Now showing items 21-40 of 262

    • 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.
    • Evaluation of In Vitro/In Vivo Correlations for Transdermal Delivery Systems by In Vitro Permeation Testing and Human Pharmacokinetic Studies, With and Without a Transient Heat Application

      Shin, Soo Hyeon; Stinchcomb, Audra L.; Hassan, Hazem; 0000-0001-5091-8870 (2018)
      An in vitro model that exhibits in vitro/in vivo correlations (IVIVC) is a powerful tool in biopharmaceutical drug development because it can efficiently predict drug product performance in vivo. While the concept of IVIVC has been utilized mostly for oral dosage forms, demonstrations of IVIVC with in vitro permeation testing (IVPT) for transdermal delivery systems (TDS) are emerging. The objective of this work was to evaluate IVIVC for TDS using two model drugs, nicotine and fentanyl, with different physicochemical characteristics (e.g. log P). Additionally, the effect of heat exposure (42 ± 2 °C) on the rate and extent of TDS drug delivery was evaluated. IVPT studies using excised human skin and in vivo pharmacokinetic (PK) studies in human subjects were conducted under harmonized study conditions and designs to evaluate IVIVC. The correlations were evaluated in multiple ways, including a single point comparison of parameters such as steady-state concentration and heat-induced increase in partial AUCs, as well as a point-to-point correlation (Level A IVIVC). Level A IVIVC was examined using multiple approaches. A strong IVIVC was consistently observed for nicotine TDS in presence and absence of heat, suggesting the utility of IVPT as a tool to evaluate and predict in vivo performance of nicotine TDS. The IVIVC results for fentanyl were relatively weaker, especially when IVIVC for heat effects were examined, with greater in vivo heat effects observed compared to the in vitro heat effects. A separate study evaluating IVIVC for fentanyl TDS without a heat exposure component and utilization of some PK parameters obtained directly from study subjects yielded improved IVIVC results. The findings from the present research work suggest that IVPT data generally shows good predictability of in vivo performance of TDS at normal temperature conditions. However, the usefulness of IVPT for assessing and predicting external factors such as heat, especially for lipophilic drug molecules, may have some limitations that could be further improved.
    • Budgetary Impact of TRICARE Antidiabetic Drug Formulary Changes

      Hung, Anna; Mullins, C. Daniel (2018)
      Background: There is guidance on how to conduct budget impact analyses (BIA) from the International Society of Pharmacoeconomics and Outcomes Research (ISPOR). However, there is a growing need to ensure that budget impact models are valid, accurate, and usable to payers. In 2016, the Defense Health Agency implemented antidiabetic formulary changes to the TRICARE pharmacy benefit. The goal of this dissertation is to predict and validate the budget impact of these formulary changes. Objective: The specific objectives of this study were to: i) estimate the annual financial consequences of antidiabetic formulary changes from the TRICARE payer perspective using TRICARE claims data over three years; ii) assess the face validity, internal validity, and predictive validity of the model; and iii) identify and compare cost drivers of both the budget and the budget impact identified through the model versus the empirical analysis. Methods: Following the ISPOR BIA guidance, a budget impact model was created in Microsoft Excel®. The counterfactual was predicted using autoregressive integrated moving average models. One year after the formulary changes, actual utilization was used to determine the realized budget and compare this to what was predicted in the budget impact model. Cost drivers were determined through one-way sensitivity analyses, subgroup analyses, and the comparison of utilization versus price in the model versus the empirical analysis. Results: In the year after the formulary changes, the model predicted a lower budget impact than what was realized ($24 million in savings versus $49 million in savings). Meanwhile, the model predicted a higher annual budget than what was realized ($686 million versus $609 million). The higher-than-predicted savings was largely due to lower utilization seen in the empirical analysis compared to the model. The antidiabetic drug classes that contributed most to these savings were the dipeptidyl peptidase-4 inhibitors, glucagon-like receptor-1 agonists, and sodium-glucose cotransporter 2 inhibitors. Conclusion: Future budget impact models should be validated by waiting at least one year and comparing model predictions to what is realized. The end user of the model should also be involved in the process of creating the model.
    • Mechanisms of PrrF-Mediated Iron regulation in Pseudomonas aeruginosa

      Djapgne, Louise; Oglesby, Amanda G. (2018)
      Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that infects people with compromised immune systems. P. aeruginosa is highly resistant to multiple antibiotics, in part due to its ability to organize into robust biofilm communities. Iron is required for P. aeruginosa virulence and biofilm formation, but iron can also be toxic to the bacteria. Therefore, iron acquisition and utilization are tightly regulated in response to iron availability. Iron homeostasis is maintained in part by the production of two small RNAs (sRNAs), PrrF1 and PrrF2, which repress the expression of iron containing proteins in iron-depleted conditions. PrrF1 and PrrF2 are encoded in tandem on the P. aeruginosa genome, allowing the expression of a longer heme-regulated sRNA named PrrH. Due to the unique sequence of this sRNA, PrrH is hypothesized to regulate distinct mRNAs involved in heme metabolism. Our lab previously showed that the locus for the PrrF and PrrH sRNAs is required for virulence in an acute murine lung infection model, yet the mechanisms guiding PrrF and PrrH regulation of virulence have not yet been determined. In this work, we identified Hfq, an RNA-binding protein that stabilizes many bacterial sRNAs, as a potential PrrF-and PrrH-binding protein. Using gel-shift assays, I showed that Hfq has a strong binding affinity for PrrF1 and PrrF2. Moreover, I showed that Hfq increases the annealing rate of PrrF with one of its mRNA targets, antR. I identified nucleotides required for PrrF interaction with antR in vitro and in vivo, and I showed that PrrF1 and PrrF2 regulation of antR is redundant. PrrF regulation of antR was previously shown to promote the production of key virulence-related metabolites, including the Pseudomonas quinolone signal (PQS). Accordingly, we showed that the PrrF1 and PrrF2 sRNAs have redundant function in the production of PQS and structurally related metabolites. I further showed that Hfq binds to the PrrH sRNA in vitro, and I performed preliminary in vitro analysis of the PrrF sRNAs with additional mRNA targets involved in iron metabolism and biofilm formation. Lastly, I demonstrated that iron and PrrF allow for increased P. aeruginosa biofilm formation in the presence of certain antibiotics. Altogether, these studies established the mechanisms of PrrF regulation of a key virulence trait, and they provided the basis for future work into the biochemical and genetic basis of PrrF-mediated virulence in P. aeruginosa.
    • Applications of Quantitative Proteomics and Phosphoproteomics to Study the Development of Resistance to Targeted Therapy in Cancer

      Awasthi, Shivangi; Shapiro, Paul, Ph.D.; Goodlett, David Robinson, 1960-; Guha, Udayan (2018)
      Targeted inhibition of protein kinases is a major approach to treat cancer. However, the effectiveness of kinase inhibitors is limited due to intrinsic and acquired resistance mechanisms that promote the progression and survival of cancer cells. The objective of this dissertation is to use liquid chromatography coupled to mass spectrometry (LC MS) based quantitative proteomics to identify potential biomarkers of resistance and response to molecularly targeted therapies in cutaneous melanoma and lung adenocarcinoma in vitro. For the first part of this thesis, I conducted a proteomic analysis of the acquired drug resistance to extracellular signal-regulated kinase (ERK1/2) pathway inhibitors in a melanoma cell line model. A combination of immunoblot assays, global label-free bottom-up proteomics, phosphoproteomics and pathway analysis was used to characterize the differential protein expression in drug resistant melanoma cells. Examination of the quantitative data pointed to an invasive and metastatic phenotypic signature in the resistant cells. We also identified and verified the overexpression of β-catenin and Caveolin-1 (CAV-1) in MEK1/2 and ERK1/2 inhibitor resistant cells. These findings suggest that these proteins have a role in the development of resistance and may represent novel targets for co-therapy. For the second part of this thesis, I have utilized a multiple reaction monitoring (MRM) based targeted proteomic technique to verify previously identified potential biomarkers of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) response in lung adenocarcinoma. Published global phosphoproteomic data were used to select a list of phosphotyrosine peptides (pY) and MRM based relative and absolute quantitative methods were developed to measure their expression in TKI sensitive and resistant lung adenocarcinoma cells. Modified immuno-MRM assays were optimized using heavy labelled synthetic peptide standards which identified the targets with good reproducibility and repeatability. The results indicated that of the 11 chosen sites, EGFR-pY1197 can be used as potential biomarker of EGFR TKI sensitivity, regardless of the EGFR TKI used. Overall these data advance our understanding of the mechanisms of targeted therapy resistance and highlight candidate biomarkers of resistance and sensitivity.
    • Learn and Apply Paradigm to Optimize Pharmacotherapy in Neonatal Abstinence Syndrome Using Pharmacometrics

      Liu, Tao; Gobburu, Jogarao; 0000-0002-9943-2131 (2017)
      Every one hour a baby with neonatal abstinence syndrome (NAS) is born in the United States. NAS is a clinical syndrome of opiate withdrawal in infants exposed to drugs either prenatally in the form of maternal use (non-iatrogenic), or postnatally in the form of medical therapy (iatrogenic). The syndrome is comprised of a combination of central nervous system, digestive system and autonomic system abnormalities that result from uninhibited excitatory neurotransmitter release from the neurons. Between 2000 and 2009, a 3-fold increase in the use of opiate drugs among pregnant women led to an increase in NAS and associated higher health care costs. Currently, morphine is the first line pharmacotherapy for NAS. Pharmaceutical companies have no incentive to invest in therapy optimization for NAS, and the current dosing strategies vary from hospital to hospital. This research is based on a virtual consortium between the Center for Translational Medicine at the University of Maryland Baltimore, Johns Hopkins Medical Institute and Thomas Jefferson University Hospital. The purpose of this research is to optimize the morphine dosing strategy in NAS patients who require pharmacotherapy by using a pharmacometrics approach with the "learn and apply" philosophy. First, a comprehensive morphine pharmacokinetic model that accounts for prognostic factors, such as body size and age, was developed in neonates. The results suggested a uridine diphosphate glucuronic acid dependent morphine clearance during the first week of life. Second, an exposure-response (ER) relationship between morphine plasma concentrations and modified Finnegan scores was built, and the model prediction was evaluated for the primary and secondary clinical outcomes, such time-on-treatment and total morphine dose. Lastly, different morphine dosing strategies were simulated based on the ER relationship and then optimized dosing strategies were proposed. The proposed dosing strategies will minimize suffering due to the withdrawal symptoms and ultimately lead to an earlier hospital discharge.
    • Exploring Protein Dynamics and Folding with Constant pH Molecular Dynamics Simulations

      Yue, Zhi; Shen, Jana; 0000-0002-4231-7474 (2017)
      Solution acidity or pH is a key environmental regulator with profound impact on biological processes such as protein dynamics and functions. Over the past decade, our research group has developed a set of tools to explicitly account for solution pH in molecular dynamics simulations. In this dissertation we demonstrate the new application areas of the constant-pH molecular dynamics tools. First we use constant-pH MD alongside fixed-protonation-state simulations to explore the dynamics of a cytoplasmic heme-binding protein (PhuS) in Pseudomonas aeruginosa, a notorious opportunistic pathogen. Our results provide atomic-level information on how heme binding affects PhuS dynamics which suggests an induced-fit mechanism, in support of the recent hydrogen-deuterium exchange data. Secondly, we apply constant-pH MD to investigate the acid-induced unfolding of BBL, a small component of the ubiquitous pyruvate dehydrogenase multienzyme complex involved in carbohydrate metabolism inside mitochondria. Our data support that BBL is a barrier-limited two-state folder, an issue that has drawn intense debate among experimental groups. More importantly, our simulations reveal that acid-induced unfolding of BBL is triggered by sequential protonation of His166 and Asp162 and thereby offering atomic details unattainable via experimental means. This work is the first time the constant-pH MD in explicit solvent has been applied to protein folding studies. Next, we employ the membrane-enabled constant-pH MD to understand how proton release drives the conformational transition of the transmembrane multi-drug efflux pump AcrB, which is crucial for the intrinsic resistance of E. coli to clinically important antibiotics. Our data address the controversy regarding the proton/drug stoichiometry and reveal the details of how deprotonation of a single residue leads to a global conformational transition in AcrB. This work paves the way for understanding the complete cycle of drug transport in AcrB and validates the membrane-enabled constant-pH MD technique for mechanistic studies of proton-coupled transporters. Lastly, we benchmark the accuracy of the all-atom constant-pH MD with charge-leveling co-ion using titration simulations of five proteins (HP36, BBL, NTL9, HEWL and SNase). The average and maximum absolute errors between the calculated and experimental pKa values based on 10-ns pH-based replica-exchange simulations are 0.7 and 0.9 units, respectively. Detailed analyses indicate that limited sampling is a major source of error. This work demonstrates the all-atom constant-pH MD method a practical tool for accurate prediction of pKa's and atomically detailed studies of proton-dependent conformational dynamics. In summary, my studies offered new mechanistic insights into the various roles of protons in protein dynamics and folding that were previously not well understood. My work further established constant-pH MD as a powerful tool for revealing atomic details of proton-coupled dynamic processes.
    • Molecular Mechanisms of Osteocyte Mechanotransduction

      Lyons, James Stephen; Stains, Joseph P. (2017)
      Diseases of skeletal fragility affect >200 million people worldwide and contribute to ~9 million factures annually. Preventing bone loss and/or restoring lost bone mass is of vital importance to limiting the personal and economic impact of these diseases. The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely through regulating the secretion of sclerostin, an inhibitor of bone formation. Osteocytes sense mechanical load in the form of fluid shear stress (FSS), and respond by reducing expression of sclerostin leading to "de-repression" of osteoblastogenesis and stimulation of de novo bone formation. However, key mechanistic details of how osteocytes sense mechanical load, transduce these signals to biologic effectors, the identity of these effectors and how sclerostin bioavailability is regulated remain unclear. A widely accepted technique for mechanically stimulating cells in culture is the introduction of FSS on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to FSS. We validated the device using the biologic response of UMR-106 cells in comparison to a commercially available system of FSS. Utilizing this FSS device we show that the microtubule (MT) network plays a critical role in how osteocytes sense and respond to FSS. We define a microtubule-dependent mechanotransduction pathway that links FSS to the generation of react ROS and Ca2+ signals, leading to reductions in sclerostin in osteocytes. In Ocy454 osteocyte-like cells, we demonstrate that an intact MT network is required for FSS-induced Ca2+-influx, calcium calmodulin-dependent protein kinase II phosphorylation, and reduction in sclerostin. Further, the abundance of detyrosinated Glu-tubulin dictates the cytoskeletal stiffness of these cells. By tuning the abundance of Glu-tubulin/cytoskeletal stiffness, we demonstrate that Glu-tubulin regulates the mechano-responsive range at which FSS elicits a Ca2+ response in osteocytes. Further, we determined that the FSS-induced reduction in sclerostin requires activation of a signaling cascade that includes production of Nox2-activated ROS, which stimulates Ca2+-influx through the cation-permeable channel TRPV4 and the subsequent activation of CamKII. By developing a better understanding of this fundamental aspect of skeletal physiology, we will raise the possibility of outlining new drug targets to combat diseases of skeletal fragility.
    • Novel Lipid A Structures for Adjuvant Discovery and Development

      Gregg, Kelsey; Ernst, Robert K.; 0000-0002-1483-735X (2017)
      There is an urgent need to develop effective immunizations against infectious diseases that continue to be a major cause of morbidity and mortality worldwide. Modern subunit vaccines require immunopotentiators or adjuvants to strengthen the protective immune response. Adjuvants in human licensed vaccines include aluminum hydroxide, a TH2-biased adjuvant, and monophosphoryl lipid A (MPLA), a TH1-biased, non-toxic Toll-like receptor 4 (TLR4) agonist. MPLA is chemically derived from Salmonella minnesota R595 lipopolysaccharide and displays reduced endotoxicity. Phosphorylated hexacyl disaccharide (PHAD) is a similar, but synthetically produced, monophosphoryl lipid A adjuvant. We hypothesized that functionally diverse lipid A adjuvant structures could be rationally designed by reprogramming the lipid A biosynthetic pathway by adding or removing lipid A modifying enzymes in a process called bacterial enzymatic combinatorial chemistry (BECC). BECC lipid A molecules were engineered in an avirulent Yersinia pestis strain, purified, and screened in immortalized cell lines for TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) responses. BECC-derived structures exhibit dose-dependent TLR4-driven NF-?B activation with both human and murine TLR4 signaling complexes. Structures that displayed reduced NF-?B activity were screened to identify BECC molecules that induced cytokine secretion patterns similar to PHAD in murine splenocytes and human primary blood mononuclear cells (PBMCs), and induced similar activation markers on human monocyte-derived dendritic cells (DCs). Selected BECC molecules were tested in vivo for their ability to adjuvant the recombinant fusion protein, rF1-V, in a murine subunit vaccination model against Y. pestis, the causative agent of plague. BECC-adjuvanted vaccines elicited strong rF1-V-specific antibody titers in C57BL/6J mice with increased titers of the TH1-associated immunoglobulin, IgG2c. The BECC adjuvant groups' titers were similar to or higher than the aluminum hydroxide and PHAD control adjuvant groups. The BECC-adjuvanted and control adjuvant vaccines were protective against a lethal, intraperitoneal Y. pestis CO92- challenge, and were more protective than the unadjuvanted rF1-V vaccine control. These data demonstrate that BECC can be used to generate functionally diverse TLR4 ligands with potential for use as TH1-biasing vaccine adjuvants.
    • Evaluation of the Use of Hydroxypropyl methylcellulose acetate succinate (HPMCAS) in Enteric Coating of Tablets and Solubility Enhancement of BCS Class II Compounds

      Deshpande, Tanvi Mahesh; Hoag, Stephen W. (2017)
      Hydroxypropyl methylcellulose acetate succinate (HPMCAS), an anionic polymer, demonstrates wide applicability in drug delivery. The goal of this dissertation is twofold: Applicability of HPMCAS in aqueous enteric coating of tablets and in solubility enhancement of BCS Class II compounds. HPMCAS used for aqueous enteric coating has a tendency to aggregate and clog the spray-nozzle during the coating process. This limitation motivated us to design and develop a stable aqueous enteric coating formulation that minimizes aggregation and spray-nozzle clogging, and maintains the enteric coating profile of tablets. Results show that elevated processing temperature activates polymer particle coalescence in plasticized dispersions, forming large aggregates that clog the spray-nozzle. We successfully developed a stable formulation containing PEG 4000, sodium lauryl sulfate (SLS), and Aerosil® R972 Pharma that formed hydrogen bonds with HPMCAS (steric repulsion), provided electrostatic stabilization, and provided hydrophobicity to the aqueous coating respectively, which in turn prevented nozzle clogging and maintained the tablet's enteric profile. The second application of HPMCAS focused on spray dried dispersions (SDDs), which is one of the most successful solubility enhancement techniques for BCS Class II compounds (e.g. Itraconazole (ITZ), a model drug for this study). Selection of suitable drug carriers (polymers) was found to be crucial for solubility enhancement and for ensuring the stability of ITZ. Incorporation of surfactants with polymers could further enhance ITZ solubility by micellar solubilization mechanism. The project goal was to develop a screening methodology for selecting polymer/surfactant combinations for enhancing solubility and kinetic stability of ITZ spray-dried dispersions (SDD). 1H NMR and fluorescence spectroscopy aided in screening polymer/surfactant combinations by determining critical micelle concentration (CMC) of the system. Observations indicated that stronger surfactant/polymer interactions (inferred by the decrease in CMC) show limited solubility and kinetic stability enhancement (due to their unavailability to interact with ITZ). Further screening of excipients for SDD preparation was performed based on the supersaturation ratio and the precipitation induction time of ITZ in the presence of different surfactant/polymer combinations. ITZ/HPMCAS-HF SDD was found to be most efficient since it enhanced and maintained the solubility of ITZ by 60-fold for up to 24 h in simulated intestinal fluid.
    • Molecular Insight into the Structure, Function, and Regulation of Bile Acid Transport

      Czuba, Lindsay Christine; Swaan, Peter W.; 0000-0001-9142-5706 (2017)
      The human Apical Sodium-dependent Bile Acid Transporter (SLC10A2), also known as hASBT, plays an integral role in the enterohepatic circulation of bile acid and cholesterol homeostasis. As a member of the solute carrier family of membrane transporters, it uses the established Na+ electrochemical gradient as an energy source to reclaim bile salts from the ileum. hASBT has been identified as a promising target for the management of hypercholesterolemia, cholestatic pruritis, and as a prodrug-targeting approach for improved bioavailability of drugs. Limiting the development of such therapeutics, is an incomplete understanding of hASBT's structure. Extensive biochemical and mutagenesis studies for hASBT support a seven transmembrane model. Yet conflicting structures have emerged with the elucidation of the crystal structures of two putative homologues from Neisseria meningitidis (nmAsbt) and Yersinia frederiksenii (yfAsbt). In the absence of a physiological context, the use of their structure as models of the human transporter is limited. In addition to the discrepancy in hASBT's fold, there is limited information regarding the specific proteoforms that are relevant to the functional expression of hASBT and in its regulation. In this work we provide novel molecular insight into the structure, function, and regulation of human ASBT. We contrasted the biochemical, inhibitory, and evolutionary attributes of nmAsbt, yfAsbt, and hASBT and identified their critical differences. The fundamental differences in ion dependency, substrate specificity, and evolutionary context imply divergent structure-function relationships and negate the use of the bacterial transporters as suitable models for hASBT. Additionally, we characterized the role of tyrosine phosphorylation in regulating the functional expression and stability of hASBT. We identified Src family kinases as critical modulators and provide support for hASBT's regulation by phosphatases. As the clinical relevance of PTMs is growing, so too are the number of FDA-approved therapeutics that target these modifications. In this regard, we have made critical advances and gained valuable insight into hASBT's regulation. Finally, we have optimized the biological sample preparation methods and have significantly increased the purity of hASBT samples. When coupled with mass spectrometry analysis, these methods will identify critical proteoforms of hASBT and facilitate a global understanding of its structure-function relationship.
    • Mechanistic Evaluation of Polyethylene Oxide for Physical Barrier Type Abuse Deterrent Formulations: Techniques and In vitro methods

      Boyce, Heather J.; Hoag, Stephen W.; 0000-0001-9817-7784 (2017)
      Abuse deterrent formulations (ADF) are designed to mitigate misuse and abuse of prescription narcotics. One mechanism of an ADF is to increase the tablet strength to reduce ease of tablet comminution while increasing the difficulty of drug extraction. Polyethylene oxide (PEO) is a popular polymer used in these complex opioid products. PEO provides these abuse resistance properties while providing extended release of the opioid drug. Evaluation of the manufacturing process and material attributes of this polymer is important to improve upon the next generation of abuse deterrent products. In addition, methods to assess abuse deterrence of these products in vitro is challenging, but important for product development and generic abuse deterrent product approvals. Thus, this dissertation seeks to determine the best evaluation methods for these products and establishes an in vitro method to assess a formulation's ability to prolong nasal absorption when nasally insufflated. Key results of this study show that heating tablets with PEO will significantly increase the strength of the tablet and key variables such as PEO composition, particle size distribution, high initial tablet solid fraction, and 1 h sinter time were found to be the optimal sintering conditions. It was also demonstrated that this sintering process could be monitored by near infrared spectroscopy to predict sintering end points. Finally, the in vitro nasal insufflation studies demonstrated that for each type of physical manipulation employed (i.e. cutting, grinding, milling), discreet particle size distributions were formed regardless of formulation. One exception occurred with milling where one drug product resulted in a much greater particle size distribution than the other milled tablets. A vertical diffusion cell was then used to assess release rate of drug from the comminuted dosage form. It was demonstrated that the VDC method was discriminant with respect to particle sizes of comminuted particles and formulation variables such as molecular weight of PEO used.
    • Towards Targeted Anti-Neoplastics: The Disruption of Aberrant Protein-Protein Interactions with Low Molecular-Weight Proteomimetics

      Lanning, Maryanna Elizabeth; Fletcher, Steven; 0000-0002-9511-4435 (2017)
      Protein-protein interactions (PPIs) play pivotal roles in a range of cellular processes including proliferation, differentiation, metabolism and apoptosis. Dysregulations of certain PPIs can lead to the development and progression of human cancers. In particular, the overexpression of the anti-apoptotic BCL-2 family members, specifically Mcl-1, have been linked to pancreatic, colorectal and lung cancers as well as leukemia and lymphoma. When over-expressed, MCL-1 prevents cell death by binding and sequestering the BH3 "death" domain of its pro-apoptotic counterpart, such as Bim. Mcl-1 has become an important target for the development of novel antineoplastics. As with many, helix-mediated PPIs, several key residues are hydrophobic and located on one face of the BH3 α-helix, specifically at the i, i + 3/4, i + 7 residues. In addition to exhibiting a hydrophobic face, Asp67 on the "other" face of the Bim-BH3 helix forms a salt bridge with the protein (Arg263). In an improved effort to develop more potent and more selective agents to disrupt the MCL-1-BH3 PPI, we used structure-based design, and developed two complementary strategies: synthetic α-helix (purine based) and BH3 mimetics (naphthoate based). Both scaffolds yielded molecules that disrupted the MCL-1-BIM PPI consistently, and subsequent studies were undertaken towards second-generation molecules. The designed molecules were subjected to biological assays and further structure-activity relationship (SAR) studies to increase affinity and potency in an effort to translate in vitro activity to on target cell activity. It is expected anticipated that the SAR developed in the present study will facilitate the development of novel therapeutics capable of inhibiting Mcl-1 will be identified, which can be advanced to preclinical evaluation.
    • 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.
    • In-vitro Efficacy and Intracellular Mechanism of Riboflavin-Conjugated PEGylated Poly- L-Lysine Dendrimer

      Pak, Yewon; Swaan, Peter W. (2017)
      Chemotherapeutic drugs have advanced using different drug delivery methods to treat breast cancer specifically. This development has arisen because many classical drugs exhibit physicochemical limitations including solubility, specificity, stability, biodistribution, and therapeutic efficacy. There were numerous adverse effects associated with these limitations because chemotherapeutic drugs enter normal tissues. In order to eliminate off-target side-effect,nanoparticles were developed to target anticancer drugs to a specific carcinogenic area. As one of developing nanomedicines, dendrimers possess ability to be utilized in different administration routes and has potential to stay in the blood circulation longer while showing increased accumulation in tumor cells. Commercially available poly (amidoamine) (PAMAM) dendrimers have the potential to cause toxicity in vivo due to lack of biodegradation at sites of accumulation. Poly-L-Lysine (PLL) dendrimers are an alternative class of dendrimers that possess a biodegradable structure. PEGylated poly-l-lysine (PLL) dendrimers are known to be more favorable due to lessened cytotoxicity manifested by masking of cationic charges and avoiding uptake by Reticulo Endothelial System (RES). Using this biodegradable dendrimer, we sought to examine the effect of PEGylation as well as delivering anti-cancer drug, Doxorubicin (DOX), to a targeted internalization pathway in human breast cancer cells effectively. PEGylated PLL dendrimers also have their limitation, in which some tumor cells are not dependent upon enhanced permeability and retention (EPR) effect. As a result, riboflavin receptor, which is found to be upregulated in the exterior of breast and ovarian cancer cells, was utilized by attaching a riboflavin ligand to PEGylated PLL dendrimers in order to be actively uptaken by breast cancer cells. To target chemotherapeutic drug selectively and efficaciously, riboflavin conjugated PLL dendrimers were assessed in-vitro by investigating cytotoxicity, uptake accumulation, and intracellular colocalization. Further investigation on the endocytosis mechanism and detailed intracellular trafficking in different compartments of the cells were analyzed in order to fully understand the machinery behind delivering chemotherapeutic drugs successfully.
    • A Tale of Two Zinc Fingers: Structure and Functional Studies of CCCH type Zinc Finger Proteins CPSF30 and TTP involved in RNA Regulation

      Shimberg, Geoffrey Daniel; Michel, Sarah L. J. (2017)
      CPSF30 and TTP are non-classical zinc finger proteins (ZFS) that contain domains with a CCCH motif. CPSF30 has 5 CCCH domains and TTP has 2 CCCH domains. Both proteins are involved in RNA regulation; CPSF30 regulates pre-mRNA and TTP regulates mRNA; however, only TTP has been shown to directly bind to RNA (via its CCCH domain, targeting AU-rich sites). Given the sequence similarity between TTP and CPSF30, we hypothesized that CPSF30 directly binds AU-rich RNA sequences via its CCCH domains. To test this hypothesis, a construct of CPSF30 containing the five CCCH domains, was over-expressed and purified. Unexpectedly, CPSF30 was reddish in color, suggesting iron coordination. UV-visible, ICP-MS analysis and XAS spectroscopy revealed that the protein contains a 2Fe-2S cluster in addition to four zinc domains. The 2Fe-2S cluster utilizes a CCCH ligand set, and is the second example of this site in biology! RNA binding studies, using EMSA and fluorescence anisotropy (FA), with ?-synuclein AU-rich pre-mRNA as a target, were then performed. From these studies, we determined that (1) CPSF30 binds directly to AU-rich targets on pre-mRNA via a cooperative binding mechanism and (2) CPSF30 requires both iron and zinc coordination for RNA binding. Studies focused on Cu(I) binding to TTP will also be presented. Cu(I) is toxic in excess and there is emerging evidence that ZF sites may be target of Cu(I) toxicity. Using UV-visible and circular dichroism spectroscopies, we have determined that 3 Cu(I) ions bind to TTP and that Cu(I) binding inhibits the structure of the protein. In addition, RNA binding studies, using FA with the TNF-? AU-rich mRNA revealed that Cu(I) inhibits the TTP-RNA interaction. We propose that inhibition of TTP function by Cu(I) contributes to its mechanism of toxicity.
    • Cardiometabolic Safety of Atypical Antipsychotic Medications among Publicly Insured U.S. Youth

      Burcu, Mehmet; Zito, Julie Magno; 0000-0003-4572-0987 (2017)
      Background: The use of atypical antipsychotics (AAPs) among publicly insured U.S. youth has substantially increased in the past two decades. Furthermore, more than half of AAP-treated youth have concomitant antidepressant or stimulant use, although the cardiometabolic effects of such combinations are largely unknown. Methods: The main focus of this dissertation was to evaluate the risk of incident type 2 diabetes mellitus (T2DM) and adverse cardiovascular events in AAP-treated youth according to the concomitant use of stimulants or serotonin reuptake inhibitors (SSRI/SNRIs)-the leading antidepressant subclass. The risk of T2DM and adverse cardiovascular events were assessed using discrete time failure models. To adjust for confounding, disease risk score methodology was employed using >125 baseline and time-dependent covariates. Medication use was assessed using four time-varying exposure measures: current/former/non-use, duration of use, cumulative dose, and average daily dose. Results: In a large regionally diverse cohort of Medicaid-insured youth, AAP use was associated with an increased risk of T2DM that increased with duration of AAP use and cumulative AAP dose. Further, in AAP-treated youth, concomitant SSRI/SNRI use was associated with an additional increased risk of T2DM, which intensified with duration of SSRI/SRNI use and SSRI/SNRI dose. In a separate set of analyses that focused on youth who initiated antidepressant treatment (regardless of AAP use), an increased risk of T2DM was also observed for SSRI/SNRIs. Finally, following treatment initiation with AAPs, current AAP use was also associated with an increased risk of incident cardiovascular events that led to hospitalizations or emergency department visits. This increased risk also intensified with increasing AAP dose and when SSRI/SNRIs were used concomitantly with AAPs. By contrast, in AAP-treated youth, concomitant use of stimulants was not associated with an increased risk of T2DM or cardiovascular events. Conclusions: In view of the growing complexity of atypical antipsychotic regimens in Medicaid-insured youth and low rates of baseline metabolic monitoring in youth initiating AAP treatment, these findings suggest that complex AAP regimens should be used judiciously with appropriate cardiometabolic monitoring. Continued efforts are warranted to support Medicaid oversight policies that assure safe and effective use of complex AAP regimens in youth populations.
    • Tribological Characterization of Pharmaceutical Formulations

      Lee, Hyun Joo; Hoag, Stephen W. (2017)
      Oral sensory perception has been extensively studied in the food and pharmaceutical industry. With the growing importance of pediatric formulations, there has been much interest in the texture of medications to increase patient acceptance. Texture is one of the first interactions during ingestion and key to our psychological impression of a product. Mouthfeel of medications is a major component for patient compliance as much as taste and aesthetics. We aim to develop the classical tribological method to understand the dynamics of our testing formulations consisting of viscosity and grittiness. Rheology and tribology are used to understand the texture of oral medications and their interaction with surfaces. The presence of particles greatly influenced the outcome of viscosity and coefficient of friction. Particles increased friction between surfaces at slower sliding speeds, but may also contribute to reduce rough surfaces and increase lubrication and coating during higher sliding speeds in the hydrodynamic regime.
    • Learn & Apply Paradigm to Inform Drug Development & Optimize Clinical Therapeutics in Oncology

      Mehrotra, Shailly; Gobburu, Jogarao (2017)
      Application of learn-apply paradigm in drug development and clinical therapeutics increases efficiency and supports decision making. The current research highlights the role of pharmacometrics to inform trial design and propose individualized management of chemotherapy induced peripheral neuropathy (CIPN) in oncology. The first project focuses on learning from early clinical trial of veliparib to inform future investigations. Population pharmacokinetics and exposure-response analyses were conducted to evaluate the contribution of intrinsic and extrinsic factors on veliparib PK, and assess the adequacy of veliparib dosing for the future trial. A 28% increase in AUC with mild renal impairment increases mucositis by only 7%, thus supporting the inclusion of patients with mild renal impairment in future trials without the need of dose adjustment. Exposure-response for efficacy (objective response rate and overall survival) and safety (mucositis) along with in vitro IC50 information supported 80 mg BID dose for veliparib. Multivariate exposure-response analysis provided supportive evidence to further evaluate veliparib in patients with myeloproliferative neoplasms and with 14 day treatment duration. The second project proposes a novel strategy based on precision therapeutics for the management of CIPN in clinical setting. An indirect response model with linear drug effect was able to describe the longitudinal-CIPN data reasonably well for paclitaxel, nab-paclitaxel and ixabepilone. The model was utilized to identify an early time point of 3 months that predicted later time course of CIPN (concordance probability ~ 75%). Utilizing the dose-CIPN model, a novel strategy to use patients own early CIPN data to predict their future CIPN time course was proposed. 'CIPN management dosing card' and 'CIPN precision therapeutics tool' were developed to prospectively manage CIPN in patients who may be at risk of developing CIPN later in the therapy. For paclitaxel, nab-paclitaxel and ixabepilone, the proposed CIPN management dosing card resulted in 61%, 48% and 35% fewer patients with CIPN after 6 cycles as compared to administering cycle 3 doses for 4th, 5th and 6th chemotherapy cycle. With CIPN precision therapeutics tool, oncologists can visualize the predicted CIPN time course and tailor the dosing to manage CIPN in an individual patient based on overall benefit/risk.
    • Impact of Prescription Drug Monitoring Program Implementation and Rigor on Prescription Opioid Utilization in Medicare

      Moyo, Patience; Simoni-Wastila, Linda; 0000-0003-1323-4554 (2017)
      Background: Prescription drug monitoring programs (PDMPs) are central to the federal and state policy responses to address prescription drug abuse. PDMPs are state-run electronic databases used to track the prescribing and dispensing of controlled prescription medications. Despite their prominence, there is limited and mixed evidence of PDMP effectiveness, particularly among vulnerable populations. This study aimed to evaluate the influence of PDMP implementation and program rigor on prescription opioid utilization among disabled and older adults. Methods: A retrospective study using 2007-2012 Medicare claims and PDMP state laws from the Prescription Drug Abuse Policy System was designed to quantify associations between PDMP status or rigor and state- and individual-level opioid utilization (opioid volume, days supplied, daily morphine equivalents, number of prescriptions, daily dose ≥120mg), accounting for sociodemographic characteristics and state controlled substance laws. A PDMP composite score was developed from the total number of best practices adopted by each state (range: 0-14), classifying states according to the median score ("high PDMP rigor" and "low PDMP rigor"). Generalized linear, negative binomial, and modified Poisson regression models adjusting for clustering were applied. Results: From 2007-2012, the number of states operating PDMPs rose from 27 to 44. PDMP implementation was associated with reduced opioid volume (-2.36kg/month, 95% CI -3.44, -1.28) compared to non-PDMP states. Observed reductions were stronger in disabled adults than older adults. Annual prescription rates per 10,000 opioid-users were lower in states with low PDMP rigor (-578 [95% CI: -1006, -151]) or high rigor (-687 [95% CI: -1081, -293]) than non-PDMP states. At the individual level, PDMPs of any rigor were associated with decreased opioid utilization. There was no significant evidence that estimated associations between states with low and high rigor PDMPs were different. Conclusions: Findings suggest PDMP rigor has limited impact on individual-level opioid utilization among Medicare beneficiaries. Further studies are needed to elucidate which PDMP characteristics add value rather than adding operating cost and effort with little return.