Now showing items 21-40 of 255

    • 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.
    • Systemic Lupus Erythematosus Patients' Benefit-Risks Preferences for Corticosteroid Use

      Ng, Xinyi; dosReis, Susan (2017)
      Introduction: Striking a balance between maximizing the effectiveness of corticosteroids while minimizing its adverse effects in Systemic Lupus Erythematosus (SLE) treatment requires careful consideration of how SLE patients value improvements in their disease versus risks of treatment adverse effects. Hence, this study aimed to elicit SLE patients' benefit-risks preferences for corticosteroid use, and to examine variation in preferences. Methods: SLE patients with current/past experience with using corticosteroids were recruited from the Johns Hopkins Lupus Center, the University of Maryland Medical Center and PatientsLikeMe. Study participants completed a web-based survey that comprised three sections: 1) sociodemographic characteristics, 2) a discrete choice experiment (DCE) measuring benefit-risks preferences, and 3) self-rated disease severity using the Systemic Lupus Activity Questionnaire (SLAQ). The DCE assessed two attributes of treatment benefit (time between flares and activities limitations), and four attributes of treatment risks relating to corticosteroids (heart-related damage, bone fractures, weight gain, and unusual mood swings/irritability). Preference weights were estimated using a conditional logit model and used to calculate the relative importance scores for each attribute and maximum acceptable risk that patients would tolerate. We also evaluated variations in preferences using a latent class analysis, and a stratified analysis based on SLAQ scores. Results: When making trade-offs between the risks and benefits of corticosteroid use, our 141 study participants (95% Female, 61% White) viewed weight gain as the most important, followed by heart-related damage and activities limitations. SLE patients were willing to tolerate higher risks for improvements in activities limitations (4.5% risk of heart damage) compared to improvements in time between flares (3.1% risk of heart damage). Our latent class analysis demonstrated the relative importance of three key attributes varied significantly across patient segments: activities limitations, weight gain, and heart damage. Individuals with SLAQ score ≤ 21 (N=75) were more willing to accept treatment risks in exchange for improvements in activity limitations compared to individuals with SLAQ score > 21 (N=66). Conclusions: Our study sheds light on SLE patients' benefit-risk trade-offs for corticosteroid use. This information can be used to guide corticosteroid therapy in this population.
    • Measuring Priorities for Nonmedical Use of Stimulants among College Students to Optimize Prevention

      Ross, Melissa Mary; dosReis, Susan (2017)
      Background: According to recent estimates, approximately one third of college students have engaged in nonmedical use of prescription stimulants (NPS) which is both dangerous and illegal, yet most universities do not have programs in place to address this growing public health concern. Methods: This mixed-methods study used in-depth interviews (n=8), four focus groups (n=17), and cognitive interviews (n=5) to elicit, refine, and vet attributes for the development of two preference elicitation instruments (Phase 1) that were used in an online survey (Phase 2). The preference elicitation instruments included a best-worst scaling (BWS) instrument that assessed college students' priorities around perceived benefits and risks of NPS and a discrete-choice experiment (DCE) that measured students' preferences for components of a college-sponsored NPS reduction program. Results: Thirty and 259 college students participated in Phase 1 and Phase 2, respectively. All participants were 18-25 years old, enrolled at a public university in Maryland, and a self-reported past-year NPS user. The majority were upperclassman (71%), female (57%), and white (66%). Phase 1 yielded 12 attributes related to students' perceived academic, punitive, health, and social benefits and risks of NPS and four three-level attributes related to components of an NPS reduction program: setting, leader, discussion topics, and additional resources. In Phase 2, for the BWS, participants prioritized the benefits and risks of NPS. Better grades and meeting deadlines, followed by college expulsion and limited future career opportunities, were most important concerns when engaging in NPS. A latent class analysis (LCA) yielded four segments with different priorities for NPS. For the DCE, program setting and leader most influenced participants' preferences for a college-sponsored NPS reduction program. A LCA generated three segments with heterogeneous preferences for components of a program. Conclusions: This dissertation is the first to apply advanced preference elicitation methods to the field of substance use. We identified college students' modifiable expectancies that most influence on NPS and their preferences for the delivery of an NPS reduction program in which they would be willing to engage. These findings can inform the delivery of personalized NPS reduction programs to maximize engagement.
    • The Impact of Maryland Care Management Entity on Quality of Antipsychotic Medication Use and Clinical Outcomes

      Tai, Ming-Hui; dosReis, Susan; 0000-0002-3540-5425 (2016)
      Background: Care Management Entity (CME) using the wraparound practice is a type of care delivery model for youth with severe mental/behavioral disorders. The CME provides coordinated, child-centered services that aim to improve youth's clinical and functional outcomes. The impact of this care management on the quality of psychotropic medication treatment and outcomes following discharge is unknown. The goals of this dissertation are to examine the quality of antipsychotic use and mental health-related outcomes between CME-enrolled and non-CME youth. Methods: Data comprised Medicaid claims linked with the CME administrative records anytime from December 2009 through December 2013. A difference-in-difference design was used to estimate the odds ratio (OR) and the average marginal effect (AME) of antipsychotic use, concomitant antipsychotic use, higher-than-recommended antipsychotic dose, and metabolic monitoring among inverse probability of treatment weighting-adjusted CME and non-CME youth. To avoid loss of the sample due to missing information on length of stay (LOS) in CME services, methods for imputing LOS in CME care were conducted and cross-validated. Building on the data imputation, two-part models were used to examine the mental health-related emergency department (ED) visits, hospitalizations, and outpatient visits within one-year post discharge between CME and non-CME youth. Results: Among the 3,493 IPTW-adjusted cohort (679 CME and 2,814 non-CME youth), the CME-served youth had a significant decrease in any concomitant antipsychotic use relative to non-CME youth (AME= -3.28%, 95% CI= -4.19% to -2.38%). However, there was no difference in antipsychotic dosing or metabolic monitoring between the two groups. In the methodological study, the multiple imputation was preferred over single imputation because it produced smaller errors and similar LOS distribution. In the outcome analyses, the CME care model resulted in a lower likelihood of receiving any mental health-related service (mental health-related ED visits: OR=0.65, 95%CI=0.46 to 0.93; hospitalizations: OR=0.60, 95%CI=0.40 to 0.89; outpatient visits OR=0.46, 95%CI= 0.32 to 0.66) post discharge. Conclusions: Future optimization of this care model may benefit from 1) greater attention to improving antipsychotic monitoring, 2) stronger prescriber engagement in the wraparound practice, and 3) further research to identify potential risk factors that associated with mental health services utilization among youth in public health insurance programs.
    • Impact of Beneficiary Characteristics, Drug Plan Formulary Policies, and Environmental Factors on Medication Adherence among Low Income Beneficiaries Covered by Medicare Part D

      Shen, Xian; Stuart, Bruce C. (2016)
      Research suggests that low income individuals often fail to take medications as prescribed. Various individual characteristics have been studied in relation to adherence outcomes, however, little is known regarding how drug plan formulary policies and external environmental factors may affect individuals' medication adherence. This dissertation evaluated the independent effects of beneficiary characteristics, drug plan formulary policies, and external environmental factors on medication adherence for oral hypoglycemic agents (OHAs), statins, and renin angiotensin system (RAS) antagonists in a cohort of low-income subsidy (LIS) recipients enrolled in randomly assigned benchmark Part D plans. The data source included a random 5% sample of 2012 Medicare administrative claims and a customized dataset capturing beneficiaries' plan assignment history. Three hosts of beneficiary characteristics, including demographics, comorbidity burden, and health services utilization, were analyzed. The formulary policies of interest included non-coverage, prior authorization, and step therapy, while the environmental factors of interest were socioeconomic environment, availability of healthcare resources, health culture, evidence-based medicine practice, and quality of primary care. Results indicated that beneficiary characteristics, Part D plan, and external environmental factors all could significantly influence LIS recipients' medication adherence. Older age, male gender, use of multiple chronic medications were associated with higher medication adherence, whereas black race, Hispanic ethnicity, high comorbidity burden, and frequent hospitalizations and ER visits were inversely related to the adherence outcomes. Placing formulary restrictions on brand-name drugs could shift utilization toward generics and lower cost per prescription fill but had minimal impact on medication adherence among LIS recipients. Geographic variation in adherence rates was observed consistently across all three drug classes of interest. Those living in areas with low socioeconomic environment and poor quality of primary care were less likely to achieve acceptable levels of medication adherence than their counterparts. In conclusion, low income beneficiaries' medication adherence is influenced by multiple levels of factors. Policies aimed at improving low income population's adherence for chronic medications may consider plan- and environment-oriented programs in addition to interventions targeting at individuals' behaviors.
    • Management of Traumatic Brain Injury with Statins among Older Medicare Beneficiaries

      Khokhar, Bilal; Simoni-Wastila, Linda; 0000-0003-0143-1390 (2016)
      Background: Traumatic brain injury (TBI) is a major health concern for older adults aged 65 and older. Older TBI patients are at increased risk of primary injury (in-hospital and all-cause mortality) and secondary injury (stroke, depression, and Alzheimer's disease and related dementias (ADRD)). There is limited research regarding optimal pharmacotherapeutic options and management of TBI patients; however, several studies have highlighted statins, used to treat hyperlipidemia, as potential pharmacologic agents to reduce inflammation and improve impaired cerebral blood flow associated with primary and secondary injury. The objectives of the study are to: 1) quantify statin utilization, and 2) determine the associations between statin use and primary and secondary injury among TBI patients. Methods: Statin use (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin), primary injury, and secondary injury were examined among Medicare beneficiaries hospitalized with a TBI between 2006 and 2010. Logistic regression was used to investigate the relationship between pre-TBI statin use and in-hospital mortality, while discrete time analysis was used to investigate the relationship between statin use following TBI and all-cause mortality and secondary injury. Results: Among the 75,698 beneficiaries who met study criteria, 37,874 (50.0%) beneficiaries used a statin at least once during the study period. The most common statin used was simvastatin, followed by atorvastatin. Fluvastatin was the least used statin. Pre-TBI use of atorvastatin (odds ratio (OR) 0.88; 95% confidence interval (CI) 0.82, 0.96), simvastatin (OR 0.84; 95% CI 0.79, 0.91), and rosuvastatin (OR 0.79; 95% CI 0.67, 0.94) were associated with significant decreases in the risk of in-hospital mortality. Any statin use was associated with reduced all-cause mortality following TBI-hospitalization discharge. Atorvastatin and simvastatin use also were associated with reductions in all secondary injury outcomes. Conclusion: Tens of thousands of older adults are hospitalized annually with TBI and experience disabling primary and secondary injury; findings from these analyses have salient implications for reducing the risk of TBI complications among older adults. The evidence generated suggests that preemptive use of statins may decrease the risk of in-hospital and all-cause mortality, as well as reduce the likelihood of stroke, depression, and ADRD.
    • Inhibiting the Iron-regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa via Competitive and Non-competitive Mechanisms

      Heinzl, Geoffrey Addison; Wilks, Angela; Xue, Fengtian; 0000-0001-5291-5999 (2016)
      The discovery and development of new antimicrobials has become a top priority as resistance to known therapeutics continues to grow. While most antimicrobials target essential functions, some in the field question this historical approach and instead propose targeting virulence factors, rendering pathogens non-pathogenic. In most Gram-negative bacteria, virulence is globally regulated by iron via the ferric uptake regulator (Fur). Recent studies show that in the host, iron is preferentially acquired via heme uptake and utilization. Pseudomonas aeruginosa encodes two heme uptake systems, both of which terminate in the oxidative cleavage of heme by the iron-regulated heme oxygenase (HemO). HemO is required for the efficient utilization of heme as an iron source in P. aeruginosa. Thus, inhibiting HemO will globally reduce virulence via disrupting the utilization of heme as an iron source. Previous work identified small-molecule inhibitors of HemO via computer-aided drug design techniques, which were validated in vitro and in vivo. Several of those compounds were further explored for optimization using medicinal chemistry, biochemistry, and microbiology techniques. Compounds were synthesized, characterized, and assessed for binding, inhibitory activity in cellulo, and antimicrobial activity. Binding was analyzed by fluorescence quenching, saturation transfer difference (STD)-NMR, heteronuclear single quantum coherence (HSQC) NMR, molecular dynamics simulations, and hydrogen-deuterium exchange mass spectrometry (HXMS). Two lead compounds were shown to bind in the heme-binding site of HemO with low micromolar affinity. Another lead compound was shown to bind to a previously unidentified back site of HemO, which was identified in silico and verified with HXMS. To analyze the mechanism of back side inhibition of HemO, site-directed mutagenesis eliminated a salt bridge (D99-R188) adjacent to the back site. These mutations disrupted the essential hydrogen-bonding network in the distal pocket, as evidenced by poor stability of intermediates and altered structural dynamics. Together, these data show that inhibiting HemO with small-molecules can be achieved on two sites of the enzyme, both the heme-binding site and the newly discovered back site. Future work includes improvement of heme-binding site inhibitors, development of novel inhibitors, and confirming the antivirulent activity of HemO inhibitors in an infection model.
    • Identification and Characterization of GTP-binding Inhibitors of LRRK2

      Thomas, Joseph Michael; Smith, Wanli W. (2016)
      Parkinson's disease (PD) is one of the most common neurodegenerative disorders in aged adults, resulting from the loss of dopaminergic neurons in the substantia nigra and the accumulation of Lewy body aggregates in the brain. Currently there are no disease altering treatments for PD. Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most common contributors to both familial and sporadic PD cases, however the exact physiological role of LRRK2 is currently unclear. As a large protein (286 kDa) with both GTPase and kinase activities, LRRK2 represents a tractable target for PD intervention. The work in this dissertation focuses on the identification and characterization of GTP-binding inhibitors of LRRK2. Moreover, I further investigate the biopathological roles of LRRK2 on neural transport functions underlying neurdegeneration and test the neuroprotective effects of LRRK2 GTP-binding inhibitors. I discovered that the novel compound, 68, inhibits GTP-binding as well as kinase activities in LRRK2, and that optimized compound FX2149, a new analog of 68, shows significant improvements in blood brain barrier (BBB) penetration and in vivo efficacy in LRRK2 mutant brains. Furthermore, I demonstrate that the LRRK2 GTPase domain mutation, R1441C, impairs neural transport functions, however these impairments can be attenuated through treatment with our GTP-binding inhibitors. Lastly, I show that our GTP-binding inhibitors increase LRRK2 ubiquitination, with a preferential increase in atypical ubiquitin linkages. This work provides further insight into LRRK2 functions in the pathobiology of PD, as well as provides evidence for the applicability of GTP-binding inhibitors as pharmacological tools and potential therapeutic agents in the treatment of PD.