Poor prognosis and resistance to therapy in malignant gliomas is mainly due to highly dispersive nature of glioma cells. Aggressive infiltration in the brain parenchyma poses a serious challenge to complete tumor resection and effective treatment. Moreover, absence of valid biomarkers confounds definitive diagnosis and therapy response prediction. Identification of novel markers will require improved understanding of glioma genetics, particularly as it relates to key regulatory signals that control glioma cell migration. Since, these tumors are genetically heterogeneous, genomic explorations could improve understanding of molecular mechanisms underlying glioma migration and aid in discovery of molecular markers of clinical impact. Thus the central aim of our study is to identify novel genetic markers in patients with glioma for better prognosis, diagnosis and prediction of response to treatment. In this study we screened for genome wide copy number alterations and loss of heterozygosity in a representative glioma population of sixty patients. Alterations were detected at multiple chromosomal loci with putative tumor suppressor genes and oncogenes. In relation to molecular determinants of glioma cell migration, alterations at the Protein Tyrosine Phosphatase Receptor type Kappa (PTPRK) locus were the most frequent in our glioma population. PTPRK alterations are relevant to glioma biology as PTPRK is a cell adhesion molecule that is highly expressed in brain. However, function of PTPRK has not been described previously in gliomas. This led to our central hypothesis that PTPRK is a novel biomarker that suppresses diffusive capacity of glioma cells and thereby may improve glioma therapeutic outcome. Overexpression and knockdown experiments were performed to study PTPRK function using malignant glioma cell lines. We explored molecular mechanisms underlying PTPRK function and its effect on response to therapy. Moreover, we discovered novel PTPRK mutations by sequencing full length PTPRK transcripts in numerous glioma biopsies. Effect of these mutations on PTPRK function and response to anti-glioma therapeutics were subsequently analyzed using in vitro cell based assays. Our results suggest that PTPRK is an independent prognostic marker and a glioma tumor suppressor that is altered at both transcriptional and post-translational levels. Proteolytic processing of transmembrane PTPRK protein generates a series of fragments which are the only detectable PTPRK forms in glioblastoma. Short hairpin RNA (shRNA) mediated downregulation of PTPRK reduces glioblastoma cell migration suggesting that proteolysis of PTPRK contribute to migration of glioblastoma cells. Additionally, overexpression of wild-type PTPRK suppresses growth and migration of malignant glioma cells which correlates with inhibition of EGFR and beta-catenin signaling, and improves effect of conventional therapies for glioma. However, PTPRK mutations abrogate tumor suppressive effects of wild-type PTPRK and alter sensitivity of glioma cells to chemotherapy. These results indicate that the cell surface PTPRK protein is a tumor suppressor of prognostic and predictive value which is frequently inactivated in malignant gliomas.

Over recent decades, intranasal drug delivery has received considerable attention. The intranasal route is a non-invasive route that provides rapid drug absorption with a quick onset of action and enhanced bioavailability by avoiding hepatic first-pass metabolism and GIT degradation. The unique anatomical connections between the nasal cavity and the CNS provided by the olfactory and trigeminal nerves also makes the nasal route an interesting administration site for the purpose of CNS targeting. Epithelial transporters in the nasal mucosa modulate the uptake and efflux of substrates and their subsequent systemic absorption and/or CNS targeting. Ribavirin, a nucleoside-analog antiviral drug compound, inhibits the replication of various strains of viral encephalitis, a progressive and fatal CNS disorder, in cell culture. However, it has been reported that ribavirin was ineffective in the treatment of encephalitis following intramuscular, subcutaneous, or intraperitoneal injection. These findings suggest that ribavirin is unable to cross the blood brain barrier. The nasal route, which shows promise in targeting drugs to the brain via the olfactory region might be a potential administration route for ribavirin in the treatment of encephalitis. Ribavirin may also be a potential substrate for uptake via nucleoside transporters in the nasal mucosa. The objective of this study was to measure the permeation of ribavirin across the nasal mucosa in vitro and to evaluate the role of nucleoside transporter-mediated uptake and P-gp-mediated efflux in the total permeability of ribavirin across the nasal respiratory and olfactory tissues. The permeation of ribavirin across the nasal mucosa was investigated using bovine nasal tissues. Both respiratory and olfactory nasal tissues were exposed to ribavirin-containing solutions (concentration range 50µM - 20mM), and a non-linear uptake of ribavirin was observed in the olfactory mucosa. These results suggest the contribution of saturable transporters, specifically nucleoside transporters, in the nasal uptake of ribavirin. A linear increase in permeation with increasing ribavirin concentration was observed across the respiratory nasal mucosa which suggests a lower activity of the nucleoside transporters in these tissues. To further evaluate the role of nucleoside transporters on ribavirin intranasal uptake, NBMPR (nitrobenzyl mercaptopurine ribonucleoside) was used as an equilibrative nucleoside transporter (ENT) inhibitor. When bovine nasal tissues were exposed to solutions containing ribavirin and NBMPR, ribavirin flux was decreased by 45% suggesting that ENTs play an important role in the nasal uptake of ribavirin. The effect of P-gp-mediated-efflux on ribavirin uptake was also studied. Two inhibitors were used to evaluate the role of general energy-dependent transporters (sodium azide) and the specific role of P-glycoprotein (verapamil). Sodium azide (known to inhibit ATP synthesis and deplete cellular ATP) only resulted in a slight increase in ribavirin flux across the nasal respiratory and olfactory tissues. However, the flux of ribavirin across the respiratory nasal tissues increased significantly when 50 uM of verapamil (specific P-gp inhibitor) was included. These findings may be attributed to the effect of sodium azide to inhibit cellular ATP synthesis, and thus it might affect other transporters such as concentrative nucleoside transporters (CNTs) that utilize ATP indirectly to transport ribavirin across the cell membrane. Knowledge of the contributions of nucleoside transporters and P-gp in the nasal uptake of ribavirin is valuable in the evaluation of the potential absorption and subsequent distribution of drugs across the nasal mucosa. Reducing P-gp-mediated efflux and enhancing nucleoside transporter activity may enable the nasal administration of ribavirin to treat encephalitis.

No description available.

G protein-coupled receptors (GPCRs) play a central role in numerous biological processes, from olfaction to vision to neurotransmission and more, leading to their classification as important drug targets. Signaling through GPCRs is carried out by a number of intracellular effector proteins, such as the α subunits of heterotrimeric G proteins and Adenylyl Cyclases (AC). In this work, inhibition of protein-protein interactions to reduce GPCR-mediated cAMP formation is explored to identify compounds with potential therapeutic value. Regulators of G Protein Signaling (RGS) inhibit Gαi/o signaling, resulting in persistent AC-mediated cAMP formation. Fragment-based screening against RGS17, which has been implicated in a number of cancers, identified seven hits that bind RGS17, though only one leads to inhibition of RGS17 function in vitro. Importantly, these compounds represent a starting point for future structure-based drug discovery targeting RGS17 for the development of new chemotherapeutics. As different RGS proteins serve different functions throughout the body, achieving isoform selectivity is important for reducing potentially deleterious side effects. Analysis of the selectivity of previously described RGS4 inhibitors revealed that several are more potent inhibitors of RGS14. This work also identified the RGS proteins for which inhibitors are likely to be found (RGS14, RGS4, RGS1) and those which are likely to be more difficult drug targets (RGS6 and RGS7). Finally, inhibition of AC8 as a means to decrease intracellular levels of cAMP is explored. AC1 and AC8 are robustly stimulated by CaM, so inhibition of the AC/CaM interaction was explored as a novel mechanism of AC inhibition. Biochemical and cell-based assays were developed, and a pilot screen of 1,000 FDA-approved compounds identified six capable of inhibiting the AC8/CaM interaction. This ultimately resulted in reduced AC8 activity and cAMP accumulation, validating this interaction as a druggable target. The compounds identified were not selective for AC8 over AC1, but they have pre-clinical utility in evaluating the biology of CaM-stimulated AC activity.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by two pathological hallmarks, selective loss of dopaminergic neurons and intraneuronal protein aggregation. The presence of an endogenous neurotoxin has been implicated in the pathogenesis of the disease, to explain the observed neurodegeneration. Dopamine (DA) has been indicated to be an endogenous neurotoxin as DA readily undergoes auto-oxidation to an o-quinone capable of protein modification. However, DA is metabolized by monoamine oxidase to form the intermediate 3,4-dihydroxyphenylacetaldehyde (DOPAL) and several studies have demonstrated DOPAL to be orders of magnitude more toxic than DA. An accumulation of DOPAL may cause dopaminergic cell death via the formation of free radicals, inhibition of the mitochondrial transition pore or protein modification. The hypothesis of this work is that DOPAL, a potential endogenous neurotoxin relevant to PD, is capable of protein modification and protein cross-linking through reactivity with amine and thiol nucleophiles. Results demonstrate that elevated DOPAL concentrations in striatal synaptosomes will yield considerable protein modification. In addition, DOPAL was demonstrated to be highly reactive towards amine nucleophiles in comparison to thiol nucleophiles. However, DOPAL was demonstrated to mediate protein cross-linking through reactivity with protein thiols subsequent to modification of amines, indicating DOPAL to be a bifunctional electrophile. Furthermore, a novel isolation procedure was developed, and through a proteomics-based approach, twelve proteins were identified to be relevant to PD and susceptible to DOPAL modification. This research demonstrates increased concentrations of DOPAL lead to significant cellular consequences (i.e. protein modification) and implicate DOPAL as a potential neurotoxin relevant to the pathogenesis of PD.

The objective of this research was to estimate prophylactic G-CSF effectiveness among patients on the extensive margin, whose treatment decisions are most likely to be affected by policy changes intended to alter the G-CSF treatment rate. Using the national Surveillance, Epidemiology and End Results (SEER)-Medicare Linked Database, we studied patients 66 years or older diagnosed with Non-Hodgkin's Lymphoma (NHL) and on anthracycline in one of the 13 SEER registry areas from 1994-2002. Prophylactic G-CSF use was designated if a patient had a G-CSF claim within the first five days of the first chemotherapy cycle. The dependent variable of neutropenia hospitalization (NH) was identified within 6 months of diagnosis and was further specified as NH incidence within 21, 42, 63 and 126 days after anthracycline initiation in sensitivity analyses. Multivariate regression estimates were used to examine whether treated patients actually benefited from G-CSF. Instrumental variable estimates using local area prophylactic G-CSF treatment rates as instruments were used to estimate whether increases in the G-CSF utilization rate could lead to further reductions in the rate of neutropenia hospitalization. We found only 9.85% of study patients had prophylactic G-CSF. After adjustment for patient demographic and clinical risk factors, multiple regressions indicated prophylactic G-CSF significantly reduced NH events within six months of diagnosis date for the patients who received G-CSF (OR=0.595, 95% CI=0.384-0.922). This estimate of G-CSF's effect may be biased low from the true values of Average Treatment effect on the Treated (ATT) because patients may be selected into treatment based on unobservable risk factors. Chow F-statistics showed our instrumental variable of area prophylactic G-CSF treatment rate described a statistically significant portion in the variation of G-CSF use (F=60.46, P Among elderly NHL patients on anthracycline-based chemotherapy, our multiple regression estimates suggest that patients treated with prophylactic G-CSF reduced their neutropenia risk within six months of diagnosis date. The effect of prophylactic G-CSF on neutropenia hospitalization among marginal patients whose choices varied with local area G-CSF treatment rate was negative. Substantial G-CSF treatment benefits within the first cycle of chemotherapy were available for patients on the extensive margin. Higher treatment rates may be guaranteed to improve patient short-term benefits from G-CSF.

Solid polymer-drug dispersions are used to prepare and stabilize amorphous forms of poorly soluble drugs as a means of improving drug solubility, dissolution and bioavailability. Despite many reports on this subject, solid dispersion dissolution mechanisms have not been well understood. An early study was reported by Simonelli, Mehta and Higuchi (SMH) in 1969 and has served as a model for dispersion dissolution behavior. These authors proposed a dissolution model (SMH) which gave good agreement between their experimental results and model predictions for one drug and one type of PVP. Few researchers have applied this traditional approach (SMH) in a systematic fashion to solid dispersion systems. One difficulty is obtaining parameters needed for predictions such as polymer diffusion coefficient, diffusion layer thickness or other pertinent parameters. In this work, a general model has been developed based on the concepts in the traditional approach (SMH) and simulations with this model were performed to show how dispersion dissolution rates change with system variables. Such simulations showed underestimation of dissolution rates resulted when compounds had low solubility. In this work, solid dispersion dissolution behavior was studied systematically with a homologous compound series (alkyl-p-aminobenzoate esters, or PABA esters) and three polyvinylpyrrolidone (PVP) molecular weights (K15, K30 and K90). The PABA esters with varying solubility used in this study were methyl PABA (MePABA), ethyl PABA (EtPABA), propyl PABA (PrPABA) and butyl PABA (BuPABA). Six solid dispersions for each PABA ester and PVP (weight ratios of PVP:PABA ester 20:1, 10:1, 6:1, 3:1, 4:1 and 2:1) were prepared by a solvent evaporation method. Solid dispersions were obtained and their amorphous character confirmed by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). Intrinsic dissolution rates for these dispersions were obtained in water with a rotating disc dissolution system. Both dissolution rate of drug (PABA ester) and carrier (PVP) were measured to obtain more information on which to evaluate the release behavior. Measuring the dissolution of the polymer (dispersion agent) and drug is unique in this work and has not been done in most other reported studies. For the more soluble PABA esters (i.e., MePABA, EtPABA and PrPABA), as drug loading increased, PABA ester dissolution rates first increased and then decreased to that of the pure drug for PVP K15 and K30 dispersions. For K90 systems, drug dissolution rates were below pure drug rates and increased steadily as drug loading increased, eventually reaching that of the pure drug. On the other hand, PVP dissolution rates decreased constantly as drug content increased for all three PVP grades. However, the decrease in polymer dissolution was more pronounced for the lower molecular weight PVPs (K15 and K30) than the higher molecular weight PVP (K90). Comparison of drug and polymer dissolution behavior indicated that congruent release of both components occurred when drug loading was low (< 15%). As drug loading increased, more deviation from congruent release behavior was observed. For BuPABA, the least soluble PABA ester, precipitated BuPABA solid accumulated on the disc surface during dissolution. PABA ester relative dissolution rates were calculated and compared with the predictions from the developed general model (based on assumptions in the traditional approach). Such predictions correlated well with experimental results at high drug loadings (i.e., >25%) but at low drug loadings (i.e.,

Volume has been suggested as a surrogate quality indicator for breast cancer surgeries by several researchers. It is crucial to understand the underlying reasons as to why there is a disparity in utilization of high volume hospitals. However, the studies that investigated the mechanism underlying the disparity in high volume hospital utilization are very limited. The objectives of this study include: 1) examine the relationship between geographic differential distance and utilization of high volume hospitals; 2) investigate other demographic, socioeconomic and clinical factors that may affect patients' utilization of high volume hospitals. Multivariate logistic regressions were used to evaluate factors that impact patients' utilization of high volume hospitals. The study results showed that geographic distance is a significant factor that impedes patients' utilization of high volume hospitals, independent of patients' clinical, demographic, and socioeconomic characteristics. It was also found that white, non-Hispanic women, patients with higher education level are more likely to be admitted in high volume hospitals compared to low volume hospitals. These factors are also significant to patients' choice of medium vs. low volume hospitals. Geographic proximity is an important factor that affects patients' choice of hospital, and directing more patients to high volume hospitals should anticipate negative effects, such as increasing the cost of seeking care at high volume hospitals. Alternative strategies need to be developed to improve surgical outcomes without increasing patients' traveling related cost, such as enhancing the network between high volume hospitals and low volume hospitals, establishing radiation centers in rural areas.

Physiologically based pharmacokinetic (PBPK) models, also known as recirculation models, consist of a series of tissue and organ blocks linked together by blood circulation, mimicking the anatomical structure of mammalian body. Each tissue is divided into vascular, interstitial, and intracellular sub-compartments. Linear system analysis (LSA)-recirculation models differ from the classical PBPK model in that they characterize each organ or tissue with a unit impulse response in the framework of input-output convolution relationship rather than systems of differential equations. Target-mediated disposition (TMD) is a phenomenon where drug disposition is influenced by capacity-limited binding to a target, resulting in dose-dependent events, such as a decrease in drug clearance with increasing dose level. Erythropoiesis stimulating agents such as recombinant human erythropoietin (EPO) and Continuous Erythropoietin Receptor Activator (C.E.R.A.) exhibit TMD where their disposition and anti-anemic activity are mediated by their interaction with EPO receptor (EPOR). The objectives of this work were: 1) to develop a minimal, receptor-based LSA-recirculation model, 2) to apply the developed model in analyzing the effect of bone marrow (BM) ablation on C.E.R.A. elimination kinetics, and comparing EPO and C.E.R.A. interaction with EPOR in vivo, 3) to investigate the efficiency of the experimental design used to achieve the previous objective for estimation of the developed model parameters, and 4) To identify the physiological conditions at which TMD-compartmental models approximate TMD-recirculation models. A literature review of LSA- recirculation models is provided in Chapter 2. In Chapter 3, receptor-based, LSA-recirculation model was mathematically formulated, and applied to analyze C.E.R.A. pharmacokinetics studied in adult sheep with normal and ablated BM using a tracer interaction method (TIM). In Chapter 4, the model developed in Chapter 3 was further applied to analyze EPO and C.E.R.A. TIM data collected in adult sheep. A comprehensive, sensitivity analysis was performed in Chapter 5. In Chapter 6, statistical moments of linearized receptor-based compartmental and recirculation models were computed; and simulation of plasma drug concentrations, and receptor profiles in both structures were presented. The developed model, together with the TIM, was able to quantitatively assess the interaction of C.E.R.A. with hematopoietic and non-hematopoietic EPOR population and provide a mechanism based explanation for C.E.R.A.'s slower elimination and greater erythropoietic activity in vivo compared to EPO, despite its lower affinity to EPOR. The TIM detected a saturable interaction between C.E.R.A. and non-hematopoietic EPOR which contradicts the behavior of EPO. The TIM experimental setting is adequate for estimation of the developed model parameters. TMD-recirculation models reduce to TMD-compartmental models under conditions of well-perfused target tissue, comparable drug initial distribution volume and target tissue extracellular volume, negligible non-receptor mediated clearance, and rapid equilibrium between venous and arterial blood drug concentrations, small extracellular volume, reduced cardiac output, low receptor pool concentration, and high drug-receptor equilibrium dissociation constant.

Cancer is a group of diseases caused by uncontrolled cellular proliferation and dissemination. After heart disease, cancer is the second most common cause of death in the United States. Main treatment approaches for cancer are surgery, radiotherapy, chemotherapy, and immunotherapy approaches. However, cancer cells have ability to develop resistance to conventional chemotherapy thus lowering the efficacy of those chemotherapeutic agents including doxorubicin (DOX). DOX has been used for the treatment of various cancers. It is usually administered via continuous intravenous infusion. Nevertheless, the use of soluble DOX is often limited by its low therapeutic index. It has been reported that DOX-induced cardiotoxicity is a life-threatening adverse effect and DOX is also a potent vesicant that can cause tissue necrosis following injections. Therefore, this dissertation investigated alternative delivery approaches for DOX including systemic and local delivery systems for enhancing antitumor efficacy while reducing the side effects of free DOX. The first part of this research aimed at developing a formulation capable of actively targeting DOX to tumors. Advances in nanotechnology have provided new ways to delivering DOX into the body and to tumor sites. Among all active targeting ligands developed to date, cRGD peptide (cyclic arginylglycylaspartic acid) occupies a unique position owing to its inherent safety, biocompatibility, and targeting ability. Thus, cRGD was used here to decorate the surface of DOX-loaded PLGA-PEG nanoparticles (NPs) using two independent crosslink reactions, EDC-NHS and thiol-maleimide reactions. The results showed that the different modification reactions yielded NPs of similar size (110-140 nm diameter). All formulations exhibited provided similar burst release phases (of DOX) over the first 12 h followed by sustained release for up to 200 h. For in vivo antitumor activity, C57BL/6J mice carrying melanoma tumors were administered with cRGD-modified DOX-loaded PLGA-PEG NPs (equivalent to 8 μg DOX) by intravenous injection once every other day for up to four doses. Tumor volumes and survival were recorded. The toxicity of this therapy was examined using serum biomarkers including bilirubin, alanine aminotransferase (ALT), and aspartate transaminase (AST). Histopathology of organs (heart, lung, spleen, liver and kidney) was evaluated using hematoxylin and eosin staining (H&E) after euthanizing the treated mice. The results indicated that the cRGD-modified DOX-loaded PLGA-PEG NPs using PLGA-PEG-maleimide polymers (cRGD-DOX-M) demonstrated higher antitumor activity as compared to other groups (p < 0.05). Finally, administration of cRGD-modified DOX-loaded PLGA-PEG NPs had no significant effect on total bilirubin, serum ALT, serum AST levels or animal weight (P > 0.05). There were no signs of tissue damage in any of the tested organs as evaluated by H&E staining. The second part of this dissertation proposed to evaluate the therapeutic effect of combining chemotherapy and immunotherapy in a murine melanoma model. In this study, DOX-loaded PLGA-PEG NPs and anti-programmed death 1 (anti-PD-1) antibodies were chosen as the model of chemotherapy and immunotherapy, respectively. Anti-PD-1 antibodies have shown a great deal of promise in the treatment of melanoma in the clinic. In this study, DOX-loaded PLGA-PEG NPs were administered IV at a dose of 8 µg of DOX/dose per mouse once every other day (total of four injections). Mice in combination treatment groups were also administered with 200 µg of anti-PD-1 solution via intraperitoneal (IP) injection every 3 days for five doses. The combination therapy demonstrated higher antitumor efficacy in vivo as compared to control, soluble DOX, monotherapy of DOX-loaded PLGA-PEG NPs or anti-PD1 solution (p<0.05). Moreover, in vivo safety studies were investigated, and the results suggested that the combination therapy was safe. Lastly, DOX-loaded PLGA-PEG millirods were successfully fabricated by a hot-melt extrusion technique and characterized for in vitro release. It was demonstrated that DOX released from the millirods could be controlled by coating with polylactide (PLA). The locally implanted uncoated DOX- loaded PLGA millirods provided significantly greater antitumor activity against melanoma tumors in mice compared to naïve group and PLA-coated DOX-loaded PLGA millirods. Antitumor activity of the millirods was related to the release profile of DOX from the millirods. PLA-coated DOX-loaded millirods exhibited slower release of DOX compared to uncoated DOX-loaded millirods which probably explains the shorter survival time of mice treated with this formulation. Moreover, skin samples from tumor-free mice were also analyzed. The results demonstrated that uncoated and PLA-coated DOX-loaded millirods could be administered peritumorally without causing local skin necrosis. In conclusion, the novel systemic delivery system and local delivery system of DOX presented here have the potential to be used as alternative approaches for cancer therapy.