Neutrophil serine proteases (NSPs) play an important role in the innate immune system. However, when the balance between NSPs and their endogenous protease inhibitors (PIs) is disrupted, they also play a critical role in the pathogenesis of inflammatory lung disease. Excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, endogenous PIs counteract these effects by inactivating NSPs. In inflammatory lung diseases, including chronic obstructive pulmonary disease, cystic fibrosis, emphysema and acute lung injury, there are insufficient levels of endogenous PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP proteolytic activity in conditions of inflammatory lung disease, in order to restore the NSP-endogenous PI balance and decrease the inflammatory response. The Kerns laboratory previously demonstrated that heparin derivatives substituted with structurally unique aromatic residues bind with high affinity and selectivity to select Glycosaminoglycan-binding proteins, and more recently, using the neomycin, kanamycin and apramycin aminoglycosides as chemical scaffolds a panel of N-arylacyl O-sulfonated aminoglycosides was prepared as novel structural mimics of heparin. The hypothesis guiding the study presented here is that structurally unique N-arylacyl O-sulfonated aminoglycoside derivatives will selectively bind and modulate the function of NSPs both in vitro and in vivo, thus representing novel lead structures for future development of a new class of therapeutic agents capable of modulating excessive NSP activity in the lung. To this end, the first objective was to screen the recently synthesized panel of N-arylacyl O-sulfonated aminoglycosides for their ability of inhibit each of the NSPs. The inhibitory profile of each N-arylacyl O-sulfonated aminoglycoside with respect to HNE, CatG and Pr3 was characterized to determine if one N-arylacyl O-sulfonated aminoglycoside could inhibit multiple NSPs. Furthermore, the mechanism of protease inhibition of two lead N-arylacyl O-sulfonated aminoglycosides, identified in the initial screen, was elucidated using CatG as the representative NSP. The N-arylacyl O-sulfonated aminoglycosides were also evaluated for their ability to inhibit the proteolytic activity of the three NSPs in a cell based assay that evaluates the ability of test compounds to inhibit NSP-mediated detachment of A549 lung epithelial cells from the surface of a 96-well plate. After concluding the in vitro analysis of the panel of N-arylacyl O-sulfonated aminoglycosides, one lead compound that inhibited all three of the NSPs was further evaluated in vivo to determine if this class of compounds exhibits any overt toxicity and if so at what concentrations, and secondly if our lead compound is able to decrease LPS-induced acute inflammation in the lung. The results of these studies validate the approach of using N-arylacyl O-sulfonated aminoglycosides to target the three NSPs as a new therapeutic method for the treatment of inflammatory lung diseases.

Type 2 diabetes (T2D) has been studied for decades. Many risk factors of T2D have been identified, but few studies were designed to investigate the pharmacokinetics/ pharmacodynamics (PK/PD) risk factors preceding the onset of T2D. Moreover, although the disease progression of T2D has received considerable attention, little is known about the disease development of T2D. It is important to understand the temporal changes of the risk factors of glucose and insulin kinetics during the development of T2D for a better understanding of the etiology of T2D. The objectives of this work are: 1) to develop a population-based glucose-insulin PK/PD model and identify the PK/PD risk factors preceding the onset of T2D, 2) to develop a methodology to evaluate the development of T2D, 3) model the time-course of the disease development based on the disease development variables (DDVs) derived from repeated intravenous glucose tolerance tests (IVGTT) and oral glucose tolerance tests (OGTT). The central hypothesis is that the development of T2D can be described and characterized by the glucose-insulin kinetics by employing a population-PK/PD based disease development analysis. To summarize, a glucose-insulin kinetic model was developed and presented in Chapter 2. The pharmacokinetics/pharmacodynamics (PK/PD) risk factors preceding the onset of T2D were investigated using a population-based Bayesian nonlinear hierarchical model. In Chapter 3, a methodology describing the disease development of T2D was developed based on four important DDVs of T2D, namely fasting blood glucose (FBG), fasting serum insulin (FSI), homeostatic model assessment of insulin resistance (HOMA-IR) and body mass index (BMI). These DDVs were investigated for their temporal patterns and relationships to the time-course of the development of T2D. The proposed model enables a quantitative, time-based evaluation of the development of T2D in this high risk population. In Chapter 4, the DDVs derived from repeated IVGTTs were evaluated. By applying the mixed effect analysis, important DDVs were identified as potential new biomarkers of T2D. Chapter 5 is an extension of application of the disease development analysis based on the DDVs derived from OGTTs. Chapter 6 is the conclusions and future works of this thesis. The proposed population model of glucose-insulin kinetics has demonstrated that pharmacokinetic differences exists for the high risk population and can be helpful for prediction of T2D. By applying the proposed disease development analysis, the time-dependency and temporal patterns of the DDVs can be identified. An examination of the temporal changes in DDVs for the glucose-insulin system before the diagnosis of the disease provides a quantitative evaluation of the pathophysiological evolution of T2D and is valuable in predicting T2D.

Pyronaridine/Artesunate (PA) 3:1 fixed dose combination is a novel artemisinin based combination therapy (ACT) for the treatment of acute uncomplicated Plasmodium falciparum or Plasmodium vivax malaria. There are limited published data on the pharmacokinetics of pyronaridine in humans, an area of importance to achieve optimal therapeutic outcome. Chapter 1 of this thesis contains a general review of malaria and pyronaridine basic pharmacokinetic properties. In Chapters 2 and 3, population pharmacokinetic models of pyronaridine in healthy and malaria-infected adult subjects as well as pediatric malaria patients are presented. Pyronaridine pharmacokinetics were best described by a two compartment model with first order absorption and elimination from the central compartment. Covariates of malaria infection, body weight, and age significantly explained pyronaridine pharmacokinetic variability in the adult population; whereas in the pediatric population model, an allometric scaling approach was utilized to incorporate the effect of body weight, age and formulation were retained as significant covariates. Chapter 2 addresses the differences in the pharmacokinetic parameters between healthy subjects and malaria-infected patients, and the lack of such differences between healthy Korean and Caucasian subjects as well as between malaria-infected Asian and African patients. In addition, Chapter 3 places particular focus on describing any formulation-specific effects associated with the granule formulation, in order to confirm the weight-based dosing of Pyramax® granules. Chapter 4 assessed the relative bioavailability of pyronaridine of the tablet and granule formulations and revealed a lack of any clinically relevant formulation-related difference in pyronaridine exposure with the geometric mean ratios and 90% confidence intervals for pyronaridine primary and secondary outcomes of interest being fully within the no relevant difference range of 80 – 125%. Finally, in Chapter 5, Generalized Estimating Equations (GEE) and logistic regression models were used to investigate the statistical association of the likelihood of liver enzyme elevations with pyronaridine pharmacokinetic parameter values in healthy and malaria-infected adult subjects. It aimed at investigating the higher observed likelihood of liver enzyme elevations in healthy subjects and addressed the shortcomings which may have influenced the significance of the results.

Cocrystals have two or more neutral components (drug and cocrystal former) in the same crystal lattice which are held together via non-covalent bonds. Since the presence of the cocrystal former alters the energy of the drug molecules and their physicochemical properties, the drug dissolution characteristics can be manipulated by cocrystallization. A diffusion-convection-reaction (DCR) model has been developed to predict cocrystal intrinsic dissolution rates from the rotating disk system. In this work, the DCR model was used to further analyze the dissolution characteristics of 1:1 and 2:1 cocrystals. The effects of diffusion, convection and complexation kinetics and equilibrium were evaluated. In addition, the sublimation properties of cocrystals were also studied in order to evaluate the importance of solute-solute and solute-solvent interactions during solid dissolution. In the first study, the phase-solubility study was performed for the acetaminophen(ACE)-2,4-pyridinedicarboxylic acid(PDA) and thymol(THY)2-4,4’-dipyridyl(DP) cocrystals. The aqueous dissolution rates of cocrystals ACE-PDA, THY2-DP, acetaminophen(ACE)-theophylline(THP) and theophylline(THP)-salicylic acid(SA) and their individual components were measured at various disk rotation speeds. The DCR model could predict their dissolution rates and the convection effect with proper model parameters. Moreover, the dissolution rate was predicted to be proportional to the apparent solubility (Sapp), emphasizing solubility as the dominant factor in a cocrystal’s reactive dissolution even with possible variation in reaction kinetics. In the second study, cocrystal dissolution was further analyzed by the DCR model for unionized 1:1 AB and 2:1 A2B cocrystals. For model analysis purpose, the model parameters were varied by several orders of magnitude to describe the dependencies of cocrystal dissolution more accurately. The congruent dissolution behavior can be predicted if all dissociated species are equally diffusive. If all diffusing species have the same diffusion coefficient (D), the cocrystal dissolution rate is independent of the complexation equilibrium and proportional to Sapp or D0.66, as expected. Otherwise, it is dependent upon the complexation equilibrium and all diffusivities. The dissociation kinetics appeared to not affect cocrystal dissolution rates, but it can affect the maintenance of reaction equilibrium in the diffusion layer. The third study focused on the ACE-PDA cocrystal dissolution into various media. Its dissolution rates in 0.05 M NaCl and 0.5 M pH 3.5 phosphate buffer showed that the presence of ions can affect diffusivities of cocrystal components. In pH 1.8-7 media, the cocrystal dissolved faster with higher medium pH or more concentrated buffer solutions, due to more PDA ionized at solid surface. The DCR model predicted the dissolution rate with adjusted diffusion coefficients. It also simulated that the medium effect on cocrystal dissolution rate arose from the change in apparent solubility. In the fourth study, the dissolution and sublimation characteristics of two thymol cocrystals (THY2-DP and thymol(THY)2-1,2-di(4-pyridyl)ethylene (BPE)) were evaluated and compared with their individual components. The sublimation rate was taken as the rate of weight loss at constant temperature. It was found that both cocrystals were less soluble than their components but they sublimed faster than the cocrystal formers and more slowly than thymol. This indicates that after forming cocrystals, the escaping tendency of thymol was reduced while that of cocrystal formers were increased. But during dissolution, the solute-solvent interaction also plays a role. Water may interact with cocrystal formers more than with the cocrystal complexes, enhancing the dissolution.

Glioma is the most common and aggressive type of primary intracranial tumors. The poor prognosis of glioma patients has not changed since decades despite the advancements in diagnostic tools and treatment strategies. The inability to accurately predict the survival and response to anticancer therapy emerges from several factors including the high heterogeneity of the tumor and the inadequacy of the currently applied world-health organization (WHO) classification system. Both factors result in high variability in the clinical outcome due to variable sensitivity to treatment. Thus, molecular classification represents an important strategy for better categorization of glioma patients and for their stratification to anticancer therapy. Our high-throughput screening analysis for the identification of genetic aberrations in the glioma study population revealed high frequency of chromosomal instabilities in glioma specimens. This indicates that DNA-repair mechanisms are defective which may have contributed to gliomagenesis and progression. Furthermore, DNA-repair represents an integral interplayer in the determination of glioma response to anti-neoplastic agents due to the fact that the majority of the currently applied agents possess their cytotoxicity via DNA-damaging actions. TDP1 has been implicated in the resistance to various types of anticancer agents in vitro, including radiation and topoisomerase poisons due to its ability to repair various types of DNA lesions. Moreover, it has been found to be overexpressed in different kinds of cancers, however, its relevance in glioma has not yet been studied. In this work we show that TDP1 is overexpressed in patients with malignant glioma compared with non-tumor cases. An ascending increase of TDP1 protein expression with a correlation with glioma grade is evidenced in the astrocytic lineage and glioblastoma multiforme samples expressed the highest levels. Moreover, we show an association between high TDP1 transcript levels and the poor prognosis of glioma patients. These findings suggest that TDP1 plays an important role in gliomagenesis; however, the underlying molecular mechanisms need to be identified. For an exploration of the predictive value of TDP1 in malignant glioma the correlation between TDP1 level and the sensitivity of malignant glioma cell lines to anticancer therapy has been investigated. We show that manipulating TDP1 level alone in malignant glioma cell lines is not sufficient to modulate their response to treatment. TDP1 overexpression or knockdown resulted in changes in the transcript levels of several DNA-repair genes including MGMT, topoisomerases and the base excision repair genes PARP-1, PNKP and XRCC1. This hindered the ability to characterize the role of TDP1 to modulate the in vitro sensitivity of malignant glioma cell lines to topoisomerase poisons and temozolomide. Nonetheless, this emphasizes the importance of the comprehensive role of several DNA-repair genes for a finalized DNA-repair process to determine the sensitivity of tumor cells to DNA-damaging anticancer agents. Finally, we tested the ability of inhibiting TDP1 enzyme activity to potentiate or synergize the cytotoxicity of topoisomerase poisons using small-molecule ligands. We show that treatment of malignant glioma cell lines with a combinational therapy of a small-molecule TDP1 inhibitor and a topoisomerase poison enhances their sensitivity to the latter drug but with minimal efficacy. As a conclusion, the characterization of TDP1 in glioma is a novel finding that can aid in enhancing the diagnosis and prognosis of patients. However, its role as a predictive biomarker for better stratification of patients to therapy needs further investigation.

Amphotericin B (AmB) is an amphiphile antifungal agent composed of lipophilic and hydrophilic structures and is known to aggregate in aqueous solution. The effect of substrate aggregation on the degradation kinetics of aqueous AmB was studied. Aggregation state of AmB (0.0108 mM) in 10.0%v/v methanol aqueous solutions were pH dependent. The dissociation equilibrium constant (Kd) values suggested that monomeric form was predominant in acidic and alkaline condition and aggregated form appeared predominantly in neutral condition. At methanol concentration above 35.0%v/v, 0.0108 mM AmB in reaction mixtures presented in a monomeric form regardless of pH. The degradation pathways of AmB were found to be pH-dependent. The effect of oxidants, antioxidants, oxidation initiators and chelators suggested that AmB was susceptible to oxidation in acidic and neutral pH regions which led spectral changes associated with the heptaene moiety. In basic conditions (pH > 9), AmB underwent hydroxide-catalyzed ring-opening lactone hydrolysis. A degradation model describing substrate loss was constructed based on the kinetics of substrate loss. The pH-rate profile displayed three regions: specific acid-catalyzed degradation at pH below 4, a specific basic-catalyzed hydrolysis at pH above 9 and a pH-independent degradation in the neutral pH range 4 – 9. The effect of methanol on degradation kinetics in the neutral pH region indicated that aggregated AmB was more susceptible to oxidative degradation than monomeric AmB.

The herbicide atrazine is one of the most commonly used pesticides in United States. Atrazine was banned in the European Union in 2005 because of its ubiquity in drinking water; however, in The United States more than 75 million pounds of atrazine are used annually, especially in the Midwest. Atrazine has many adverse health effects including enhancing developmental, immunologic endocrine alterations. Studies have reported that exposure to atrazine causes dopaminergic toxicity and mitochondrial dysfunction; these cellular changes have been linked to an increase in the incidence of Parkinson's disease. The objective of this study is to characterize atrazine effect on the respiratory and olfactory mucosae with specific attention to the potential for atrazine transfer to the brain via the olfactory system. Uptake of atrazine was investigated across excised nasal mucosal tissues equilibrated in Krebs's buffer (KRB) or in a co-solvent system containing propylene glycol (PG), similar to the commercial herbicide product. Active uptake pathways were probed using 2,4-dinitrophenol (2,4-DNP) as a metabolic inhibitor. Brightfield microscopy was used to assess the effects of ATZ exposure on the tissues. ATZ was found to be transported across the nasal tissues in a manner consistent with passive diffusion, and 2,4-DNP did not reduce the overall uptake of ATZ. Microscopy results showed erosion of the epithelial surface following exposure to ATZ-PG-KRB when compared to control and ATZ -KRB. These results suggest a negative effect of the ATZ co-solvent formulations on nasal tissues with the potential for increased systemic and CNS exposure.

Barriers to effective pulmonary drug delivery are inherent to the lung physiology and present a challenge when attempting to bypass the natural defenses against inhaled particles. Inhaled particulate aerosols that deposit in the conducting airways of the lungs can become trapped in the respiratory mucus layer where they are then subjected to rapid clearance by the mucociliary escalator. The details of particle behavior after deposition and before this clearance are not well understood, however. Several physical processes may influence particle behavior on the mucus, including a penetration of particles into the mucus layer or a lateral transport of particles across the lung surface. Particles which deposit onto the respiratory mucus are subject to a number of forces which dictate these behaviors in the lung and may influence the retention of particles in the lungs and the efficacy of pulmonary drug delivery. The goal of this thesis was to investigate the behavior of microparticles deposited on a synthetic mucus model designed to mimic the bulk viscoelastic and surface tension properties of conducting airway mucus. Studies were conducted to determine the effects of mucus surface properties and particle physicochemical properties on the submersion and lateral mobility of single particles and the spreadability of particle-laden droplets at the air-liquid interface. Synthetic mucus gels were developed with viscoelastic properties that mimicked those of non-diseased tracheobronchial mucus. Infasurf, a calf lung surfactant extract, was spread onto the gel surfaces and compressed in a Langmuir trough to attain physiologically relevant surface tensions (~30-34 mN/m) and to analyze surfactant behavior on viscoelastic subphases. Microparticles were aerosolized onto the model mucus surface and imaged by brightfield microscopy at varying surface tensions on gels of varying viscoelastic properties to determine the extent of capillary submersion, in the fluid interface. Lateral transport of microparticles across the surfactant interface was quantified using particle tracking techniques. Finally, the spreading patterns of surfactant-laden droplets containing model drugs or particles were monitored by time-course imaging. Studies revealed that key physicochemical properties, including particle size and hydrophobicity, influenced particle submersion and mobility on the mimetic surfaces. Submersion, transport, and droplet spreadability were all inhibited with increasing gel viscoelastic properties, suggesting that such inhibition would be expected by healthy or disease tracheobronchial mucus. While low surface tensions promoted microparticle submersion into the subphase, the lateral transport and droplet spreadability were inhibited on gels with pre-existing surfactant films. The extent of droplet spreading could be enhanced by adding surfactant to the droplets. Overall, these studies aid our understanding of particle behavior and their fate at lung-like fluid surfaces, which has implications for both pulmonary drug delivery and pulmonary toxicity. Particulate aerosols which are designed to be smaller and more hydrophilic would experience improved mobility in the lungs and potentially gain the ability to submerge through the thick and highly viscous mucus barriers of diseased lungs. In healthy lungs, improved submersion could lead to greater particle retention, reduced mucociliary clearance, and a more effective delivery of drug to the epithelium. Lastly, by adding surfactant to drug containing liquid aerosol droplets, the deposition and distribution of drug could be improved in the peripheral regions of the obstructed lungs of cystic fibrosis patients. Results from these studies provide new knowledge that can be used to predict the behavior of aerosols deposited in the lungs and can aid the design of aerosols for drug delivery applications.

Nanoparticles have many proposed advantages for use in nasal drug delivery systems. Nanoparticles can improve uptake and efficacy and lower toxicity compared to the drug alone. In order to study the transport behavior of nanoparticles across nasal tissues, the uptake of non-biodegradable, fluorescently-labeled, carboxylate-modified polystyrene nanoparticles was measured. These 40 nm particles are spherical in shape and loaded with fluorescein, a fluorescent dye that can be measured spectrophotometrically, to determine the number of particles that entered the nasal tissues. In order to identify the pathways involved in the uptake of these particles, different pharmacologic inhibitors were also included in the nanoparticle transport studies. The results indicate that the nanoparticles enter the nasal tissues using several endocytosis mechanisms, namely, macropinocytosis, clathrin-mediated endocytosis, and caveolin-mediated endocytosis. These findings suggest that more than one endocytic pathway is involved in the uptake process in the nasal tissues, and these multiple pathways may help to increase the total nanoparticle uptake in the nasal tissues.

Transarterial chemoembolization (TACE) has been practiced in patients for over 30 years and describes the infusion of chemotherapeutic agents followed by embolic particles. This infusion is normally performed by selecting tumor feeding arteries with a catheter under image guidance. The overall goal of TACE is to deliver a high dose of drug directly to a tumor, prevent drug clearance, and induce ischemic necrosis of the tumor. The limitations for current beads system including non-biodegradable and biodegradable beads used for TACE are low drug loading and only water soluble drug can be loaded in beads. The drug loading methods used in current beads system were ion-exchange method and expanding-loading-shrinking method, but these methods didn't allow loading high drug content (up to 10% drug loading) and water insoluble drug. The other limitation for current biodegradable beads used for TACE only had narrow size range beads. In those instances where treatment is not complete or the tumor recurs, physicians would like to be able to access a tumor on multiple occasions in order to administer additional TACE treatments as needed. It may not be possible to re-enter the feeding artery once this artery had been occluded by non-biodegradable beads. For overcoming above limitations, the goal in this study is developing a new biodegradable bead which should have wide size range, achieve high drug loading and high drug loading efficiency, and load water soluble and water insoluble drug. Extrusion/spheronization technology was chosen for drug loading method. It must be noted that not every polymer can be successfully extruded and spheronized. Oxidized cellulose (OC) was chosen in this study, which is biodegradable polymer. OC was evaluated as new excipient for extrusion/spheronization in this study. Differential scanning calorimetry (DSC) and dynamic vapor sorption analysis were used to compare the interaction and distribution of water within MCC and OC. The amounts of nonfreezing and freezing water in hydrated samples were determined from melting endotherms obtained by DSC. The moisture sorption profiles were analyzed according to the GAB equations. The adsorbed monolayer was not statistically different for MCC and OC after accounting for the amorphous content of the polymers. These results suggest that OC can act as a “molecular sponge,'' and thus aid in the production of beads by extrusion and spheronization. A composite central design was used to evaluate the influence of spheronizer speed, spheronizer time and water level (granulation liquid,) on pellet yield and sphericity. All factors as well as the interactions between water level and spheronizer speed were significant (P<0.05) for sphericity. And water level was significant (P<0.05) for pellet yield. The water insoluble drug, methotrexate (MTX), was used in this study. The drug content of OC and OC/carbopol beads was up to 40% and drug loading efficiency was 100%. The swelling ratio of the MTX loaded OC/carbopol beads were up to 200%, and the swelling ratio was decreased when drug content was increased. Comparing to commercial embolization produce Contour SE, OC and OC/carbopol beads were significantly more compressible. Recoverability of OC/carbopol beads is close to Contour SE. The beads stability increased with an increase in the MTX content. 100-900 μm beads could be delivered through from 18G to 23G needles. The release method involved the use of a T-Apparatus where the drug experiences an element of diffusion through a static environment. This method was developed to resemble the in -vivo situation in embolization procedures more closely. Release results showed from 57% to 78% MTX was released from OC/carbopol beads in 6 days depending on the drug content. OC as new pelletization aid can be used to produce beads by using extrusion/spheronization. The new biodegradable OC base beads have wide size range, achieve high drug loading (up to 40%) and high drug loading efficiency, and are able to load water soluble and water insoluble drug. Physical and mechanical properties of MTX loaded OC base beads match the requirement for catheter deliverability. The result of release study showed slow release. The biodegradable OC base beads are suitable for TACE.