Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases.

The objective of this investigation was to evaluate quantitative electroencephalography (EEG) as a measure of CNS stimulation. The reproducibility and sensitivity of quantitative EEG was compared to neuroendocrine, mood, and psychomotor performance measures. The study was conducted in two parts. The first part investigated the inter- and intra-individual variability associated with a series of pharmacological response measures under baseline (no drug) conditions. It was an open-label pilot study in which eight healthy male volunteers underwent a series of tests (EEG, visual continuous performance task (CPT), a finger tapping task, and self-rated mood scales) repeated eight times over a 12 hour period on three occasions, one week apart. The second part evaluated the sensitivity of quantitative EEG to dextroamphetamine (DA) compared to other response measures. It was a double-blind, placebo-controlled, four-period crossover study in eight healthy male volunteers. Subjects received 5 mg, 10 mg, or 20 mg DA or placebo orally, and underwent the same series of tests as well as blood collection for serum prolactin and DA determination, eight times over a 12 hour period. A GC method allowing quantitation of 2ng/mL DA in serum was developed. The greatest between-day, within-day, and intrasubject variability was associated with quantitative EEG. Learning effects were observed for the psychometric tests, and first session effects were apparent for several of the tests including the EEG. EEG response to DA was observed only in the 3 subjects who had baseline alpha activity greater than 35%. There was a statistically significant decrease in serum prolactin levels after DA administration, with the largest decrease observed after the 5 mg dose. Mood scales showed that 3 of 9 subjects experienced dysphoria after DA dosing. The effect on mood was generally greater as the dose increased. One subject was discontinued from the study because he experienced intense dysphoria after the 5 mg dose. Doses could not be distinguished based on the results of the psychometric tests. Effects on mood, serum prolactin levels, and performance as measured by CPI and finger tapping were not correlated with the EEG changes observed. Pharmacokinetic evaluation showed that the rate of DA absorption appears to decrease as the dose increases. Quantitative EEG conducted under our study conditions and study population was not more sensitive for the assessment of CNS stimulation than the other response measures evaluated. The sensitivity may be improved by screening volunteers to select subjects with higher background alpha activity.

In 1962, Owen, Dietz, and Camiener reported the isolation of a new antitumor antibiotic from the culture filtrate of Streptomyces sparsogenes. The structure of the crystalline antibiotic, named sparsomycin, remained elusive until 1970, when Wiley and MacKellar reported results of spectroscopic and degradation studies which elucidated the structure. In addition to the molecular structure, investigators have examined the mechanism of action, toxicity, and related analogues, striving to establish sparsomycin or a synthetic analogue's usefulness as an effective chemotherapeutic agent. The initial pharmacological evaluation of sparsomycin revealed it possessed activity against KB human epidermoid carcinoma cells, a variety of gram-negative and gram-positive bacteria, and fungi. This broad spectrum of activity prompted a closer examination of the biochemical mechanisms. These studies revealed sparsomycin interfered with protein synthesis by inhibiting peptide bond formation near the enzyme peptidyl transferase. Ottenheijm, Liskamp, and Tijhuis reported the first total synthesis of sparsomycin in 1979, which provided access to greater quantities of the material for investigational use. Sparsomycin was selected for use by cancer patients in phase I clinical trials, but was found to cause ocular toxicity which hindered its development as an antitumor agent. In an effort to reduce or eradicate the toxic effects while maintaining the antitumor activity, analogues of sparsomycin were prepared. Using the sparsomycin analogues which were synthesized, studies were performed to determine the effect alteration of key structural parameters had on the efficacy of the compounds. Previous investigators examined analogues which incorporated modifications of the uracil ring, the unique mono-oxodithioacetal moiety, and the stereochemical configuration of the chiral centers. Vince and Lee reported there was an apparent requirement for the Q-configuration at the asymmetric carbon atom. Overall, however, the small number of sparsomycin analogues prepared and evaluated limited the definitive statements concerning the functional groups required for antitumor activity. In order to expand and clarify the structure-activity relationships, three series of new sparsomycin analogues were prepared for this project. The compounds of Series I and II, distinguished by the inclusion or exclusion of a hydroxymethyl functional group, were designed to elucidate the effect on activity of replacing the mono-oxodithioacetal side chain of sparsomycin with 4-substituted benzyl groups. The Series III analogues, which excluded the hydroxymethyl functional group, featured a 4-substituted benzyl amide group in place of the mono-oxodithioacetal moiety of sparsomycin, and were designed to investigate the potential interaction of an amide oxygen in contrast to the sulfoxide oxygen of sparsomycin. The target compounds synthesized for this project were experimentally examined to quantitate their effects on [75Se]-selenomethionine incorporation as an 125 cell growth, indirect measurement of protein synthesis, and 5-125I-iodo- 2-deoxyuridine incorporation as an indirect measurement of DNA synthesis in bone marrow, P388 lymphocytic leukemia, and P815 mastocytoma cells. The results for the Series I and II analogues indicated the removal of the hydroxymethyl functional group as seen in sparsomycin affected activity to varying degrees depending upon the assay and the type of cells used. The results for the Series III compounds suggested the removal of the hydroxymethyl functional group and substitution of the mono-oxodithioacetal side chain of sparsomycin with a substituted benzyl amide moiety was not beneficial for activity. Finally, examination of the collective data revealed that the bromobenzyl-substituted analogues consistently imparted the greatest inhibitory activity, while the methoxybenzyl-substituted analogues displayed the least. The methyflnnzyl and the unsubstituted benzyl compounds were intermediate in inhibitory potency. The activity may correspond to the lipophilic and electronic characteristics of the substituents on the benzyl moiety of the analogues. It appears that the bromobenzyl-substituent of hydrophobic and electron withdrawing character is optimal for inhibitory activity, and conversely, the methoxybenzyl substituent of hydrophilic and electron donating character is least desirable.

The objective of this thesis was to understand the regulation of islet Cl⁻ current by cAMP. This current, known as Icl,islet flew is the first Cl⁻ channel current characterized in pancreatic 𝛃 cells. Icl,islet has been hypothesized to modulate insulin secretion through changes in islet electrical activity. Both 5 𝛍M forskolin and 100 𝛍M IBMX (3-isobutyl-1-methylxanthine), agents that increase intracellular cAMP, were shown to activate an outwardly-rectifying ionic current in HIT cells that closely resembled Icl,islet. The current was blocked when iodide was substituted for external Cl⁻ or when the Cl⁻ channel blocker niflumic acid was applied to cells. In contrast, removal of [Na⁺]O did not inhibit the current. In many cells, Cl⁻ current activated and then spontaneously deactivated following cAMP stimulation, suggesting the possibility that the channel desensitizes to [cAMP]i. Exposing cells to multiple cAMP activators revealed that Cl⁻ current declined because it became refractory to increased [cAMP]i. The implication of these results to islet physiology is discussed.

Human immunodeficiency virus (HIV) and opioid abuse have been described as synergistic epidemics. Pharmacologically, it has been found that opioids have the capacity to enhance HIV infection and replication. Research has shown that activation of the mu-opioid receptor (MOR) elevates the expression of the HIV-1 entry co-receptor CXCR4 on T-lymphocytes in the peripheral nervous system, thus allowing for enhanced viral entry and invasion. Although the exact mechanism for opioid modulation of CXCR4 expression and subsequent exacerbation of HIV is unknown, several hypotheses exist. One hypothesis is that MOR and CXCR4 are functionally interacting through the formation of a heterodimer. This hypothesis is supported by studies substantiating the ability for MOR and CXCR4 to form heterodimers with other GPCRs, and the finding that MOR and CXCR4 were co-expressed in several central and peripheral regions including immune cells. To test this hypothesis, a series of bivalent ligands containing both a mu opioid receptor (MOR) antagonist and a CXCR4 antagonist pharmacophore was designed and synthesized to understand the pharmacological role of the putative CXCR4-MOR heterodimer in opioid exacerbated HIV progression. These bivalent ligands were evaluated for their binding and functional activities in radioligand binding, antibody binding, [35S]GTPγS, and calcium mobilization assays. In these assays, the bivalent ligands were shown to maintain binding and functional activities in both MOR and CXCR4 monoclonal cell lines. In addition, these bivalent ligands were evaluated for their ability to block HIV entry in a reverse transcriptase assay, and for their ability to inhibit morphine exacerbated HIV invasion in an LTR-luciferase assay. In these assays, the bivalent ligands were shown to inhibit HIV entry in a dose dependent manner. However, due to experimental limitations in our morphine exacerbated reporter system, the ability for the bivalent ligands to inhibit viral entry upon morphine co-exposure was not fully validated. Finally, molecular modeling approaches were utilized to visualize the putative binding modes of the bivalent ligands in a constructed MOR-CXCR4 heterodimer model. Overall, these studies have provided a solid basis for the utility of bivalent ligands in studying MOR-CXCR4 interactions and their involvement in opioid potentiated HIV progression. Further studies are ongoing to optimize the bivalent ligands construct and explore new analyses to evaluate their ability to block opioid modulation of the virus.

Spectral imaging is a powerful technique which uses the wavelength to identify/quantify the exact location and amount of the molecules. It facilitates the identification of materials and studying their properties through analyzing the way they interact with light. The study of light interaction with elements is called spectroscopy; spectroscopy examines how light behaves in the target and recognizes materials based on their spectral signatures. Spectral signatures can be compared to fingerprints which can be used to identify a person; spectral signatures can be used to identify materials. Therefore, we hypothesize that identifying the exact location and quantity of molecules present in the given cells samples can be done by using a spectral imaging system. In this study, we identify the exact UV-Vis and fluorescence spectra of organic substances including Rhodamine 6G, Doxorubicin and UV-Vis spectra inorganic compounds including silver (Ag), gold (Au) nanoparticles (NPs). After that, we used the Q-TOF LC/MS system to quantify the maximum and minimum detectable concentrations of Rhodamine 6G and Doxorubicin by checking the chemicals spectrum based on the molecular weight. In addition, we used HPLC system to quantify the chemicals basing on their UV spectrum. Forwards, we used spectral imaging system to determine the exact amount and location of the molecules within cells samples. For Rhodamine 6G and doxorubicin, we started with the minimum detectable concentration by Q- TOF and consider it as a maximum limit with spectral imaging. And for NPs we used the maximum concentration for the analysis. Using the spectral imaging we were able to vii detect the exact location of Rhodamine 6G which was in the cytoplasm, Doxorubicin in the nucleoplasm, and NPs in both. Furthermore, spectral imaging was able to detect much lower concentrations of Rhodamine 6G and Doxorubicin by the spectrum in comparison to Q-TOF LC/MS.

acase@tulane.edu / 1 / Alejandra E. Marks

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.