treatment of triple negative breast cancer

      Stern, Sydney; Wang, Hongbing, Ph.D.; 0000-0002-1479-605X (2022)
      Triple negative breast cancer (TNBC) affects 10-20% of all breast cancer cases and is associated with suboptimal outcomes due to drug resistance and/or intolerable side effects. The absence of targetable sites leaves cytotoxic chemotherapy the standard of care. Cyclophosphamide (CPA) and doxorubicin (DOX) are among the most used chemotherapeutic agents for TNBC. CPA, an alkylating prodrug, requires hepatic metabolic conversion to the rate-limiting metabolite, 4-hydroxy-cyclophosphamide (4-OH-CPA), primarily via cytochrome P450 (CYP) 2B6. Additionally, a portion of CPA is metabolized by CYP3A4 leading to a neurotoxic byproduct, chloroacetaldehyde, and an inactive byproduct, dechloroethyl-CPA. The constitutive androstane receptor (CAR, NR1I3), a nuclear receptor, regulates the expression of CYP2B6. Therefore, activation of CAR leads to preferential induction of CYP2B6 and subsequent bioactivation of CPA. On the other hand, DOX is often associated with dose-limiting cardiotoxicity. Mounting evidence suggests that this cardiotoxicity is in part attributed to the production of oxidative stress. It has been demonstrated that the activation of the nuclear factor erythroid 2-related factor (Nrf2) acts as a mediator in the protection against DOX-induced cardiotoxicity. Nrf2 regulates various antioxidant proteins and genes, such as heme-oxygenase 1 (HO-1), by binding to cis-acting antioxidant response elements in the promoter region of target genes, protecting against oxidative stress and inflammation. Our hypothesis was that dual activation of CAR and Nrf2 enhances the bioactivation of CPA while reducing DOX-mediated cardiotoxicity and improving the efficacy:toxicity ratio of CPA/DOX-based treatment for TNBC. CN06 was identified as a novel CAR and Nrf2 dual activator via high throughput screening and a chemical modification approach. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we demonstrated that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Overall, these results indicate CAR and Nrf2 as a promising therapeutic strategy to improve the therapeutic index of CPA/DOX in the treatment of TNBC.
    • The Role of Constitutive Androstane Receptor in the Bioactivation of Oxazaphosphorines

      Wang, Duan; Wang, Hongbing, Ph.D. (2013)
      Prodrugs are pharmaceutical substances that are administered in an inactive form and are subsequently converted to the active therapeutic moiety through bioactivation. Among them, oxazaphosphorines represent a major class of anti-cancer prodrugs, with cyclophosophamide (CPA) and ifosfamide (IFO) being the most widely used ones. Because of the increased polypharmacy in oxazaphosphorine-based chemotherapy, drug-drug interactions have been raising concerns. Hepatic CYP2B6 and CYP3A4 differentially contribute to the activation and inactivation of CPA and IFO and many clinically used drugs and environmental compounds can influence the expression of these enzymes. Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR)are important regulators of CYP2B6 and CYP3A4 expression. Activation of PXR induces both isozymes, while selective activation of CAR leads to preferential induction of CYP2B6 over CYP3A4 in human liver. Since CPA is predominantly bioactivated by CYP2B6 while deactivated through CYP3A4, we hypothesized that a combination of CPA and CAR activators may enhance the therapeutic effect of CPA by preferential induction of CYP2B6 over 3A4. The hypothesis was tested in an innovative hepatocyte-hematopoietic cell co-culture system that was demonstrated to be a useful in vitro model for studying the biotransformation and therapeutic effects of CPA and potentially other prodrugs in an environment that closely mimics in vivo conditions.Based on this system, CITCO was proved to preferentially induce CYP2B6 over CYP3A4 and subsequently enhance CPA anti-cancer activity. Similar trends were observed using rodent primary hepatocytes and corresponding CAR activators. Because CITCO is not clinically available, a panel of FDA approved drugs together with 800 Chinese herbal medical products were screened for preferential induction profile of CYP2B6 over CYP3A4. Matrine (Figure S1), the major component of two Chinese medical products was found to selectively induce CYP2B6 over CYP3A4 and enhanced CPA therapeutic effect in vitro in the co-culture system. The autoinduction profile and the underlying mechanism of CPA and IFO were also addressed. Both compounds induced CYP2B6 and CYP3A4 at mRNA and protein levels in human primary hepatocyte. While both drugs were proved to be PXR activators which indiscriminately induce CYP2B6 and CYP3A4, CPA but not IFO was demonstrated to be an indirect CAR activator.
    • The Role of the Constitutive Androstane Receptor in Cyclophosphamide-based Treatment of Lymphomas

      Hedrich, William Dominic; Wang, Hongbing, Ph.D.; Hassan, Hazem (2018)
      Cyclophosphamide (CPA) is an alkylating prodrug which has been utilized extensively in combination chemotherapies for the treatment of cancers and autoimmune disorders since its introduction to the market in the late 1950s. The metabolic conversion of CPA to its pharmacologically active metabolite 4-OH-CPA is catalyzed primarily by cytochrome P450 (CYP) 2B6. CPA is also subject to metabolism by CYP3A4 to an inactive metabolite, N-dechoroethyl-CPA (N-DCE-CPA), and a neurotoxic byproduct, chloroacetaldehyde. CPA is the backbone of the frontline chemotherapeutic regimen used for the treatment of non-Hodgkin lymphoma which combines the CHOP (CPA-doxorubicin-vincristine-prednisone) regimen with rituximab, a monoclonal CD20 antibody. The constitutive androstane receptor (CAR, NR1I3), an orphan nuclear receptor, is recognized as the key mediator of xenobiotic-induced expression of CYP2B6, Importantly, mounting evidence suggests that activation of hCAR leads to preferential induction of CYP2B6 over CYP3A4 which suggests that selective hCAR activation may enhance CPA bioactivation and enhance the efficacy:toxicity ratio of CHOP chemotherapy for NHL. CHOP chemotherapy has been associated with severe and cumulative cardiotoxicity arising from the doxorubicin component of the regimen and it is recommended that lymphoma patients with existing heart conditions avoid treatment with the full CHOP combination. Recently, it has been demonstrated that Nrf2 (nuclear factor (erythroid-derived 2))-like 2, NFE2L2) plays a key role in governing doxorubicin-induced cardiotoxicity. Nrf2 regulates the expression of important antioxidant genes and proteins which protect tissues from damage due to oxidative stress and inflammation. It has been shown both that insufficient Nrf2 expression results in hypersensitivity to doxorubicin cardiotoxicity and that stimulation of Nrf2 by small molecule activators can provide protection from doxorubicin-mediated toxicity. Our hypothesis was that hCAR activation will increase hepatic expression of CYP2B6 while having a negligible impact on other genes responsible for the disposition of CHOP drugs. Further, activation of Nrf2 in cardiac tissue may provide protection against cardiotoxicity induced by the doxorubicin component of CHOP. Together, these gene expression alterations will lead to augmented antineoplastic activity of CHOP in target lymphoma cells while alleviating the untoward cardiotoxicity associated with this regimen. This hypothesis was tested with a variety of methods including a novel hepatocyte-lymphoma-cardiomyocyte cell co-culture system as an in vitro model for studying the biotransformation of CPA and therapeutic effects of CHOP as well as the off-target toxicity in healthy tissues in an environment that closely resembles the in vivo condition. Using this system we successfully demonstrated that activation of hCAR with small molecule activators can significantly increase the anticancer activity of the CHOP regimen in lymphoma cells. Additionally, activation of Nrf2 in cardiomyocytes in co-culture significantly reduced the doxorubicin-induced cardiotoxicity of CHOP. Utilizing a hCAR-transgenic mouse model, we were able to show in vivo that the combination of a selective hCAR activator alongside CHOP significantly increases the anticancer activity of CHOP in a lymphoma tumor xenograft study. Taken together, these results implicate hCAR and Nrf2 as drug targets for facilitating CHOP-based treatment of lymphomas. We were able to identify several compounds from the NIH Chemical Genomics Center Pharmaceutical Collection which activate both hCAR and Nrf2 and have provided preliminary evidence for their utility in CHOP-based lymphoma treatment.