Screening Method for the Identification of Compounds That Activate Pregnane X Receptor.

The pregnane X receptor (PXR) is a nuclear receptor found mainly in the liver and intestine, whose main function is to regulate the expression of drug-metabolizing enzymes and transporters. Recently, it has been noted that PXR plays critical roles in energy homeostasis, immune response, and cancer. Therefore, identifying chemicals or compounds that can modulate PXR is of great interest, as these can result in downstream toxicity or, alternatively, may have therapeutic potential. Testing one compound at a time for PXR activity would be inefficient and take thousands of hours for large compound libraries. Here, we describe a high-throughput screening method that encompasses plating and treating HepG2-CYP3A4-hPXR cells in a 1536-well plate, as well as reading and interpreting assay (e.g., luciferase reporter gene activity) endpoints. These cells are stably transfected with a human PXR expression vector and CYP3A4-promoter-driven luciferase reporter vector, allowing the identification of compounds that activate PXR through cytochrome 450 3A4. We also describe how to analyze the data from each assay and explain follow-up steps, namely pharmacological characterization and quantitative polymerase chain reaction (qPCR) assays, which can be performed to confirm results from the original screen. These methods can be used to identify and confirm hPXR activators after completion of a compound screening. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput assay to identify hPXR activators Basic Protocol 2: Quantitative high-throughput screening a compound library to classify hPXR activators Basic Protocol 3: Performing pharmacological characterization and qPCR assays to confirm hPXR activators.

Targeting CAR and Nrf2 improves cyclophosphamide bioactivation while reducing doxorubicin-induced cardiotoxicity in triple-negative breast cancer treatment.

Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC), although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular coculture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show 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. Furthermore, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as potentially novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy/toxicity ratio of CPA/DOX-containing chemotherapy.

Cell-Based Assays to Identify ERR and ERR/PGC Modulators.

The estrogen-related receptor alpha (ERRα, NR3B1) is an orphan nuclear receptor which plays a role in endocrine disruption, energy homeostasis, and cancer prognosis. One of the unique features of this transcription factor is the interplay with its cofactors. For instance, certain modulators require the presence of proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) alongside ERRα. Therefore, identification of ERRα agonists and antagonists require examination of this nuclear receptor alone and together with PGC-1α. In this book chapter, we describe the step-by-step protocol of a multiplex luciferase assay designed to identify ERRα agonists, antagonists, and toxicity in one quantitative high-throughput screening assay using two different stable cell lines.

Characterization of human pregnane X receptor activators identified from a screening of the Tox21 compound library.

The pregnane X receptor (PXR; NR1I2) is an important nuclear receptor whose main function is to regulate enzymes within drug metabolism. The main drug metabolizing enzyme regulated by PXR, cytochrome P450 (CYP) 3A4, accounts for the metabolism of nearly 50% of all marketed drugs. Recently, PXR has also been identified as playing a role in energy homeostasis, immune response, and cancer. Due to its interaction with these important roles, alongside its drug-drug interaction function, it is imperative to identify compounds which can modulate PXR. In this study, we screened the Tox21 10,000 compound collection to identify hPXR agonists using a stable hPXR-Luc HepG2 cell line. A pharmacological study in the presence of a PXR antagonist was performed to confirm the activity of the chosen potential hPXR agonists in the same cells. Finally, metabolically competent cell lines - HepaRG and HepaRG-PXR-Knockout (KO) - were used to further confirm the potential PXR activators. We identified a group of structural clusters and singleton compounds which included potentially novel hPXR agonists. Of the 21 selected compounds, 11 potential PXR activators significantly induced CYP3A4 mRNA expression in HepaRG cells. All of these compounds lost their induction when treating HepaRG-PXR-KO cells, confirming their PXR activation. Etomidoline presented as a potentially selective agonist of PXR. In conclusion, the current study has identified 11 compounds as potentially novel or not well-characterized PXR activators. These compounds should further be studied for their potential effects on drug metabolism and drug-drug interactions due to the immense implications of being a PXR agonist.

Human constitutive androstane receptor agonist DL5016: A novel sensitizer for cyclophosphamide-based chemotherapies.

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC50 value of 0.66 µM and EMAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.

Identification of Compounds That Inhibit Estrogen-Related Receptor Alpha Signaling Using High-Throughput Screening Assays.

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.

Identification of Modulators That Activate the Constitutive Androstane Receptor From the Tox21 10K Compound Library.

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor involved in all phases of drug metabolism and disposition. However, recently it's been implicated in energy metabolism, tumor progression, and cancer therapy as well. It is, therefore, important to identify compounds that induce human CAR (hCAR) activation to predict drug-drug interactions and potential therapeutic usage. In this study, we screen the Tox21 10,000 compound collection to characterize hCAR activators. A potential novel structural cluster of compounds was identified, which included nitazoxanide and tenonitrozole, whereas known structural clusters, such as flavones and prazoles, were also detected. Four compounds, neticonazole, diphenamid, phenothrin, and rimcazole, have been identified as novel hCAR activators, one of which, rimcazole, shows potential selectivity toward hCAR over its sister receptor, the pregnane X receptor (PXR). All 4 compounds translocated hCAR from the cytoplasm into the nucleus demonstrating the first step to CAR activation. Profiling these compounds as hCAR activators would enable an estimation of drug-drug interactions, as well as identify prospective therapeutically beneficial drugs.

Identification of Estrogen-Related Receptor α Agonists in the Tox21 Compound Library.

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the ß-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

Outcome differences in adolescent blunt severe polytrauma patients managed at pediatric versus adult trauma centers.

BACKGROUND: Previous research suggests adolescent trauma patients can be managed equally effectively at pediatric and adult trauma centers. We sought to determine whether this association would be upheld for adolescent severe polytrauma patients. We hypothesized that no difference in adjusted outcomes would be observed between pediatric trauma centers (PTCs) and adult trauma centers (ATCs) for this population. METHODS: All severely injured adolescent (aged 12-17 years) polytrauma patients were extracted from the Pennsylvania Trauma Outcomes Study database from 2003 to 2015. Polytrauma was defined as an Abbreviated Injury Scale (AIS) score ≥3 for two or more AIS-defined body regions. Dead on arrival, transfer, and penetrating trauma patients were excluded from analysis. ATC were defined as adult-only centers, whereas standalone pediatric hospitals and adult centers with pediatric affiliation were considered PTC. Multilevel mixed-effects logistic regression models assessed the adjusted impact of center type on mortality and total complications while controlling for age, shock index, Injury Severity Score, Glasgow Coma Scale motor score, trauma center level, case volume, and injury year. A generalized linear mixed model characterized functional status at discharge (FSD) while controlling for the same variables. RESULTS: A total of 1,606 patients met inclusion criteria (PTC: 868 [54.1%]; ATC: 738 [45.9%]), 139 (8.66%) of which died in-hospital. No significant difference in mortality (adjusted odds ratio [AOR]: 1.10, 95% CI 0.54-2.24; p = 0.794; area under the receiver operating characteristic: 0.89) was observed between designations in adjusted analysis; however, FSD (AOR: 0.38, 95% CI 0.15-0.97; p = 0.043) was found to be lower and total complication trends higher (AOR: 1.78, 95% CI 0.98-3.32; p = 0.058) at PTC for adolescent polytrauma patients. CONCLUSION: Contrary to existing literature on adolescent trauma patients, our results suggest patients aged 12-17 presenting with polytrauma may experience improved overall outcomes when managed at adult compared to pediatric trauma centers. LEVEL OF EVIDENCE: Epidemiologic study, level III.

A Preliminary Analysis of Compassion Satisfaction and Compassion Fatigue With Considerations for Nursing Unit Specialization and Demographic Factors.

Compassion fatigue (CF), or vicarious traumatization, is a state of physical/emotional distress that results from caring for those experiencing pain. We sought to characterize levels of CF in intensive care unit (ICU) and oncology nursing populations with subanalyses comparing specific personal/professional demographic factors. The Professional Quality of Life (ProQOL) scale, a validated tool for assessing CF, burnout (BO), and compassion satisfaction (CS), was distributed to the ICU and oncology divisions of a community hospital. Demographic data and ProQOL scale scores were collected and compared within specialty and gender subgroups. Two-sample t tests and regression analyses were used to compare groups. Statistical significance was defined as p < .05. A total of 86 nurses submitted completed surveys able to be analyzed. Levels of CS were significantly lower (p = .023) and levels of BO were significantly higher (p = .029) in ICU nurses than in oncology nurses. Male nurses exhibited significantly higher CS (p = .001) and significantly lower BO (p = .021) and CF (p = .014) than female nurses. Intensive care unit nurses and female nurses from both ICU and oncology specialties may be at increased risk for developing a poorer overall ProQOL and CF.

Identifying environmental chemicals as agonists of the androgen receptor by using a quantitative high-throughput screening platform.

The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy's disease and Klinefelter's syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR ß-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation.

Big children or little adults? A statewide analysis of adolescent isolated severe traumatic brain injury outcomes at pediatric versus adult trauma centers.

BACKGROUND: The appropriate managing center for adolescent trauma patients is debated. We sought to determine whether outcome differences existed for adolescent severe traumatic brain injury (sTBI) patients treated at pediatric versus adult trauma centers. We hypothesized that no difference in mortality, functional status at discharge (FSD), or overall complication rate would be observed between center types. METHODS: All adolescent trauma patients (aged 15-17 years) presenting with isolated sTBI (head Abbreviated Injury Scale [AIS] score ≥3; all other AIS body region scores ≤2) to accredited Levels I to II trauma centers in Pennsylvania from 2003 to 2015 were extracted from the Pennsylvania Trauma Outcome Study database. Dead on arrival, transfer, and penetrating trauma patients were excluded from analysis. Adult trauma centers were defined as non-pediatirc (PED) (n = 24), whereas standalone pediatric hospitals and adult centers with pediatric affiliation were considered Pediatric (n = 9). Multilevel mixed effects logistic regression models and a generalized linear mixed models assessed the adjusted impact of center type on mortality, overall complications, and FSD. Significance was defined as a p value less than 0.05. RESULTS: A total of 1,109 isolated sTBI patients aged 15 to 17 years presented over the 13-year study period (non-PED, 685; PED, 424). In adjusted analysis controlling for age, shock index, head AIS, Glasgow Coma Scale motor, trauma center level of managing facility, case volume of managing facility, and injury year, no significant difference in mortality (adjusted odds ratio, 0.82; 95% confidence interval [CI], 0.23-2.86; p = 0.754), FSD (coefficient, -0.85; 95% CI, -2.03 to 0.28; p = 0.136), or total complication rate (adjusted odds ratio, 1.21; 95% CI, 0.43-3.39; p = 0.714) was observed between center types. CONCLUSION: Although the optimal treatment facility for adolescent patients is frequently debated, patients aged 15 to 17 years presenting with isolated sTBI may experience similar outcomes when managed at pediatric and adult trauma centers. LEVEL OF EVIDENCE: Epidemiologic study, level III; therapeutic study, level IV.

Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.

Quantitative high-throughput identification of drugs as modulators of human constitutive androstane receptor.

The constitutive androstane receptor (CAR, NR1I3) plays a key role in governing the transcription of numerous hepatic genes that involve xenobiotic metabolism/clearance, energy homeostasis, and cell proliferation. Thus, identification of novel human CAR (hCAR) modulators may not only enhance early prediction of drug-drug interactions but also offer potentially novel therapeutics for diseases such as metabolic disorders and cancer. In this study, we have generated a double stable cell line expressing both hCAR and a CYP2B6-driven luciferase reporter for quantitative high-throughput screening (qHTS) of hCAR modulators. Approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection were screened employing both the activation and deactivation modes of the qHTS. Activators (115) and deactivators (152) of hCAR were identified from the primary qHTS, among which 10 agonists and 10 antagonists were further validated in the physiologically relevant human primary hepatocytes for compound-mediated hCAR nuclear translocation and target gene expression. Collectively, our results reveal that hCAR modulators can be efficiently identified through this newly established qHTS assay. Profiling drug collections for hCAR activity would facilitate the prediction of metabolism-based drug-drug interactions, and may lead to the identification of potential novel therapeutics.

Identification of novel activators of constitutive androstane receptor from FDA-approved drugs by integrated computational and biological approaches.

PURPOSE: The constitutive androstane receptor (CAR, NR1I3) is a xenobiotic sensor governing the transcription of numerous hepatic genes associated with drug metabolism and clearance. Recent evidence suggests that CAR also modulates energy homeostasis and cancer development. Thus, identification of novel human (h) CAR activators is of both clinical importance and scientific interest. METHODS: Docking and ligand-based structure-activity models were used for virtual screening of a database containing over 2000 FDA-approved drugs. Identified lead compounds were evaluated in cell-based reporter assays to determine hCAR activation. Potential activators were further tested in human primary hepatocytes (HPHs) for the expression of the prototypical hCAR target gene CYP2B6. RESULTS: Nineteen lead compounds with optimal modeling parameters were selected for biological evaluation. Seven of the 19 leads exhibited moderate to potent activation of hCAR. Five out of the seven compounds translocated hCAR from the cytoplasm to the nucleus of HPHs in a concentration-dependent manner. These compounds also induce the expression of CYP2B6 in HPHs with rank-order of efficacies closely resembling that of hCAR activation. CONCLUSION: These results indicate that our strategically integrated approaches are effective in the identification of novel hCAR modulators, which may function as valuable research tools or potential therapeutic molecules.

Regulation of the human hydroxysteroid sulfotransferase (SULT2A1) by RORα and RORγ and its potential relevance to human liver diseases.

The retinoid-related orphan receptors (RORs) were postulated to have functions in tissue development and circadian rhythm. In this study, we revealed a novel function of RORα (NR1F1) and RORγ (NR1F3) in regulating the human hydroxysteroid sulfotransferase (SULT2A1), a phase II conjugating enzyme known to sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, and related androgens. A combination of promoter reporter gene assay and EMSA and chromatin immunoprecipitation (ChIP) assays showed that both RORα and RORγ transactivated the SULT2A1 gene promoter through their binding to a ROR response element found in the SULT2A1 gene promoter. Interestingly, this ROR response element overlaps with a previously reported constitutive androstane receptor response element on the same promoter. Down-regulation of RORα and/or RORγ by small interfering RNA inhibited the expression of endogenous SULT2A1. In primary human hepatocytes and human livers, we found a positive correlation between the expression of SULT2A1 and RORs, which further supported the regulation of SULT2A1 by RORs. We also found that the expression of RORα and RORγ was impaired in several liver disease conditions, such as steatosis/steatohepatitis, fibrosis, and hepatocellular carcinoma. The positive regulation of human SULT2A1 by RORs is opposite to the negative regulation of Sult2a1 by RORs in rodents. In summary, our results established SULT2A1 as a novel ROR target gene. The expression of RORs is a potential predictor for the expression of SULT2A1 as well as disease conditions.

A novel xenobiotic responsive element regulated by aryl hydrocarbon receptor is involved in the induction of BCRP/ABCG2 in LS174T cells.

Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17ß-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR-responsive element (AhRE5) located -2357/-2333bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug-drug interactions.

Effects of commonly used excipients on the expression of CYP3A4 in colon and liver cells.

PURPOSE: The objective of this investigation was to assess whether common pharmaceutical excipients regulate the expression of drug-metabolizing enzymes in human colon and liver cells. METHODS: Nineteen commonly used excipients were evaluated using a panel of experiments including cell-based human PXR activation assays, real-time RT-PCR assays for CYP3A4 mRNA expression, and immunoblot analysis of CYP3A4 protein expression in immortalized human liver cells (HepG2 and Fa2N4), human primary hepatocytes, and the intestinal LS174T cell models. RESULTS: No excipient activated human PXR or practically induced CYP3A4. However, three excipients (polysorbate 80, pregelatinized starch, and hydroxypropyl methylcellulose) tended to decrease mRNA and protein expression across experimental models. CONCLUSION: This study represents the first investigation of the potential role of excipients in the expression of drug-metabolizing enzymes. Findings imply that some excipients may hold potential for excipient-drug interactions by repression of CYP3A4 expression.