Relevant Degree Programs
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G+PS regularly provides virtual sessions that focus on admission requirements and procedures and tips how to improve your application.
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are important anti-retroviral drugs indicated in combination therapy of human immunodeficiency virus-1 (HIV-1) infection. NNRTI therapy is associated with pharmacokinetic drug interactions, the underlying mechanisms of which are poorly understood. The present study investigated the effects of NNRTIs on the activity of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), key transcriptional factors regulating the expression of various drug-metabolizing enzymes and transporters. The experimental approaches included cell-based luciferase reporter gene assays, in vitro competitive ligand binding assay, nuclear translocation analysis by confocal imaging, coactivator recruitment assays, and target gene expression determination in primary human hepatocytes. Rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, were identified as agonists of PXR and inducers of cytochrome P450 3A4 (CYP3A4), a target gene of PXR. By comparison, rilpivirine, etravirine, and efavirenz, but not nevirapine or delavirdine, were indirect activators of the wild-type isoform of CAR and inducers of cytochrome P450 2B6, a target gene of CAR. Among the NNRTIs investigated, only efavirenz activated the SV23 and SV24 splice variants of CAR, indicating that NNRTIs activated CAR in a drug-specific and isoform-selective manner. To further understand PXR regulation by rilpivirine, the role of microRNA in rilpivirine activation of PXR was investigated. MicroRNAs are small non-coding RNAs that are post-transcriptional regulators causing mRNA degradation or translational repression by binding to complementary regions in target mRNA. Bioinformatic analysis (www.microRNA.org) predicted sequence complementarity between hsa-miR-18a-5p and PXR. Reporter gene assays revealed a functional hsa-miR-18a-5p microRNA recognition element in the 3′-untranslated region of PXR. In cell-based assays, over-expression of hsa-miR-18a-5p by transfecting LS180 human colon adenocarcinoma cells with a mimic of hsa-miR-18a-5p decreased PXR mRNA and protein expression. Rilpivirine and rifampin did not affect PXR expression, but it decreased endogenous expression of hsa-miR-18a-5p in LS180 cells. In contrast, over-expression of hsa-miR-18a-5p decreased PXR mRNA expression and CYP3A4 inducibility by rilpivirine and rifampin. These data suggest that hsa-miR-18a-5p regulates PXR and contributes to drug activation of PXR. Overall, the present study shows activation of PXR and CAR by select NNRTIs and provides mechanistic understanding of NNRTI-mediated drug-drug interactions.
The rising numbers of poorly water soluble drugs found in the pipelines of the pharmaceutical industry elicit the need for the development of enabling formulations. Lipid-based drug delivery systems (LbDDS) are a versatile group of formulations including self-nanoemulsifying drug delivery systems (SNEDDS). The advantage of using LbDDS, such as SNEDDS, for poorly water soluble drugs is that typically the oral bioavailability is increased compared to traditional solid dosage forms. In order to examine the effect of lipid digestion on absorption of poorly water soluble drugs from SNEDDS the lipase inhibitor orlistat (tetrahydrolipstatin) was used. Halofantrine and fenofibrate were chosen as model drugs and for neither halofantrine nor fenofibrate digestion appeared to have an effect on the oral bioavailability in rats.The administration of halofantrine and fenofibrate in super-SNEDDS led to increasedbioavailability compared to SNEDDS when evaluated in a rat model in vivo. For both halofantrine and fenofibrate there was a significant increase of Cmax for the super-SNEDDS compared to the SNEDDS. For halofantrine no significant increase was seen in the bioavailability for the super-SNEDDS however, for fenofibrate the AUC of the super-SNEDDS was significantly larger than for the SNEDDS.The dynamic in vitro lipolysis model is typically used in the evaluation of digestibility and solubilization capacity of SNEDDS and other LbDDS. The traditional interpretation of data from the lipolysis model is that the solubilized fraction of drug is available for absorption, whereas the precipitated fraction needs to be re-dissolved in order to be absorbed which is believed to lower the bioavailability. However, for super-SNEDDS precipitation seems to beinversely correlated with oral absorption when comparing the in vitro data with the in vivo data. In order to examine precipitation of poorly water soluble drugs dosed in LbDDS invivo the gastric and intestinal content of rats was examined for precipitated crystalline drug using XRPD and PLM. The study revealed that precipitation onlywas evident in the stomach, thus precipitation in the intestinal in vitro modelmay be an artifact and may require the addition of an in vitro gastric digestion step to be predictive in vivo.
Valproic acid (VPA) is a widely prescribed broad-spectrum antiepileptic drug. Clinical use of VPA is associated with a rare, but possibly fatal, idiosyncratic hepatotoxicity. The mechanism of VPA hepatotoxicity is not known, but it may involve reactive metabolites of VPA. Using a sandwich-cultured rat hepatocyte model, the present work investigated the toxicity of two specific VPA metabolites, (E)-2,4-diene-VPA and valproyl-1-O-β acyl glucuronide (VPA-G), and their role in the hepatocyte toxicity of VPA. The overall experimental strategy was to modulate the in situ formation of these two metabolites and determine the consequences on VPA toxicity in sandwich-cultured rat hepatocytes. VPA toxicity was assessed by markers such as 2′,7′-dichlorofluorescein formation (oxidative stress), BODIPY 558/568 C12 accumulation (steatosis), lactate dehydrogenase release (necrosis), and cellular content of total glutathione (antioxidant status). (E)-2-ene-VPA, which is a β-oxidation metabolite of VPA, was also included in the experiments concerning (E)-2,4-diene-VPA, as it formed relatively large amounts of (E)-2,4-diene-VPA. Based on the modulatory experiments with phenobarbital and 1-aminobenzotriazole, in situ generated (E)-2,4-diene-VPA did not appear to contribute to VPA toxicity in sandwich-cultured rat hepatocytes. However, the results from (E)-2-ene-VPA experiments indicated the toxic potential of in situ generated (E)-2,4-diene-VPA in sandwich-cultured rat hepatocytes, when generated at high concentrations. As part of this study, a sensitive and rapid ultra-high performance liquid chromatography – tandem mass spectrometry method for the quantification of VPA-G in hepatocyte culture medium was developed, validated, and applied successfully to quantify in situ concentrations of VPA-G. From a comprehensive screening of several known inducers of uridine 5'-diphospho-glucuronosyltransferase enzymes, this study identified β-naphthoflavone, L-sulforaphane, and phenobarbital to be effective in increasing the in situ formation of VPA-G from VPA in sandwich-cultured rat hepatocytes. According to the findings with β-naphthoflavone and borneol, in situ generated VPA-G did not appear to be toxic to sandwich-cultured rat hepatocytes and was unlikely to contribute to the hepatocyte toxicity of VPA. Overall, the results of the present study add value to the existing knowledge on the role of reactive metabolites of VPA in VPA hepatotoxicity. Future studies should investigate the role of VPA-CoA thioester formation on VPA toxicity in sandwich-cultured rat hepatocytes.
Valproic acid (VPA) therapy is associated with a rare but severe hepatotoxicity. The relationships between the various pathophysiological findings of VPA-induced hepatotoxicity and the role of VPA biotransformation in the induction of hepatotoxicity have not been systematically investigated. The present thesis compared the effects of VPA, synthesized VPA metabolites, and alpha-F-VPA on markers of mitochondrial dysfunction (WST-1), cytotoxicity (LDH), oxidative stress (DCF), and glutathione (GSH) depletion in a novel model of sandwich-cultured rat hepatocytes (SCRH). The contribution of the CYP- and UGT-mediated biotransformation of VPA in VPA-induced toxicity was also examined. Time-dependent effects of VPA on GSH depletion were characterized in relation to the effects of VPA on the WST-1, LDH, and DCF markers. The effects of glutathione supplementation on the attenuation of the markers for VPA-induced toxicities were investigated. Urine samples from children on VPA therapy were assayed to correlate levels of VPA metabolites with the lipid peroxidation marker, 15-F2t-isoprostane. Lastly, the contributions of hepatic CYP-enzymes in the oxidative metabolism of VPA were characterized in human liver microsomes. Our findings in SCRH indicated that (E)-2,4-diene-VPA was the only exogenously administered metabolite tested that was consistently more toxic than VPA. Consistent with this finding, alpha-F-VPA, which is resistant to bioactivation by several biotransformation pathways, was nontoxic. Chemical inhibition experiments indicated that the CYP- and UGT-mediated metabolism of VPA or the in situ generated VPA metabolites were unlikely involved in the observed VPA-induced toxicities in SCRH. Furthermore, VPA-associated GSH depletion appeared not to be a factor in the mitochondrial dysfunction, but may play a partial role in VPA-induced cytotoxicity. GSH may serve a protective role against VPA-induced oxidative stress in SCRH. In human subjects, the VPA-glucuronide or N-acetylcysteine metabolites were extremely weak but statistically significant predictors of lipid peroxidation in the urine of children receiving VPA. From the reaction phenotyping experiments, CYP2C9 was the major catalyst for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human liver microsomes, whereas CYP2A6 contributed partially to 3-OH-VPA formation. Overall, these findings add significant knowledge to the role of VPA and its metabolites in the induction of hepatotoxicity and how VPA is metabolized in humans.
Master's Student Supervision (2010 - 2018)
Relatively little is known about the expression, localization and regulation of rat testicular xenobiotic-metabolizing enzymes, including cytochrome P450s (CYP) and microsomal epoxide hydrolase (mEH), which are involved in the metabolism of xenobiotics including drugs and toxicants, and of endobiotics such as steroid hormones and prostaglandins. Suppression or induction of these enzymes in testis may alter the magnitude of tissue exposure to xenobiotics and endobiotics levels. In the present study, constitutive expression of various xenobiotic-metabolizing enzymes (Study 1) and their regulation by 17β-estradiol benzoate (EB) (Study 2) and an endocrine disrupting chemical, bisphenol A (BPA) (Study 3), were investigated in adult rat testis. As shown in Study 1, immunoblot analysis of testicular microsomes prepared from untreated rats revealed the presence of CYP1B1, CYP2A1, CYP17A1, NADPH-cytochrome P450 reductase (POR) and mEH, and absence of CYP1A1, CYP1A2, CYP2B1, CYP2E1, CYP2D1, CYP2D2, CYP2C6, CYP2C7, CYP2C11, CYP2C12, CYP2C13, CYP3A1, CYP3A2, CYP4A1, CYP4A2 and CYP4A3. Immunohistochemical analysis of tissue sections prepared from frozen testis indicated that CYP1B1, CYP2A1 and CYP17A1 were localized in interstitial cells, but not in seminiferous tubules, whereas mEH and POR were localized in both interstitial cells and in seminiferous tubules. In Study 2, subcutaneous (sc) treatment of EB at 0.004, 0.04, 0.4 or 4 μmol/kg once daily for 14 days suppressed testicular expression of CYP1B1, CYP2A1 and CYP17A1 at each of the dosages tested. EB also suppressed mEH and POR protein expression at dosages > 0.04 μmol/kg. In Study 3, administration of BPA at 400, 800 or 1600 μmol/kg sc once daily for 14 days decreased testicular CYP1B1, CYP2A1, CYP17A1, mEH and POR protein expression at each of the dosages tested. EB and BPA did not produce a general down-regulation of testicular protein expression because neither of these chemicals decreased calnexin protein (endoplasmic reticulum marker) levels. In summary, CYP1B1, CYP2A1, CYP17A1, mEH and POR were detected in rat testis and their expression was confined to interstitial cells (CYP1B1, CYP2A1, CYP17A1, mEH and POR) and seminiferous tubules (mEH and POR). Constitutive expression of these rat testicular enzymes was suppressed by exogenous administration of 17β-estradiol and BPA.