Now showing items 61-80 of 324

    • Impact of the Medicare Annual Wellness Visit on Geographic Variation in Dementia Diagnosis

      Hanna, Maya; Perfetto, Eleanor M. (2020)
      Background: Alzheimer's-disease-and-related-dementia (ADRD) is often misdiagnosed or diagnosis is late in disease progression. Diagnosis variations can be driven by access variations related to geographic location. To improve timely and accurate ADRD diagnosis, the Medicare Annual Wellness Visit (AWV) required a cognitive assessment, starting in 2011, which may reduce diagnosis variations. Objective: Assess impact of the AWV on geographic variation in ADRD diagnosis and outcomes. Methods: Aim 1: ADRD patients and caregivers from western, central, and eastern Maryland were interviewed to understand diagnosis-pathway geographic differences. Data were analyzed using interpretative phenomenological analysis to identify themes. Aim 2: Using the CMS Chronic Condition Data Warehouse (CCW) and HRSA Area Health Resource Files, 5-year, county-level ADRD cumulative incidence were compared pre- (2006-2010) and post- (2011-2015) AWV implementation in Mid-Atlantic states. Geographically-weighted, generalized linear models assessed the association between the AWV and ADRD cumulative incidence, controlling for demographic and access measures. Aim 3: A retrospective cohort study using CCW was conducted in newly diagnosed ADRD individuals. Health care utilization (HCU) was compared for individuals with an AWV pre-diagnosis versus no AWV. Difference-in-difference models assessed 10-month outcomes between exposure groups. Results: Aim 1: Average time from first doctor visit (concerning signs/symptoms) to ADRD diagnosis was 3.3, 2, and 5.3 years for western, central, and eastern regions, respectively. Aim 2: AWV participation was not significantly associated with increased 5-year ADRD cumulative incidence. The association between AWV and 5-year ADRD cumulative incidence varied by county with stronger associations clustered in eastern Virginia, Maryland, and Delaware. Aim 3: Receiving an AWV pre-diagnosis was associated with increased HCU [ED (rate ratio [RR]: 1.20, 95% confidence interval [CI]: 1.11, 1.30), hospitalizations (RR: 1.26, 95% CI: 1.13, 1.40), and outpatient (RR: 1.08, 95% CI: 1.04, 1.12)]. Conclusions: Longer times to ADRD diagnosis were observed in rural versus urban regions. The AWV demonstrates the potential to minimize geographic differences by increasing diagnosis rates and HCU. Due to low and variable participation during early implementation, the true impact of the AWV is yet to be established. It may take longer to see impacts on longer-term outcomes.
    • Effects of Static Growth on P. aeruginosa Iron Homeostasis and Virulence

      Brewer, Luke; Oglesby, Amanda G.; Kane, Maureen A. (2020)
      Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogens that causes life-threatening, antimicrobial resistant infections in vulnerable patient populations, including patients with cystic fibrosis, cancer, and chronic wounds. During infection, P. aeruginosa requires iron to maintain critical aspects of its metabolism, and possesses numerous virulence factors and iron uptake mechanisms that allow it to compete for this essential metallo-nutrient in the iron-limiting host environment. These systems are tightly regulated by iron-responsive regulatory mechanisms, which ensure adequate uptake while preventing iron toxicity. Because of the essential role of these regulatory mechanisms in maintaining iron homeostasis, they are considered a promising approach for treating P. aeruginosa infections. One prominent regulator of P. aeruginosa iron homeostasis is the PrrF small RNA (sRNA) regulator, which is essential for virulence in acute murine lung infection. Unfortunately, the exact contribution of PrrF to P. aeruginosa pathogenesis has not yet been elucidated. Moreover, our current understanding of PrrF and other iron regulatory mechanisms is largely based on studies using shaking and highly aerated cultures, which are not likely representative of microbial communities in vivo. To address these gaps, the work in this thesis utilizes proteomic, metabolic, and genetic approaches to determine the impact of static growth on iron-responsive regulatory mechanisms in P. aeruginosa, including PrrF sRNAs. We demonstrate that iron regulation paradigms in P. aeruginosa are dramatically altered in static conditions, due in part to changes in PrrF activity. Notably, we identify type 6 secretion systems (T6SS) as a target of enhanced iron regulation in P. aeruginosa in static conditions, and demonstrate that this altered regulation is caused by changes in the production and activity of the PrrF-regulated quorum signaling molecules, 2-alkyl-4(1H)-quinolones (AQs). Furthermore, we demonstrate that altered AQ activity may modulate clinically-significant interactions with other opportunistic pathogens, such as S. aureus, In turn, the work described herein has broad implications for the study of P. aeruginosa infections, and highlights the need to further probe essential P. aeruginosa iron homeostasis mechanisms in static conditions.
    • Metabolism-based Alterations of Constitutive Androstane Receptor (CAR) Activity and Downstream Effects

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

      Honick, Moshe Avraham; Polli, James E. (2019)
      Despite their potential for improving the oral bioavailability of poorly water soluble drugs, spray dried dispersions (SDDs) have properties that make them challenging to formulate. The objective of this dissertation was to elucidate composition and process factors for favorable SDD performance and to develop fast-, medium-, and slow-release formulations for an IVIVC study. Itraconazole (ITZ) was used as a model poorly soluble drug and hypromellose acetate succinate (HPMCAS) was used as a carrier polymer for the SDDs. Film casting proved to be a useful screening method for demonstrating the feasibility of producing amorphous SDDs of ITZ and HPMCAS as well as for rank ordering the grades of HPMCAS (i.e. HPMCAS-L > HPMCAS-M > HPMCAS-H) in terms of in-vitro dissolution performance. Producing solid oral dosage forms of ITZ-HPMCAS SDDs proved challenging due to the low particle size, poor flowability, and low bulk density of the SDDs. Initial tableting on a Natoli hand-operated press showed that drug release from tablets containing SDDs of ITZ and HPMCAS-L were very sensitive to small differences in compaction pressure and porosity. Interestingly, the same sensitivity was not observed in SDDs of ITZ and HPMCAS-M. Using a compaction simulator, reproducible fast-, medium- and slow-release tablet formulations of ITZ and HPMCAS SDDs was developed by varying polymer grade (HPMCAS-L, HPMCAS-M), slugging pressure (20, 40 MPa), and compaction pressure (70, 85, 100 MPa). The performance of SDDs was further evaluated by comparing the compaction behavior of ITZ-HPMCAS SDDs and physical mixtures of ITZ and HPMCAS. Although the compressibility of both the SDDs and physical mixtures were similar, the SDDs had a greater tendency to laminate, especially at higher compression speeds. Tablets of SDDs containing ITZ and HPMCAS-L were particularly prone to lamination compared to the SDDs containing HPMCAS-M or HPMCAS-H. Interestingly, when the SDDs were not laminated they had a greater tensile strength than tablets produced with the physical mixtures. In conclusion, there are significant challenges associated with formulating SDDs of ITZ and HPMCAS. In addition to elucidating composition and process factors impacting performance, fast-, medium-, and slow-release formulations for an IVIVC study were developed.
    • The Design and Development of Dual MCL-1/BCL-2 and HDM2/Bcl-2 Protein Family Inhibitors Using a Polypharmacology Approach

      Drennen, Brandon; Fletcher, Steven (2019)
      Apoptosis, a cellular process that leads to cell death, is a vital signaling pathway for maintaining homeostasis. Intracellular-activated apoptosis is regulated by the B-cell lymphoma 2 (BCL-2) family of proteins, which encompasses two classes of proteins: the pro-apoptotic and anti-apoptotic members. Apoptosis is controlled by a protein-protein interaction (PPI) between the two members. Specifically, the anti-apoptotic proteins’ surface hydrophobic binding groove binds to the α-helical Bcl-2 homology 3 (BH3) domain of the pro-apoptotic proteins, thus inhibiting apoptosis. During apoptotic conditions, BH3 activator proteins are expressed and disrupt the PPI, initiating apoptosis. During tumorigenesis, the anti-apoptotic proteins are overexpressed and capture the activator proteins before they can act, progressing tumor development. A strategy developed to overcome this oncogenic transformation is BH3 mimicry, the design of small molecules that behave like BH3 activators to free the pro-apoptotic proteins. Though potent BH3 mimetics have been synthesized, cytotoxic and resistance issues have arisen. Specifically, BCL-XL inhibition causes thrombocytopenia within patients and BCL-2 inhibition causes resistance mechanisms to emerge that involve the upregulation of MCL-1. Presently, there are no potent dual inhibitors of BCL-2 and MCL-1 to overcome these issues. Additionally, p53 has been shown to regulate apoptosis through the Bcl-2 family by either direct interactions or increasing their expression. P53 is rapidly degraded due to the overexpression of HDM2, a ubiquitin ligase, within cancer cells. The PPI between p53 and HDM2 resembles the PPI between the members of the Bcl-2 family. Also, Venetoclax (BCL-2 inhibitor) and idasanutlin (HDM2 inhibitor) act synergistically in combination therapies. Thus, we followed a polypharmacology approach to synthesize dual BCL-2/MCL-1 and dual HDM2/Bcl-2 family inhibitors. We were able to create potent dual MCL-1/BCL-2 indazole inhibitors (Ki MCL-1 < 1.50 µM, BCL-2 < 0.050 µM, BCL-XL > 10.00 µM), dual HDM2/Bcl-2 family pyrazole and imidazole inhibitors (Ki MCL-1 < 0.050 µM, HDM2 < 25.00 µM), HSQC-confirmed nicotinate-based MCL-1 inhibitors (Ki MCL-1 < 3.00 µM) and a new alpha-helix mimetic scaffold for disrupting PPIs. Further optimization of these inhibitors is planned, along with cell viability studies. Overall, these inhibitors can serve as starting points for future experiments and polypharmacology designs.
    • Novel Cholinergics for Treatment of Central Nervous System Disorders

      Johnson, Chad; Coop, Andrew; 0000-0001-7584-3000 (2019)
      Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder thought be caused by a combination of characterized by genetic, biological, environmental, and psychological factors. It can be accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and diminished quality of life. Between 2-7% of adults with this disorder die by suicide. In addition, almost half of patients who are treated initially with an SSRI do not achieve complete remission, and nearly a third after four different treatment regimens (nimh.nih.gov). While counseling and antidepressant medication can be effective treatments, current selective serotonin re-uptake inhibitors (SSRI's) take weeks before therapeutic effects are observed. This "delay" period of action is not well understood and presents a significant challenge for medical professionals in the management of major depression. Mechanisms of anti-depressants have been a major focus of both current/past research in hopes of developing more effective and faster acting drugs. Directly related to this, clinical data (nimh.nih.gov) that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine had rapid anti-depressant effects in humans--likely mediated through an antimuscarinic effect. Unfortunately, scopolamine can produce cognitive impairment including memory disturbances due to its anticholinergic properties. Since major depressive disorder is associated with deficits in cognition, this would produce an undesired additive effect that would only exacerbate the problem. It is our goal to identify a muscarinic antagonist that may be able to relieve depression and have little to no effect on memory or cognition. The 3-exo-1-azabicyclo[2.2.1]heptane, 1-azabicyclo[2.2.2]octane, 1-azabicyclo[3.2.1]octane, and N-methyltetrahydropyidine 3 (and 4)-substituted-1,2,4-oxadiazoles appear to be excellent chemical scaffolds for the generation of potent muscarinic agonists/antagonists. In order to probe the orthosteric site of the mAChRs we designed a large library of compounds and evaluated them via a battery of pharmacological assays to confirm both their antidepressant and cognitive effects. This resulted in the identification of lead compound (CJ2100) that showed potent antidepressant activity without cognitive impairment. (Supported by NIMH Grant 107499)
    • Advancing Drug and Biomaterial Design with Constant pH Molecular Dynamics Simulations

      Tsai, Cheng-chieh; Shen, Jana (2019)
      Molecular dynamics (MD) simulation is a valuable tool for investigating motions of macromolecular systems; however, solution pH, a critical factor for biological and chemical processes, is neglected in conventional MD simulations. To address this weakness, our group developed various continuous constant pH molecular dynamic (CpHMD) tools. In this dissertation, applications and new protocols that utilize CpHMD for drug and biomaterial design are presented. β-secretase 1 (BACE1, implicated in Alzheimers disease) and Src kinase (implicated in cancer) were utilized as model systems for drug design applications. In chapter 2, we applied CpHMD to understand the binding of BACE1 with two small-molecular inhibitors. We discovered that, despite the structural similarity of the two inhibitors, the titration behavior of the protein active site differs and this difference dramatically impacts the protein-ligand interactions and consequently the inhibitor affinity. Further, we tested a new protocol that combines CpHMD titration with free energy simulations to construct the pH-dependent binding free energy profiles. The resulting data showed excellent agreement with experiment and identified one potential allosteric site in BACE1. Next, in chapter 3, we simulated Src kinase to test the capability of CpHMD tostudy kinases. Starting from the crystal structure of the inactive Src, CpHMD can capture the conformational activation along the major inactive states without introducing any biasing potential or mutation. Starting from chapter 4, we studied the chitosan-based hydrogel systems to explore the detailed molecular mechanisms that give rise to macroscopic materials properties. We examined the flexibility of chitosan chains under different environmental conditions such as pH and salt concentration. Simulation data revealed that in addition to electrostatic screening, salt ions enhance the chain flexibility by interrupting the intra-molecular hydrogen bonds and thereby shifting the conformational populations between extended and bent states. In chapter 5, the atomic-level mechanism of pH-responsive chitosan-based hydrogels with switchable mechanical properties was investigated. Our data suggested that the electrostatic crosslinks are formed through the pH-dependent salt-bridge interactions between the chitosan glucosamines and surfactant headgroups. The pKa difference between the chitosan crystallite and the surfactant-bound chitosan is a key for the persistent but erasable gradient in the structural and mechanical properties between the two crosslinked regions. In summary, my work provids insights that will contribute to drug and biomaterial design and highlights the usefulness of CpHMD in these fields.
    • Developing a patient-driven cost-effectiveness analysis of pharmacological treatments for patients with chronic hepatitis C

      Mattingly II, T. Joseph; Mullins, C. Daniel; 0000-0001-7786-5780 (2019)
      Background: Innovations in hepatitis C virus (HCV) drug therapy included in comparative clinical effectiveness evaluations focus on sustained virologic response (SVR) without consideration of socioeconomic or psychological outcomes. This study aimed to identify and prioritize variables important to patients and determine the impact of patient-centered parameters on the cost effectiveness of HCV treatments. Methods: An individual-based state-transition model was developed with the guidance of a patient-centered multi-stakeholder advisory board and patient-only Delphi panel. The model was used to perform a patient-driven cost-effectiveness analysis (CEA) of direct acting antivirals (DAAs) over 10 and 20 year time horizons from both health sector and societal perspectives. The patient-centered model and CEA results were then compared with recently published HCV CEAs. Results: Patients identified treatment effectiveness, longer life, fear of complications, financial issues, quality of life, and impact on society as important factors to include. Fear of harming others was considered more important than physical symptoms in terms of patient-reported problems caused by HCV. Total infected life-years (ILYs) and work days missed were reduced in the treatment group for both 10 and 20 year health sector models in addition to quality-adjusted life-year gains. Compared to no treatment, the incremental cost-effectiveness ratio for treatment would be $3,464/ILYs avoided, $715/work day missed, and $39,086/QALY gained. When costs of absenteeism, presenteeism, and patient/caregiver time were included, the DAA intervention was cost-saving at both 10 and 20 years. Very few traditional economic models for HCV treatments attempt to capture the indirect and non-medical costs or outcomes that may impact HCV patients. Conclusions: Treatment was cost-effective from a health sector perspective and cost-saving when including non-health costs such as patient/caregiver time and productivity. Compared to published HCV CEAs that focused mainly on SVR, our patient-centered CEA provides results that reflect the outcomes of interest informed by direct patient engagement.
    • Application of Machine Learning Algorithms for Predicting Missing Cost Data

      Rueda, Juan-David; Slejko, Julia F.; 0000-0002-0907-7106 (2019)
      Objective: To compare new alternatives to estimate health care costs in the presence of missing data using methods based on machine-learning (ML). Introduction: Costs must be correctly estimated for value assessment and budget calculations. Problems arise when they are not correctly estimated. Sometimes costs can be biased and lead to wrong decisions that affect population health. Cost estimation is a challenging task and it is more challenging in the presence of missing data. Methods: We used Surveillance, Epidemiology, and End Results program (SEER)-Medicare including patients with multiple myeloma newly diagnosed from 2007-2013. We explored the problem of missing data using different approaches creating artificial missing data. We hypothesized that the use of ML techniques improves the prediction of mean medical total costs in the presence of missingness. ML methods included support vector machines, boosting, random forest, and classification and regression trees. First, we analyzed the problem considering only one dimension, when one variable is missing in a cross-sectional scenario, using generalized linear models as a comparator against ML. Then, we added time as a factor for missingness, utilizing reweighted estimators against ML. Finally, we explored the different levels of censoring and determined how each censoring level affected our cost estimations. In this case, we created multiple linear spline models to establish the effect of censoring on the bias of the estimator. Results: We demonstrated that ML algorithms had better prediction when data were missing completely at random and missing at random. All the methods performed badly in the missing not at random scenario. In the second aim, we showed that ML-based methods predict just as well as reweighted estimators for the five-year total cost of a patient with multiple myeloma. Lastly, we found that ML methods are consistent and robust at low and moderate levels of censoring; however, we failed to prove that they are better than the reweighted estimators. Conclusions: ML-based methods are a good alternative for the prediction of missing cost data in the case of cross-sectional and longitudinal data.
    • Effect of Cadmium Exposure on the Transport System of Organic Cation Transporters and Multidrug and Toxin Extrusion Proteins (OCTs/MATEs)

      Yang, Hong; Shu, Yan, Ph.D. (2019)
      The universal pollution by cadmium (Cd) in our agricultural land and the prevalence of cigarette smoking make the environmental Cd exposure an unneglectable human health concern. While the mechanism of cadmium accumulation has been extensively studied, no explicit mechanism has been reported regarding the elimination of cadmium from the body. On the other hand, whereas Cd exposure has been correlated with a variety of diseases, little is known pertaining to its effect on drug disposition and response in patients. Thus, we aim to delineate the mechanism of cadmium elimination and detoxification and to gain new insights into its effect on xenobiotic disposition and response. The OCTs/MATEs transport system are pair of transporter proteins highly expressed at the basolateral and apical membrane of hepatocytes and renal proximal tubules respectively. Recently, Cd has been identified as a substrate of OCTs, while we determined that MATEs could reduce the toxicity of Cd by serving as its efflux transporters in vitro. In addition, knockout of Mate1 in mice kidney resulted in higher renal toxicity in both chronic and acute Cd intoxication studies. We found that Cd was an inducer of OCT activity while an inhibitor towards MATEs in cells. Consistently, Cd exposure could lead to accumulation of the substrates of these transporters in mouse liver and kidney. Being focused on human (h) OCT2 and MATE1, our mechanistic studies revealed that hOCT2, as compared to hMATE1, was more active in trafficking between the plasma membrane and the cytoplasmic storage pool. Cd exposure could trigger the formation of a protein complex consisting of AKT2, calmodulin and AS160, which could then selectively facilitate the phosphorylation of AKT2 at T309, and initiate the translocation of hOCT2 to the plasma membrane. Altogether, our findings have identified MATE transporters as new contributors for Cd detoxification, and provided foundation to uncover environmental Cd as a previously unrecognized factor for the broad variation in drug disposition and response.
    • Medical Costs of Alpha-1 Antitrypsin Deficiency-associated Chronic Obstructive Pulmonary Disease in the United States

      Sieluk, Jan; Mullins, C. Daniel; 0000-0002-1833-0273 (2018)
      Objectives: The objective of this study was to isolate the healthcare resource utilization and economic burden attributable to the presence of a genetic factor among Chronic Obstructive Pulmonary Disease (COPD) patients with and without Alpha-1 Antitrypsin Deficiency (AATD), twelve months before and after their initial COPD diagnosis. Methods: Retrospective analysis of OptumLabs® Data Warehouse claims (OLDW; 2000 – 2017). The OLDW is a comprehensive, longitudinal real-world data asset with de-identified lives across claims and clinical information. AATD-associated COPD cases were matched with up to 10 unique non-AATD-associated COPD controls. Healthcare resource use and costs were assigned into the following categories: office (OV), outpatient (OP), and emergency room visits (ER), inpatients stays (IP), prescription drugs (RX), and other services (OTH). A generalized linear model was used to estimate total pre- and post-index (initial COPD diagnosis) costs from a third-party payer’s perspective (2018 USD) controlling for age, gender, race/ethnicity, census region, augmentation therapy use, oxygen use, insurance type, year of COPD diagnosis, and Charlson Comorbidity Score. Healthcare resource utilization was estimated using a negative binomial regression. Results: The study population consisted of 8,881 patients (953 cases matched with 7,928 controls). The AATD-associated COPD cohort had higher expenditures and use of OV and OTH services, as well as OV, OP, ER, RX, and OTH before and after the index date, respectively. Adjusted total cost ratios for AATD-associated COPD patients as compared to controls were 2.036 [95% CI: 1.601 – 2.590] and 1.976 [95% CI: 1.550 – 2.517] while the incremental cost difference totaled $6,861 [95% CI: $3,025 - $10,698] an $5,772 [95% CI: $1,940 - $9,604] per patient before and after the index date, respectively. Conclusions: Twelve months before and after their initial COPD diagnosis, patients with AATD incur higher healthcare utilization costs that are double the cost of similar patients without AATD. This study also suggests that increased costs of AATD-associated COPD are not solely attributable to augmentation therapy use. Future studies should further explore the relationship between augmentation therapy, healthcare resource use, and other AATD-associated COPD expenditures.
    • Advances in Mass Spectrometric Structural Biology Techniques for Pattern Recognition Receptor Ligands of Microbial Origin

      Oyler, Benjamin; Goodlett, David Robinson, 1960- (2018)
      Pattern recognition receptors (PRR) are the innate immune system’s first-line sentinels for distinguishing “self” from “non-self.” Many molecules found in the bacterial cell wall are PRR ligands, including lipopolysaccharide (LPS), cardiolipin (CL), and peptidoglycan (PGN). Molecular structural biology techniques are essential for determining both basic cell biology and host-bacteria interactions through ligand-receptor binding mechanisms. Recent interest in designer PRR ligands or PRR ligand mimetics for use in drug discovery pipelines have given this research more translational value as well. Mass spectrometry (MS) has the unique capability to derive primary structures of ions as well as monitor many different ions in complex mixtures. Several different advances in PRR ligand structure analysis were achieved in this dissertation. First, chemical structure of an LPS-derived vaccine was determined using a top down tandem MS approach. Several different instrumental configurations and methods were employed to demonstrate complementarity of data and broad applicability of the approach. Second, CL from a newly discovered Actinomycete marine sponge symbiont was analyzed and compared to CL from a terrestrial Firmicute to generate hypotheses about host-bacterium interactions. This was the first molecular analysis of any secondary metabolites from this species of bacteria. Third, PGN subunits (muropeptides) from Rickettsia typhi were analyzed in a data dependent global LC-tandem MS approach. This was the first example of PGN structure discovery for R. typhi and the first example of this approach applied to PGN structure elucidation for any Rickettsiae species. All of these developments will help to advance PRR-ligand interaction research – an emerging and promising field for development of novel disease treatment and prevention approaches. Modulation of the innate immune response to bacterial insult is a challenging task without a clear understanding of underlying molecular mechanisms and how they might be manipulated by medicine. One key step in this process is development of sensitive and specific chemical analysis methods fit to acquire unequivocally interpretable data. While all of the methods described herein were applied to specific biological problems, their applicability to other scientific questions is broad.
    • Mass Spectrometry based structural analysis and systems immunoproteomics strategies deduce specifics of host-pathogen interactions

      Khan, Mohd M.; Goodlett, David Robinson, 1960- (2018)
      The innate immune system is the first line of defense against pathogens. Pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) sense and sample pathogen-associated molecular patterns (PAMPs). On the host myeloid cell surface, the proinflammatory Gram-negative bacterial outer membrane component lipopolysaccharide (LPS, also known as endotoxin) activates the innate immune system via TLR4. Intracellularly, LPS is detected by the noncanonical inflammasome through caspase4/5/11. In the present work, mass spectrometry (MS)-based top-down structural analysis of LPS uncovered major determinants of molecular pathogenesis, and MS-based systems immunoproteomics elucidated specific features of the immune response against endotoxin. We used targeted proteomics to profile the host response to the pathogens Escherichia coli, Staphylococcus aureus, and Burkholderia cenocepacia, and we discovered significant temporal changes in the macrophage secretome. Additionally, we identified global changes in protein secretion in TLR4- and caspase11- stimulated macrophages. Finally, we observed bacterial proteomic rewiring within the biofilm forms of Burkholderia, possibly explaining the observed lowering in sensitivity to antibiotics.
    • "Zoom in" on protein functions through integrated mass spectrometry

      Li, Wenjing; Kane, Maureen A.; 0000-0002-5387-7004 (2018)
      Mass spectrometry (MS) has been a powerful tool in cracking the protein codes for human biology regarding to their structure and function. Rich information can be collected through MS either at protein level (native/intact, top-down) or peptide level (middle-down and bottom-up), however integration of these approaches in order to generate a comprehensive view of the protein(s) has been underutilized. In this thesis, strategic integration of MS platforms was developed for two protein systems with the aim of elucidating the fundamental molecular function related to protein-ligand or protein-protein interactions toward fulfilling the potential of MS-based platforms for application in drug discovery. In the first study, the integration of native top down mass spectrometry coupled with ion mobility analysis provided extensive structural information to understand gold finger protein complex that formed with the exchange of zinc. Native top down analysis identified the stoichiometry, binding residues and preferable binding sites when gold replaces zinc in the parent nonclassical zinc finger protein tristetraprolin (TTP). The subtle difference in conformation were monitored by ion mobility simultaneously. The heterogeneity of gold fingers that were reflected by MS-based assays was not obvious by other conventional assays, suggesting the unique analytical power of MS for in-depth drug-target investigations. In the second study, bottom-up and native MS were applied on interrogation of the role of cellular retinol binding protein, type I (CrbpI) in maintaining retinoid homeostasis. The endogenous level of CrbpI was confidently quantified through customized bottom-up assays, which demonstrated a positive correlation with active metabolite retinoic acid (RA). Further investigation focused on the interaction between CrbpI and its biological ligands (retinol and retinal), and the RA-producing enzyme retinal dehydrogenase 1(Raldh1), using native mass spectrometry. These studies contributed fundamental information toward elucidating the role of CrbpI in facilitating RA biosynthesis, and also suggested its potential as a disease marker.
    • Atrial Fibrillation Risk-Stratification Schemes: Improving Patient-Centeredness and Precision

      Oehrlein, Elisabeth Maria; Perfetto, Eleanor M. (2018)
      Background: Despite treatment-guideline recommendations and availability medications to reduce stroke risk, widespread underutilization of oral anticoagulants (OACs) has been previously documented among individuals with atrial fibrillation (AF). Younger age and female gender are important in light of evidence that these groups, in particular, may not receive optimal AF care. The objective of this dissertation was to identify: 1) What are the barriers to patients initiating OACs? 2) Are providers aware of and using the RSSs and do disparities exist by age and gender? 3) Are RSSs predictive of stroke and OAC initiation among subpopulations (women and <65 years of age)? Methods: In Aim 1, we invited patients and health care providers (HCPs) to participate in in-depth interviews. In aims 2 and 3, we conducted retrospective cohort studies using Optum’s Clinformatic Data Mart (2008-2016). We used logistic regression to calculate odds ratios and 95% confidence intervals to identify whether RSSs were associated with OAC initiation and whether disparities exist by age or gender in aim 2. For Aim 3, we used a discrete time approach to estimate the risk of ischemic stroke associated with RSSs. Separately, we tested whether incorporating risk factors identified in the literature as predictive of ischemic stroke improved prediction among women and patients ≤65 years. Results: Themes from qualitative interviews include: specialists heightened perception of stroke risk compared to generalists and comorbidities/characteristics absent from RSSs also factor into risk consideration. The proportion of patients initiating OACs was only approximately 30%. CHADS2, but not CHA2DS2-VASc, scores corresponded with higher odds of OAC initiation. We found no statistically significant differences between odds of initiating OACs among OAC-recommended males/females or age categories. Among women and those ≤65 years, all CHA2DS2-VASc scores >1 and CHADS2 scores >0 were significant predictors of stroke. Prognostic models developed within subpopulations were no better at predicting stroke than existing RSSs. Conclusions: RSSs are associated with ischemic stroke among newly diagnosed females and <65 years of age patients. Initiation of OAC treatment was consistently low. More research is needed to more clearly understand why RSSs might not be followed and why OACs are not initiated.
    • Innovation of Vancomycin Treatment in Neonates Via A Bayesian Dose Optimization Toolkit For Adaptive Individualized Therapeutic Management

      Pastoor, Devin DeForest; Gobburu, Jogarao (2018)
      Personalized medicine continues to gain momentum as a topic for discussion, yet directly linking patient-level decision support to more advanced analytical techniques, such as nonlinear mixed effects modeling, is not being practiced in most hospitals. Current practice for Vancomycin therapy uses dosing nomograms to determine the dosing regimen for patients. For simplicity, these nomograms stratify patients into bins based on some combination of weight, serum creatinine, and/or age to adjust starting regimens. Yet, studies across the US and Europe have shown as few as 37% of neonates achieve recommended target concentrations using such nomograms. The purpose of this research was to develop a bayesian decision support toolkit to provide adaptive, individualized dose recommendations for neonates. First, a bayesian nonlinear mixed effect model was developed and qualified for predictive forecasting in individual patients. Second, this model was used to develop a novel algorithm for dose individualization. Finally, a web application was developed to allow clinicians to provide decision support for clinicians involved in vancomycin dosing decisions. The proposed strategy can decrease the number of patients improperly dosed up to 90%, drastically reducing the chance for treatment failure, toxicity-related adverse events, and resistance development.
    • Lower Gastrointestinal Tract Delivery of a Tetra-specific Antibody ABAB-IgG1 for the Treatment of Clostridium Difficile Infection (CDI)

      Jiang, Bowen; Stephen W. Hoag; 0000-0003-0913-6468 (2018)
      More than 246 protein or peptide therapeutics are on the market, including 47 monoclonal antibodies approved since early 1980s. Although most protein therapeutics are administered parentally, treatments for gastrointestinal diseases such as Clostridium difficile infection (CDI), ulcerative colitis and Crohn’s Disease would benefit from an oral delivery system that can target biologics to a site in the GI tract, with less systematic exposure and therefore less systematic toxicity, given the targets of disease are in the GI tract. This research focused on local delivery of a novel antibody therapeutic, ABAB-IgG1, for the treatment of CDI. The antibody was fabricated into a multi-particulate delivery system comprised of nonpareil beads and functional polymers using spray coating techniques. A proof-of-concept study was conducted using BSA as a model protein. BSA was first spray layered onto beads, then coated with pH sensitive polymers. There was no significant change in BSA conformation and aggregation profiles after the spray layering process. BSA multi-particulates were stable for at least 1 month stored at 4 ⁰C. In vitro dissolution testing showed that the enteric coated BSA beads remained intact in acidic media, while releasing BSA in higher pH buffers. A Design of Experiments strategy was used to understand how the formulation and process parameters impacted antibody stability during spray coating process and during accelerated stability studies. The formulation of novel structured antibody ABAB-IgG1 was also optimized based on the conformational and colloidal stabilities using various high throughput biophysical characterization techniques. The multi-particulate delivery system of ABAB-IgG1 was evaluated both in vitro and in vivo, and showed the feasibility of delivering ABAB-IgG1 to the lower GI tract using the multi-particulate system. The multi-particulate delivery system, which is well-studied for small molecule drugs, can be adopted to biologics without modification of existing fluid bed processing equipment, which implies the possibility of efficient scale up of this technique in existing industrial-scale equipment.
    • Novel WNT/beta-catenin Signaling Pathway Inhibitors for the Treatment of Metabolic Disorders

      Obianom, Obinna N.; Shu, Yan, Ph.D. (2018)
      The WNT/β-catenin signaling (β-cat) pathway is critical for embryonic development and tissue homeostasis. For this reason, alterations in the β-cat pathway are associated with many ailments including metabolic disorders, which may result from defects in the energy metabolism. The contribution of β-cat pathway to energy metabolism has become a subject of many investigations following the identification of polymorphisms in β-cat pathway components that predispose individuals to type-2-diabetes. Current evidence suggests that downregulation of the β-cat pathway activity may help treat metabolic disorders. Given these findings, the overarching goal of this thesis was to discover and develop novel β-cat pathway inhibitors and to examine their efficacy on glucose and lipid metabolism. We started with an FDA approved anthelmintic, pyrvinium, which is a potent inhibitor of the β-cat pathway. Our results showed that pyrvinium improved glucose tolerance by inhibiting glucose output, hepatic lipid accumulation and activating the AMPK pathway. Despite these beneficial effects, pyrvinium is unsuitable for repurposing to use orally in the treatment of metabolic disorders due to its almost zero bioavailability and other unspecific toxic effects in mice at higher doses. Based on the structure of pyrvinium, we decided to discover new potent β-cat pathway inhibitors with lower toxicity and improved bioavailability. Our screening of more than 150 newly synthesized pyrvinium derivatives led to the discovery of YW1128 as such a candidate having the aforementioned properties. Administration of YW1128 led to decreased lipid accumulation and improved glucose tolerance in the mice fed with high fat diet. Previous studies had suggested a critical role of hepatic β-cat pathway in determining the whole body metabolic homeostasis. So we next sought to achieve a selective delivery of the new derivatives to the liver without having significant disposition in other tissues. We performed a proof-of-concept study where we took advantage of high expression of organic cation transporter 1 (OCT1) in the liver to modify the compounds that were not specifically permeable to OCT1 expressing cells. We inserted a biguanide, which is a major backbone of several OCT1 substrates, into these compounds and showed that they became highly permeable to cells overexpressing OCT1. This suggests that insertion of the biguanide moiety into YW1128 may be an approach to improve its selective liver targeting. In conclusion, this thesis uncovered the efficacy by small molecule inhibition of β-cat pathway in the treatment of metabolic disorders and established that incorporating a biguanide moiety to the compounds may serve as a strategy to achieve selective liver targeting.
    • Utilization and Cost of the Biologic Disease Modifying Anti-rheumatic Drugs among Medicare Beneficiaries with Rheumatoid Arthritis

      Gaitonde, Priyanka; Shaya, Fadia T. (2018)
      Background: Disease modifying anti-rheumatic drugs (DMARDs) are essential for symptom control among rheumatoid arthritis (RA) patients. Biologic DMARDs are expensive and typically used among moderate to severe RA patients. The prevalence of RA is higher among Medicare beneficiaries compared to the rest of the population in the U.S (2% vs. 0.6%). The coverage rules of Medicare, in addition to access factors and patient preferences, may influence the use of facility-administered, infusible biologics (Part B covered) and home-administered self-injectable/oral biologics (Part D covered). However, there is limited information about utilization patterns of biologic DMARDs by route of administration and their impact on Medicare spending overall. The goals of this dissertation were to identify patient factors and healthcare expenditure associated with biologic DMARD use by route of administration among Medicare beneficiaries with RA. Methods: The study population consisted of Medicare beneficiaries with RA from the 5% random sample of the Chronic Conditions Warehouse database from 2006-2015. First, the study analyzed patient-level factors associated with biologic DMARD use by route of administration using generalized estimating equations. Second, adherence (PDC>80%), discontinuation, and switching patterns for biologic DMARDs were measured, accounting for patient level-factors , using logistic regression, Cox proportional hazards models, and chi-square analyses, respectively. Third, the study compared annualized average healthcare costs of patients who were adherent to versus non-adherent to biologic DMARDs. Results: Among Medicare beneficiaries diagnosed with RA who received DMARD treatment (n=46,002), 71.8% (n=33,028) used traditional DMARDs, and among biologic DMARD users (n=12,931), twice as many used infusible biologics (18.3%, n=8,436) compared to self-injectable/oral (9.9%, n=4,538). Beneficiaries who were low-income subsidy (LIS) recipients i.e. who had lower out-of-pocket costs for using biologics, (compared to non-LIS) had 4.54 times higher odds of using self-injectable/oral biologics (95% CI: 4.2 - 5.0) and 5% lower odds of discontinuing biologic DMARDs (OR=0.94, 95% CI: 0.91-0.97). The total healthcare cost was lower among adherent compared to non-adherent infusible biologic DMARD users ($33,797 vs. $90,181; p<0.001) and among adherent vs. non-adherent self-injectable/oral biologic DMARD users ($64,977 vs. $80.908; p<0.05). Conclusions: Adherence and cost savings generated, as a result, varied by the biologic DMARD route of administration. Additionally, beneficiaries' LIS status was associated with the route of administration used and the discontinuation rates, indicating an association with their out-of-pocket costs. These findings are relevant to the discussion about the proposed transition of Part B covered infusible medications under the Part D which is projected to increase the beneficiary out-of-pocket cost. The evidence on adherence can also be used for value-based insurance design involving RA biologics. Future research could leverage the findings from this study to additionally explore how biosimilar products may impact overall biologic utilization and RA treatment spending.
    • Longitudinal Patterns of Early Mental Health Service Utilization in a Medicaid-insured Birth Cohort and the Impact of Continuity of Care on the Quality of Pediatric Mental Health Treatment

      Pennap, Dinci; Zito, Julie Magno (2018)
      Background: The prevalence of pediatric mental health (MH) diagnosis and treatment have expanded in the U.S. We assessed the longitudinal patterns of incident diagnosis and new psychotropic medication use in a Medicaid-insured birth cohort. Additionally, continuity and quality of MH service utilization were assessed in a publicly-insured pediatric population. Quality care was defined by the 2009 Children's Health Insurance Program Reauthorization Act (CHIPRA) mandated children's health care quality measures. Methods: We applied longitudinal designs to Medicaid claims data from a Mid-Atlantic state (2007-2014). Using Kaplan-Meier estimators we assessed the cumulative incidence of MH service use in a cohort of newborns (aim 1). We assessed the association between relational patient-provider continuity of care and: 1) emergency department (ED) visits or hospitalizations in the 12 months following first MH diagnosis among 3-16 year olds (aim 2); and 2) the quality of follow-up care among 6-12 year old new users of ADHD medications (aim 3), using logistic regression models. Quality was defined as having ≥1 follow-up outpatient visit in the 30 days following medication initiation and ≥2 follow-up visits in the 270 days after the first follow-up visit, with a total medication supply of ≥210 days. Results: By age 8, 19.7% and 10.2% of the birth cohort (n=35,244) had received a MH diagnosis or psychotropic medication, respectively. Among medication users, 80.5% received monotherapy, 16.4% received 2 medication classes, and 4.3% received ≥3 medication classes concomitantly for ≥60 days. Compared to children with high CoC, the odds of ED visits was significantly higher among youths with low CoC [Odds Ratio(OR)=1.27; 95% CI=1.13-1.41] and low CoC was associated with greater odds of hospitalization [OR=1.17; 95% CI=1.06-1.29]. Compared to those with low CoC, children with higher continuity of care had greater odds of meeting CHIPRA initiation- [OR=1.41; 95% CI=1.25-1.60] and continuation-phase [OR=1.45; 95% CI=1.29-1.64] visit-based measures. Conclusions: Early exposure to psychotropic medications and prolonged duration of use have implications for long-term safety, highlighting the need for safety and outcomes research in pediatric populations. Our findings suggest a need for more research in the areas of quality assessment and continuity of care among youths with mental health conditions.