RSS 1.0Theses and Dissertations School of Pharmacy
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Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses database.
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Regulation of retinoid homeostasis by cellular retinol-binding protein, type 1Retinoic acid (RA) is the main active metabolite of Vitamin A, an essential diet-derived nutrient. RA signaling regulates cell differentiation, proliferation and apoptosis. RA levels are tightly regulated throughout the body via the expression and activity of catabolic and biosynthetic enzymes, and chaperone proteins, including cellular retinol binding protein, type 1 (CRBP1). CRBP1 binds to retinol and retinal, protecting them from non-specific oxidation, and facilitating their delivery to the appropriate enzymes for RA biosynthesis. CRBP1 has been shown to be decreased in disease states that display dysfunctional proliferation and differentiation, including cancers. Reduction of CRBP1 levels directly correlates with reduction in RA and restoration of CRBP1 expression has been shown to increase RA levels and positively impact RA-dependent outcomes. Research on the role of CRBP1 in disease has been limited because of its low abundance and poor immunogenicity. We have developed a targeted, bottom-up proteomics approach for absolute CRBP1 quantitation in complex biological matrices and have utilized this assay to answer important biological questions regarding the role of CRBP1 in regulating RA and RA-mediated signaling. While proper RA homeostasis is essential for biological processes throughout the body, the research in this thesis has focused on its role in the small intestine, heart, and lung. In the small intestine, RA plays an essential role in regulating the gut immune response. In instances of cellular stress in the intestine, RA levels are decreased. We have employed our CRBP1 quantitative assay, along with retinoid metabolite quantitation and quantitative gene expression, to systemically probe the mechanism of disrupted retinoid signaling in intestinal disease via an in vitro model of the small intestine. Proper RA levels are also necessary for growth and development, including heart and lung morphogenesis, and have also been shown to be disrupted in many diseases, such as heart failure and lung cancer. Using a global CRBP1 knock-out mouse model, we have also explored the in vivo effect of loss of CRBP1 on retinoid signaling via multi-omics analysis. Together these studies will help further our understanding of the mechanisms and impact of CRBP1 loss in diseases of the intestine, heart, and lungs.
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Method Optimization of a New Automated Platform for Proteome-Wide Structural BiologyProteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein HOS and dynamics requires integrated approaches, including mass spectrometry (MS), which has evolved into an indispensable tool for proteomics research. One approach readily integrated with MS is protein footprinting. In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change. The method can modify thousands of proteins in a single experiment leading to structural information across the proteome. IC-FPOP modifies proteins on the microsecond timescale making the method suitable to study fast biological processes. However, the single cell flow system developed for initial IC-FPOP experiments had temporal limitations motivating the design for a higher throughput platform. My research describes the development of a new platform for IC-FPOP entitled Platform Incubator with XY Movement (PIXY). PIXY permits IC-FPOP to occur in a sterile system using a temperature-controlled stage top incubator, peristaltic pumps for chemical transport, mirrors for laser beam guidance, and a mobile stage for XY movement. Automated communication amongst the entire PIXY system was made possible using LabVIEW software which allows the analysis of one sample in only 20 seconds. Well over 2000 proteins in HEK cells can be oxidatively modified by IC-FPOP in PIXY. This allows for a greater amount of structural information to be obtained. The capabilities of this high throughput platform permit other cell based experimental applications including fluorescent imaging and time-dependent solution transfer. PIXY’s ability to accommodate automated time points and subsequent changes over time make it a powerful tool for probing protein biochemistry in the native cellular environment.
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Effect of Excipients on the Performance of Spray-dried Amorphous Solid Dispersion (ASD) in TabletsAmorphous solid dispersions (ASD) are a proven method of improving the solubility and bioavailability of poorly soluble drugs. Immediate-release tablets are frequently used as the final dosage form for ASDs. The selection of polymers and excipients is critical for the manufacturability and bioavailability of ASD tablets. ASDs were prepared by spray drying; ASD tablets were then generated using a compaction simulator. We first studied the impact of polymer types and drug-polymer ratios on bulk powder properties, morphologies, and compaction behaviors of ASDs. Itraconazole (ITZ) and indomethacin (IND) were used as model drugs, and two polymers were used: hydroxypropyl methylcellulose acetate succinate (HPMCAS) and polyvinylpyrrolidone (PVP). The results indicated that the tabletability increased with decreasing drug loadings, except for ITZ-PVP ASDs. Multivariate analysis revealed that particle surface area was the most significant factor influencing the tensile strength of ASD tablets. Secondly, the contact angle and surface free energy of ITZ ASD tablets containing different HPMCAS grades and drug loadings were evaluated using a Drop Shape Analyzer. A larger contact angle was correlated with a higher dissolution rate, suggesting that contact angle could be a high throughput tool for screening ASDs formulations. Lastly, we investigated the influence of fillers such as microcrystalline cellulose, lactose, mannitol, and starch on drug release and stability of ITZ-HPMCAS ASDs. We discovered that the dissolution performance and physical stability of tablets were influenced by the choice of filler. The results and inferences drawn from this research will provide valuable insights into ASD formulation development downstream tablet production.
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The Regulatory Role of the Cytoplasmic Heme Binding Protein PhuS in Pseudomonas aeruginosaPseudomonas aeruginosa is an opportunistic pathogen that requires iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either system is eventually sequestered by the cytoplasmic protein PhuS, which specifically shuttles heme to the iron-regulated heme oxygenase HemO. Furthermore, a conformational rearrangement upon heme binding is necessary for the protein-protein interaction with HemO and a ligand switch between the heme coordinating ligands (His209 and His212) was proposed ot be required for translocation of heme to HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron- responsive sRNAs prrF1, F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, can also modulate PrrF and PrrH levels in response to heme. The dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host. Additional biophysical, genetic and metabolic approaches have been conducted to determine the role of the PhuS heme coordinating residues regulate the mutual exclusivity of heme and DNA binding and the resulting effects on PrrF and PrrH expression.
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Development of the Lennard-Jones Parameters for the Polarizable Classical Drude Oscillator Force FieldThe quality of Force Fields (FF) determines accuracy of observations made through molecular simulations. Accuracy of such simulations may be achieved by explicit inclusion of electronic polarization, such as via the implementation of the Drude harmonic oscillator, as in Drude Polarizable FF. Although the Drude Polarizable FF spans a large range of biomolecules including proteins, nucleic acids, lipids and carbohydrates, an expansion of its small molecule FF is essential, given the vastness of chemical space. Such an expansion must be accompanied by the optimization of van der Waals (vdW) interactions, in the context of the Lennard-Jones (LJ) formalism. Optimization of the LJ parameters is a multivariate and multi-objective problem and is one of the most challenging aspects of FF optimization. Through this thesis, we have developed a method that utilizes the sampling power of Latin Hypercube Design (LHD) and learning abilities of Deep Neural Network (DNN) to overcome some of these challenges. The model is trained on empirical pure solvent/crystal properties of a selected set of “training set” compounds, where the final selection is based on the quality of both gas phase and condensed phase properties. The optimized LJ parameters are validated for transferability on “validation set” compounds, while their ability to reproduce other experimental thermodynamic properties such as hydration free energy and dielectric constants, are also verified. Chapter 1 of this thesis presents an introduction to underlying concepts of FFs, with a major focus on polarizable FFs. Chapter 2 details development of the method, using four different chemical classes, i.e., alkenes, 3 and 4 membered rings and nitriles. Chapter 3 updates the method developed in chapter 2, addressing the challenge of parameter correlation. Chapter 4 applies the updated method to another chemical class (alkynes), while Chapter 5 concludes the thesis and is a discussion of the challenges associated with empirical FF development with a focus on LJ parameters. Overall, the method developed through this thesis addresses the most challenging aspect of FF development, i.e., LJ parameter development, implemented in a manner that could be utilized in context of development of both additive and polarizable FF.
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Design, Development, and Characterization of Gallium (III) Salophen Metallotherapeutics Targeting Heme Sensing and Iron Acquisition Pathways in Pseudomonas aeruginosaThe development of new antibiotics is outpaced by the rise in multi-drug resistant (MDR) bacteria, creating a global health problem. Pseudomonas aeruginosa, one such bacterium, is labeled as a “critical priority” pathogen by the WHO for its resistance to treatment and prevalence in hospital-acquired infections and immunocompromised patients where it is often life threatening. Adding to this problem, most new discoveries are derivatives of existing antibiotic classes rather than new strategies. Newer approaches targeting bacterial pathways critical to infection but not survival outside the host are expected to exert less selective pressure and slow resistance onset. One such strategy is interfering with bacterial iron uptake and utilization, as iron is a key micronutrient with several iron-regulated virulence traits used to counter iron-sequestering defense mechanisms of the host. P. aeruginosa can shift between the acquisition of labile iron stores and the more abundant heme-bound iron at various stages of infection, so inhibitors targeting these pathways must account for this adaptability. One such approach to targeting iron utilization in several forms is the use of gallium, which mimics ferric iron in ionic size and charge but cannot undergo critical redox processes, thus causing toxicity in the bacteria that acquire it under the guise of iron. This work describes the synthesis and characterization of Gallium Salophen (GaSal) and subsequent analogs targeting heme and iron acquisition pathways in P. aeruginosa. In this characterization, GaSal binds to a hemophore, HasAp, secreted by P. aeruginosa, and inhibits an extra-cytoplasmic function (ECF) signaling cascade with the outer-membrane receptor HasR, which is critical for sensing and adapting to host heme levels. GaSal is simultaneously a substrate for uptake, independent of its effect on HasAp. Using a combination of cell-based assays as well as in vitro target characterization and finally preliminary animal infection studies, GaSal and subsequent derivatives are shown to be promising new developments targeting several points in the iron uptake and utilization pathways of P. aeruginosa. Continued developments aim to retain such activity and include several routes towards further optimization and development as a therapeutic.
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Impact of Undertreatment of Depression on Suicide and Suicide Attempt among Children and Adolescents: A Simulation Study with Microsimulation and Agent-Based ModelsBackground: Depression is a strong risk factor for suicide, but undertreatment of depression is common among children and adolescents. The impact of undertreatment of depression on suicidal behaviors in this population is largely unknown due to the limitations of conventional data sources and methods. This dissertation research aims to overcome these challenges by using simulation models to answer two questions: 1) Is undertreatment of depression associated with increased risk of suicidal behaviors? 2) Do interventions that reduce undertreatment of depression lower the risk of suicidal behaviors? Methods: A microsimulation model simulated the 1-year suicide rate and suicide attempt risk with 12-, 36-, 52-week antidepressant treatment and no treatment in children and adolescents with depression. Modified Poisson regression estimated the suicide rate ratios and suicide attempt risk ratios for 12-, 36- and 52-week treatment compared with no treatment. An agent-based model simulated the potential impact of the following interventions in preventing suicide and suicide attempt in a synthetic population of children and adolescents: 1) depression screening (i.e. reducing untreated depression); 2) reducing attrition during depression treatment (i.e., increasing the proportion who complete the first 12 weeks of treatment); 3) suicide intervention (i.e., screen and treat individuals who need suicide care) among depressed individuals; 4) universal suicide intervention in medical settings. Results: Compared with no treatment, 12-, 36- and 52-week antidepressant treatment was significantly associated with decreased suicide rate and risk of suicide attempt. Depression screening could reduce the risk of suicide attempt (-0.64% (95% Credible Interval (CI): -1.13%, -0.11%)) only when 80% untreated depression was reduced. Universal suicide intervention showed a significant decrease in the risk of suicide attempt, which increased with the screened proportion (20%: -0.68% (95% CI: -0.87%, -0.55%), 50%: -1.47% (95% CI: -1.61%, -1.77%), 80%: -2.89% (95% CI: -4.57%, -2.31%). The other interventions did not show a significant effect in reducing the risk of suicide attempt in the population. Conclusion: Antidepressant treatment for at least 12 weeks may reduce risk of suicidal behaviors. Universal suicide intervention in medical care settings may be more effective in reducing suicidal behaviors compared with interventions that reduce undertreatment of depression.
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The Role of CAR and Nrf2 Dual Activation in doxorubicin/cyclophosphamide-based treatment of triple negative breast cancerTriple negative breast cancer (TNBC) affects 10-20% of all breast cancer cases and is associated with suboptimal outcomes due to drug resistance and/or intolerable side effects. The absence of targetable sites leaves cytotoxic chemotherapy the standard of care. Cyclophosphamide (CPA) and doxorubicin (DOX) are among the most used chemotherapeutic agents for TNBC. CPA, an alkylating prodrug, requires hepatic metabolic conversion to the rate-limiting metabolite, 4-hydroxy-cyclophosphamide (4-OH-CPA), primarily via cytochrome P450 (CYP) 2B6. Additionally, a portion of CPA is metabolized by CYP3A4 leading to a neurotoxic byproduct, chloroacetaldehyde, and an inactive byproduct, dechloroethyl-CPA. The constitutive androstane receptor (CAR, NR1I3), a nuclear receptor, regulates the expression of CYP2B6. Therefore, activation of CAR leads to preferential induction of CYP2B6 and subsequent bioactivation of CPA. On the other hand, DOX is often associated with dose-limiting cardiotoxicity. Mounting evidence suggests that this cardiotoxicity is in part attributed to the production of oxidative stress. It has been demonstrated that the activation of the nuclear factor erythroid 2-related factor (Nrf2) acts as a mediator in the protection against DOX-induced cardiotoxicity. Nrf2 regulates various antioxidant proteins and genes, such as heme-oxygenase 1 (HO-1), by binding to cis-acting antioxidant response elements in the promoter region of target genes, protecting against oxidative stress and inflammation. Our hypothesis was that dual activation of CAR and Nrf2 enhances the bioactivation of CPA while reducing DOX-mediated cardiotoxicity and improving the efficacy:toxicity ratio of CPA/DOX-based treatment for TNBC. CN06 was identified as a novel CAR and Nrf2 dual activator via high throughput screening and a chemical modification approach. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we demonstrated that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Overall, these results indicate CAR and Nrf2 as a promising therapeutic strategy to improve the therapeutic index of CPA/DOX in the treatment of TNBC.
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Biorelevant In Vitro Dissolution Models to Evaluate Poorly Soluble DrugsBiorelevant media have been devised to mimic the composition of fluids present in the stomach and intestine in fasted and fed states. They are increasingly being used in pharmaceutical product development and to predict in vivo drug dissolution. Since biorelevant media are more complex, their fabrication has been described as challenging and much remains to be understood about the mechanisms by which they are able to enhance dissolution of poorly soluble drugs. The first aim of this work was to assess the repeatability and reproducibility of current biorelevant media and determine the sources of variability when biorelevant media are used to perform dissolution across different study conditions for two model poorly soluble drugs (i.e., ibuprofen and ketoconazole). The effect of volume on small-volume dissolution using biorelevant media was also predicted. Results indicated favorable interday repeatability, favorable interanalyst repeatability, and favorable interlaboratory reproducibility. Commercial media showed greater interlaboratory reproducibility than “from scratch” media. From a nested and then crossed statistical analysis of variance (ANOVA), the rank-order importance of sources of variation overall were location > operator (nested in location) > day > fabrication method > residual. An algorithm to predict the effect of volume on biorelevant media dissolution in high, intermediate, and low solubility scenarios proved to be accurate in 13 of 16 cases. The second aim of this work was to predict dissolution into fasted and fed state biorelevant media and further devise a new model to predict the food effects on dissolution. Solubility studies, intrinsic dissolution studies, particle size analysis, and high-performance liquid chromatography (HPLC) were used to predict dissolution rate as well as dissolution and solubility enhancement of three model poorly soluble drugs (i.e., griseofulvin, ketoconazole, and ibuprofen) in fasted and fed state gastric and intestinal media over their surfactant-free counterparts. Drug dissolution rate into fed state biorelevant media was attenuated relative to drug solubility enhancement due to low colloid diffusivity. Dissolution enhancement in fasted state media was about as much as solubility enhancement due to minimal incorporation of drug into the mixed micelles. A model was also devised to predict the food effect on dissolution by considering the rate of dissolution in fed state biorelevant media over the rate of dissolution in fasted state biorelevant media and the diffusivity of colloids in each. The resulting model allowed for the prediction of a food effect on dissolution and agreed with food effects observed in vivo for the three model drugs
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Predictors and subsequent healthcare utilization associated with CDC-guideline opioid thresholds among commercially insured new chronic opioid usersBackground: The receipt and subsequent healthcare utilization surrounding new chronic opioid users (NCOUs) is multifactorial and includes clinical, demographic, and state-level factors. This study evaluated i) predictors for receipt of chronic opioid therapy informed by CDC-guideline morphine milligram equivalent (MME)/day recommendations and the short-term healthcare utilization measured by ii) total healthcare costs and iii) all-cause hospitalization after new chronic opioid use. Methods: We conducted a retrospective cohort study using IQVIA PharMetrics® Plus for Academics commercial claims with NCOUs identified between January 2014 through March 2015. NCOUs were defined as having at least 60-days coverage of opioids within a 90-day period with at least a 30-day opioid-free period prior to the date of the first qualifying opioid prescription. The short-term healthcare observation period began the 91st day or the day after last day coverage of the chronic opioid period, whichever is sooner. We placed NCOUs in one of three-tiered risk-based opioid thresholds categories: low (> 0 to < 50 MME/day), medium (≥ 50 to < 90 MME/day), and high (≥ 90 MME/day). A multinomial logistic regression was used to evaluate the impact of prescription drug monitoring program (PDMP) rigor on the receipt of respective opioid thresholds. A generalized linear model and multivariable logistic regression was utilized to evaluate the incremental total healthcare costs (ITHC) and odds of incurring a hospitalization between the thresholds, respectively. Results: A total of 16,684 NCOUs were identified. Among the NCOUs, a state with high PDMP robustness had lower odds of receiving medium (0.74; 0.62-0.90) and high (0.74; 0.59-0.92) thresholds when compared to low. When compared to low, medium and high were found to have higher ITHC, (US$, 95% Confidence Interval [CI]) $1,429 (947-1,911) and $1,775 (1,183-2,368), respectively. When compared to medium, the ITHC for high $267 (-310-844) was non-significant. When evaluating odds of all-cause hospitalization (adjusted odds; 95% CI), when compared to low, no difference was identified with medium (1.01; 0.94-1.28) or high (1.01; 0.84-1.22). Conclusion: Among NCOUs, PDMP robustness was found to decrease the odds of subsequent receipt of higher thresholds. However, short-term healthcare costs and all-cause hospitalization did not differ among the thresholds.
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Lessons Learned from the Characterization of CPSF30 – A Zinc Finger Protein Containing an Unexpected 2Fe-2S ClusterZinc Finger (ZF) proteins utilize zinc as a structural co-factor. ZFs are classed based upon the amino acid ligands that coordinate Zn. One class of ZFs is the CCCH class, which uses three cysteine and one histidine as zinc coordinating ligands. These proteins regulate RNA via a ZF/RNA binding interaction. One member of the CCCH class of ZFs is cleavage and polyadenylation specificity factor 30 (CPSF30) which contains 5 CCCH domains. In addition, CPSF30 has a ‘CCHC’ or zinc knuckle domain. CPSF30 regulates pre-mRNA processing. Experiments to determine the RNA recognition properties of CPSF30 and the role of an unusual Fe-S co-factor have been performed. A construct of CPSF30 that contains the 5 CCCH domains binds to an RNA sequence – AAUAAA – which is also called the polyadenylation signal (PAS) and present in a majority of pre-mRNA. I determined that mutations to the PAS, including some associated with human diseases, result in binding affinity changes to CPSF30 suggesting a connection between RNA binding and disease states. I isolated and characterized full length CPSF30, which contains the CCHC domain along with the 5 CCCH domains, for the first time. A major finding was that the CCHC zinc knuckle domain binds polyU RNA. Thus, CPSF30 appears to have bipartite RNA recognition. I determined that RNA recognition to these two distinct RNA sequences by CPSF30 is a competitive event and proposed a model of CPSF30/RNA binding related to alternative polyadenylation or cytoplasmic polyadenylation. The Fe-S cluster of CPSF30 was characterized. Mössbaauer and XAS spectroscopy data support a 2Fe-2S cluster with a CCCH ligand set. Reduction with dithionite followed by UV-visible and EPR spectroscopies demonstrated that the protein is redox active. Metal coupled protein oxidation/mass spectrometry indicate that ZF2 of CPSF30 is the site of the Fe-S co-factor.
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Evaluation of In Vitro-In Vivo Correlations in Topical and Transdermal Drug Delivery Systems by In Vitro Permeation Testing and Pharmacokinetic Studies for Bioavailability/Bioequivalence and Heat Effect AssessmentIn vitro-in vivo correlation (IVIVC) has gained great attention in the biopharmaceutical field because of its potential ability to predict drug performance in vivo, eliminating costly and time-consuming clinical trials. However, the regulatory guidance on IVIVC has only been established for extended-release oral dosage forms, and not for topical and transdermal formulations. An in vitro model is necessary to establish IVIVC. The present work showed that the in vitro permeation test (IVPT) using excised human skin can quantify and distinguish bioavailability (BA)/bioequivalence (BE) between a brand-name topical metronidazole gel, a bioequivalent generic gel, and a non-bioequivalent generic cream. A harmonized pharmacokinetic (PK) study in healthy human subjects was conducted to evaluate IVIVC. The scaled average bioequivalence approach was used to establish BE between the gels and distinguish the cream from the gels. The metronidazole PK study didn’t provide reproducible serum levels; therefore, IVIVC’s predictability was weak. The present work also showed IVPT’s ability to predict BA for two rivastigmine TDS under transient heat exposure. A harmonized PK study was conducted. A Level C and a Level A IVIVC were established under baseline temperature (32.0°C). IVPT demonstrated significantly increased maximum flux for both TDS with a comparable fold enhancement. However, the PK study didn’t demonstrate a consistent heat effect on maximum serum concentrations of rivastigmine under elevated temperature (42.0 °C). The IVIVC’s predictability for the heat effect was therefore weak. Encouragingly, findings suggested that IVPT is useful in assessing BE for topical drug products. For hydrophilic drugs, such as metronidazole, it is possible that IVPT may have limited predictability in vivo if significant absorption of the drug occurs via the shunt route, and/or if significant reproducible systemic absorption can’t be quantified from dosing the semisolid over a large surface area. In conclusion, IVPT was able to quantify the magnitude of heat effect on the absorption from rivastigmine TDS, and data also suggested that under baseline temperature, IVPT will likely show good predictability of TDS performance in vivo. However, experimental conditions should be further altered to validate IVPT’s usefulness in the prediction of TDS BA in vivo under the heat exposure.
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In Vitro Dissolution, Supersaturation, and Permeation to Predict In Vivo Oral Drug AbsorptionMost new drug candidates are poorly water soluble, and as such have limited absorption and bioavailability. The current research focuses on two major aspects of oral drug absorption research (i) characterization of spray dried dispersions (SDDs) of a poorly soluble drug to elucidate the factors that impact overall formulation performance and absorption, and (ii) development and utilization of an in vitro dissolution-permeation model to predict in vivo human absorption and performance of various drugs representing different Biopharmaceutic Classification System (BCS) classes. The first aim was to characterize itraconazole (ITZ) SDDs from three grades (L, M and, H) of hydroxypropyl methylcellulose acetate succinate (HPMCAS) polymer based on supersaturation kinetics and molecular interactions that contributed to the overall SDD performance and drug absorption. A combination of in vitro and solution state drug-polymer interaction studies was used. Results indicated that a high supersaturating concentration, and rate and extent of supersaturation caused the largest increase in absorption. We concluded that such stronger hydrophobic interactions between drug and polymer were relative detriment to ITZ absorption for ITZ and HPMCAS SDDs. Conventional dissolution testing has been modified in terms of in vitro design and medium composition. Biorelevant media closely simulate the composition of human gastrointestinal fluids but are challenging to prepare and contain multiple components. The second aim was to assess the similarity of dissolution profiles from biorelevant media. Results indicated favorable interday repeatability, interanalyst repeatability, and interlaboratory reproducibility suggesting this approach could be incorporated into clinically relevant dissolution models. An in vitro model that can capture the dynamic interplay between dissolution and permeation is sought for poorly soluble drugs, with potential to guide the drug development process and product life cycle management. The third aim was to characterize and utilize a dissolution-hollow fiber membrane (D-HFM) system to correlate in vitro and in vivo parameters for several BCS classes drugs. Model predictions and experimental D-HFM system studies were performed using drug solutions and drug products. Results indicated close agreement between predicted and observed drug permeation profiles, and between the D-HFM system derived in vitro and in vivo permeation constants and absorption profiles.
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Novel Measurements of Impaired Hepatobiliary and Intestinal TransportTransporters are membrane-bound proteins that play critical roles in the pharmacokinetics of endogenous substances and drugs, including their enterohepatic recirculation. The overall objective of this dissertation was to devise and apply novel measurements to assess impaired hepatobiliary and intestinal transport. The first objective was to assess transport of novel bile acid analogues within the enterohepatic circulation. Multi-fluorinated bile acids (MFBA) were assessed for their potential utility as visual probes of transport within live mice using 19F magnetic resonance imaging (MRI). Results indicate that MFBA have the potential to be used as a novel diagnostic test for bile acid diarrhea. However, due to obstacles in commercialization of MFBA using 19F MRI, we developed and assessed the use of novel nitroxide-bile acid conjugates (NBAC) as a potential alternative approach using conventional MRI. However, these compounds did not interact with intestinal or hepatic bile acid transporters. The second objective was to evaluate metformin-mediated transport concerns. First, a comprehensive literature review of metformin was assembled in a biowaiver monograph to evaluate the risks of allowing a Biopharmaceutics Classification System (BCS)-based biowaiver. Second, the in vivo metformin-mediated repression of the bile salt export pump (BSEP) was assessed. We conducted a clinical study to assess the effect of oral metformin on the pharmacokinetics of BSEP probe substrates. Results indicate that metformin increased pravastatin plasma exposure due to repression of BSEP-mediated elimination of the drug, and reduced BSEP-mediated reabsorption of conjugated primary bile acids in plasma, which are otherwise highly recycled via enterohepatic recirculation. The third objective was to evaluate potential excipient risks in allowing BCS-based biowaivers. A comprehensive assessment was conducted regarding the current regulatory framework in drug formulation and an evaluation of excipients that have been classified as absorption-modifying excipients (AME). Polysorbate 80 is recognized as an AME based on in vitro and preclinical data. Thus, in a clinical study in 12 healthy adult volunteers, we assessed the effect of oral polysorbate 80 on various drugs. Results indicate that polysorbate 80 had no impact on valacyclovir, chenodeoxycholic acid, or enalaprilat absorption.
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Healthcare Resource Utilization and Costs Associated with Antiretroviral Regimen ComplexityBackground Single tablet antiretroviral regimens (STR) offer one-pill, once-a-day dosing for people living with HIV. In commercially insured populations under 50 years of age, these formulations have been associated with better adherence and lower healthcare resource utilization and costs compared to multi-tablet regimens (MTR). The objectives of this dissertation were to detail patterns of STR and MTR use and the associated healthcare utilization, incremental costs, and overall budget impact among Medicare beneficiaries living with HIV. We relied on Andersen’s Behavioral Model, Generalized Estimating Equations (GEE), instrumental variable regression, and budget impact modeling to answer these questions. Methods Using a 5% random sample of Medicare Fee-for-Service beneficiaries from the Chronic Conditions Data Warehouse (CCW) we identified Medicare beneficiaries living with HIV from January 1, 2014 through December 31, 2019. We used generalized estimating equation to estimate trends in antiretroviral and other healthcare resource utilization. We estimated 1-year incremental total direct medical costs with an instrumental variable approach via two-stage least squares regression. We utilized these estimates of utilization and costs to build a five-year budget impact model. Results Among 8,316 Medicare beneficiaries contributing 282,258 person-months of observation, STR receipt was 24% more likely in 2019 (57%) than it was in 2014 (27%), and integrase-based regimens were predominant among both STR (80%) and MTR (74%) by 2019. Among 7,044 beneficiaries who initiated a new ART regimen, the instrumented STR variable was associated with statistically significant lower 1-year incremental total direct medical costs compared to MTR (-$13,487.70, (95% Confidence Interval: -$16,750.77 to -$10.224.63), p<.001). Our budget impact model predicted $1.8 billion in additional costs in year 5 under a scenario where current trends in ART utilization and list prices continue compared to a scenario where they remain at 2019 levels. The excess costs were driven by $2.3 billion in greater ART spending among the 147,953 beneficiaries living with HIV despite reductions in all other healthcare resource utilization categories and related savings. Conclusion These findings demonstrate the adoption and predominance of STR and integrase-based regimens among Medicare beneficiaries living with HIV. STRs were associated with significantly lower 1-year total direct medical costs compared to MTR among an understudied population. This study improves upon prior study designs and demonstrates the usefulness of prescriber preference IVs. However, the magnitude of the reduction in HcRU associated with STR may not be adequate enough to justify increases in ART costs as was observed during the study period. Though still small in number, the costs of caring for people living with HIV in Medicare will likely continue to grow as a proportion of total spending and will be markedly impacted by any policies aimed at controlling expenditures on prescription drugs.
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Patterns, Factors and Outcomes associated with Gabapentin use in Combination with Opioids and Benzodiazepines among Social Security Disability Insurance (SSDI)-eligible Medicare BeneficiariesBackground: Little is known about the patterns, factors, and public health outcomes associated with concurrent utilization of gabapentin, opioids, and benzodiazepines (GABA+OP+BZD) Objective: To examine the patterns, factors, and public health outcomes associated with concurrent utilization of GABA+OP+BZD among Social Security Disability Insurance (SSDI) eligible beneficiaries. Methods: Using a 5% sample of 2013-2016 Medicare data, we utilized a retrospective cohort design to examine the following patterns of concurrent utilization: monotherapy, dual therapy, tri-therapy, switching, augmentation, discontinuation, and continuation. Similarly, a retrospective cohort design was utilized to examine the sociodemographic and clinical factors associated with the longest concurrent medication utilization episode, defined based on the overlap of prescriptions for GABA+OP+BZD. We used a nested case control design to examine the association between concurrent utilization of GABA+OP+BZD and adverse outcomes (respiratory depression, substance and opioid related overdose, and adverse drug-related events) among disabled beneficiaries with acute pain [AP], chronic pain [CP], and mental health conditions [MH]. Results: Among disabled beneficiaries, gabapentin initiators were significantly more likely to become dual and tri-therapy users (p<0.01) and to augment therapy (50.1%) when compared to opioid (28.7%) and benzodiazepine (38.7%) users; the majority augmented within 2-months after initiating therapy. Back pain [AOR(95%CI): 1.23(1.07-1.41)], chronic pain [1.27 (1.07-1.51)], mental health [1.16 (1.02-1.33)], opioid dose [1.05 (1.03-1.06)] and duration [1.07 (1.06-1.07)], and benzodiazepine duration [1.06 (1.05-1.06)] were positive predictors of having longest concurrent use involving GABA+OP+BZD. Concurrent GABA+OP+BZD use was associated with increased odds of respiratory depression [AP: 1.35 (1.19-1.52), CP:1.24 (1.11-1.38) and MH: 1.16 (1.02-1.32)], opioid related overdose [AP: 1.43 (1.04-1.98), CP: 1.47 (1.07-2.00) and MH: 1.44 (1.04-2.00)], substance related overdose[AP: 1.77 (1.26-2.50), CP: 1.70 (1.24-2.34) and MH: 1.92 (1.31-2.82)] and adverse drug related events[AP: 1.36 (1.22-1.50), CP: 1.23 (1.10-1.36) and MH: 1.15 (1.02-1.30)]. Conclusion: Our study provides the first evidence of patterns, factors, and outcomes associated with concurrent utilization of GABA+OP+BZD. Given noted adverse outcomes associated with GABA+OP+BZD, it is imperative that the benefits and risks of co-prescribing these medications be examined comprehensively, especially for those at the greatest risk of being prescribed these medications.
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Exploiting Vulnerabilities in Cancers with Activated Extracellular Signal-Regulated Kinase (ERK1/2)Constitutively active extracellular signal–regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. ERK1/2 pathway inhibitors are important therapies for treating many cancers, however, acquired resistance to most protein kinase inhibitors limit their ability to provide durable responses. Few studies have looked at the adaptive proteome responses of BRAF/MEK dual inhibitor resistant cells, and fewer still have established the utility of allosteric inhibitors targeting the ERK2 protein. The overall goal of the current study was to identify signaling mechanisms of therapeutic resistance and further investigate a previously identified inhibitor of melanoma cell growth with putative activity with the protein ERK2. To test this, we hypothesize that vulnerable targets can be identified for therapeutic intervention, and test if alternative inhibitors exhibit clinical potential for melanoma treatment in two specific aims. In Aim 1, we characterized the global protein changes happening in constitutively-activeERK1/2 melanoma cells models that developed resistance to BRAF (PLX4032) and MEK1/2 (AZD6244) inhibitors using mass spectrometry. We additionally identified putative targets for treatment and analyzed the potential for a metabolic inhibitor, niclosamide. In Aim 2, studies were focused on elucidating the structure-activity relationship and binding mechanism of a ERK2-specific inhibitor developed in house, SF-3-030. We identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF(V600E). We then identified the mechanism of action of the inhibitor in preventing melanoma growth using mass spectrometry analysis. Overall, these studies help to elucidate how cells overcome currently used clinical therapies, what vulnerable markers can be identified and used for therapeutic intervention, and that inhibitors alternative to the clinical standard can show potential for melanoma treatment.
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Association of Patient Cost Sharing and Area Deprivation with Multiple Myeloma Treatment Receipt and OutcomesIntroduction: Advances in multiple myeloma (MM) treatment have improved survival, but there are increased concerns about treatment affordability and access. This study assessed 1) how cost-sharing assistance and area deprivation affect treatment receipt, 2) changes in the patient cost responsibility and disparities in treatment over time, and 3) how the low-income subsidy (LIS), which lowers Part D cost sharing, and area deprivation affect treatment access and survival. Methods: Using the Surveillance, Epidemiology, and End Results-Medicare database, we identified patients diagnosed with MM. The effect of cost-sharing assistance and area deprivation on treatment was estimated using multilevel logistic regression. We estimated the monthly incremental patient cost responsibility among MM patients compared to non-cancer controls and examined changes over time (2007-2011, 2012-2016). The effect of diagnosis period and area deprivation on treatment was estimated using multilevel logistic regression. The association between LIS, area deprivation, and mortality was estimated from a mixed-effects Cox proportional hazards model. We assessed whether treatment mediates the association between LIS and mortality. Results: Individuals receiving Medicare Parts A, B and D cost-sharing assistance had higher odds of receiving treatment compared with non-recipients (OR=1.21; 95%CI: 1.01–1.45). Living in the most deprived area (Quintile 5) was associated with lower odds of receiving treatment compared with the least deprived area (Quintile 1; OR=0.81; 95%CI: 0.65–0.99), but there was no difference in the other quintiles. The difference in the estimated monthly incremental patient cost responsibility between 2012-2015 and 2007-2011 was $58 [average marginal cost; 95%CI: $12–$105]). The difference in the likelihood of any treatment receipt between Quintile 1 and 5 decreased, but the difference in the likelihood of receiving a novel agent-based regimen increased. The mortality hazard was higher for LIS recipients relative to non-recipients in Quintiles 1, 3 and 4 (HR=1.50, 1.38, 1.28; p=0.0001), and there was no difference in the other two quintiles. This association was partially mediated by treatment receipt. Conclusions: The patient cost responsibility for MM care increased over time. The type of cost-sharing assistance and area deprivation affect treatment receipt, although not across all quintiles. LIS receipt did not confer a survival benefit.
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A Cluster Analytic Approach to Identify Insomnia Subtypes and Their Relationship with Economic OutcomesINTRODUCTION: Insomnia is a heterogenous condition with respect to underlying risk factors, presentation of symptoms, comorbidities, disease course, and outcomes. Consequently, individuals with insomnia may also have varying patterns of healthcare resource utilization and costs. However, the impact of insomnia heterogeneity on economic outcomes is not known. METHODS: We used an integrated claims-electronic health records dataset to identify individuals aged 18-64 with insomnia between 2009-2018. A k-modes clustering algorithm with a Jaccard coefficient similarity measure was used to identify clinically relevant insomnia subtypes based on sociodemographic, comorbidity, behavioral, life event, family history, medication use, vital sign, and insomnia symptom-related characteristics. An optimum cluster solution was chosen based on clinical interpretability and significance. Insomnia clusters were compared on baseline characteristics using Chi-square tests. Logistic regression models were used to identify the association between cluster membership and binary outcomes (inpatient hospitalization, emergency department [ED] visits). Generalized linear models were used to assess similar associations with count physician office visits, non-physician outpatient visits, prescription drug fills) and cost outcomes associated with all points of service. RESULTS: A total of 17,124 individuals with insomnia met the study inclusion criteria. The cluster analysis resulted in a five-cluster solution. The clusters were labelled as ‘Insomnia associated with obesity and hypertension’ (28.6%), ‘Insomnia associated with mental health conditions and chronic pain’ (25.4%), ‘Insomnia associated with older age, high comorbidity burden, and fatigue’ (24.6%), ‘Insomnia associated with substance use disorders’ (5.2%), and ‘Insomnia associated with overweight status, alcohol use, and low comorbidity burden’ (16.2%). Relative to the reference cluster ‘Insomnia associated with overweight status, alcohol use, and low comorbidity burden’, individuals in cluster labelled as ‘Insomnia associated with older age, high comorbidity burden, and fatigue’ displayed higher total healthcare costs (cost ratio [CR]: 1.46; 95% CI: 1.32, 1.62) primarily driven by higher inpatient (CR: 1.68; 95% CI: 1.48, 1.91) and prescription drug fill (CR: 1.49; 95% CI: 1.34, 1.65) costs. CONCLUSION: Findings from the present study can help improve our understanding about developmental trajectories for insomnia diagnosis and facilitate the design of tailored interventions that target those at the highest risk for adverse economic consequences.
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Molecular Mechanisms of Intestinal Bile Acid Transport and Immunomodulatory Potential of Bile AcidsBile acids are catabolic products of cholesterol that play an important role in the digestion of dietary facts, lipid soluble vitamins, and drugs, as well as a role in immune regulation and glucose homeostasis. They function as complex signaling molecules to prevent intracellular accumulation of bile acids and modulate bile acid and cholesterol homeostasis via activation of a nuclear receptor, Farnesoid X receptor (FXR), to repress bile acid uptake transporters and enhance bile acid efflux transporters. The human sodium dependent bile acid transporter (ASBT) is a highly regulated intestinal uptake transporter that acts as the rate limiting step in bile acid transport in the enterohepatic circulation. Targeted ASBT inhibition is currently being investigated for use in cholestasis, hyperlipidemia, chronic idiopathic constipation, and type 2 diabetes. While studies on post-translational modifications (PTMs) have revealed N-linked glycosylation and phosphorylation as regulators of ASBT, ASBT regulation is still poorly understood. The lipid-based PTM, S-acylation, is the reversible addition of an acyl chain, via a labile thioester linkage, onto cysteine residues, thereby increasing the affinity of proteins to cellular membranes. In this work, we show that human ASBT is S-acylated and that S-acylation is vital for ASBT function, cell surface expression, substrate transport kinetics, and protein stability. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Mutation of cytosolic tyrosine residues resulted in decreased ASBT S-acylation suggestive of crosstalk between both PTMs and the existence of multiple PTM-based proteoforms. Finally, we investigate functional implications of the potential acyl transferases responsible for ASBT acylation. Overall, we have provided valuable insight on human ASBT regulation and highlighted the necessity for further investigation of the impact of PTM proteoform in drug development. While understanding ASBT regulation is vital in addressing intestinal and hepatobiliary disease states, the extent as to which its substrate, bile acids, play in other molecular processes is important in fully understanding the broader relevance of intestinal bile acid transport and bile acid signaling. Bile acids have emerged as complex signaling molecules in glucose homeostasis and immune regulation where they can activate specific receptors to increase insulin secretion and exert anti-inflammatory responses from mucosal immune cells, respectively. Using immunological approaches, we provide preliminary evidence and scientific perspective on the use of bile acids in nanoformulation that aims to exploit the immunomodulatory potential of bile acids.
