Tetracyclines, originally isolated in the late 1940's and early 1950's from the mycelium of Streptomyces, have achieved major importance as therapeutic and prophylactic agents against a wide range of infections in human and veterinary medicine. They have also achieved great importance in agriculture where they are widely used to promote weight gain in livestock. Commercial production, during 1958 in the United States alone was 120 tons with a value of approximately $1 million per ton. The clinical importance of these compounds has stimulated efforts to define their mode of action as inhibitors of bacterial reproduction. Antibiotics having as many functional groups as the tetracyclines may have many modes of action. The problem facing the research worker is to determine the relative contribution of each mode of action in a given biological system as a function of antibiotic concentration. If all parts of a biological system are exposed to an equivalent antibiotic concentration, one might expect that the degree of inhibition of the various sub-component systems would be proportional to the relative stabilities of the reaction products of the antibiotic and the sub-component system. The most critical reaction from the standpoint of cellular reproduction would then represent the point of the primary biochemical lesion or the site of action. The type of reaction would be the mode of action for the biological system. For a given biological system it is probable that there is one critical reaction which is most sensitive to the antibiotic, but this reaction may not be the same for every biological system (see Snell and Cheng (l) for an excellent discussion of the difficulties of defining a mode of tetracycline action). Many investigators presently feel that the primary biochemical lesion inflicted upon susceptible bacteria by tetracyclines is a general inhibition of protein synthesis (2,3,4). Therefore it was felt that the study of several measures of inhibition, particularly cell reproduction and protein synthesis, in the presence of several tetracyclines might be a useful way of studying modes of tetracycline action. Biological activities of a large number of compounds obtained quantitatively under identical conditions and in a precise manner are required to establish structure-activity relationships. Presently available activities for tetracycline antibiotics have been summarized by Barrett (5), Boothe (6) and Plakunov (7). Many of these activities have been obtained under conditions such that the results parallel clinical activities. Thus, some compounds may not have achieved equilibrium with the test system. Some activities have been obtained for the purpose of studying antibiotic resistance while still others have been obtained in widely varying and not easily interrelatable biological test systems. Consequently, quantitative activities suitable for structure-activity relationships have not been reported for most tetracyclines. The present work was undertaken to obtain activities suitable for structure-activity relationships and which are pertinent to both bacterial reproduction and the proposed mode of action. The activities obtained should include both clinically active and “inactive” tetracycline antibiotics.

An original study was undertaken to examine the relationship of clients of Project Jump Street, Inc., a methadone maintenance program, to changes in personality factors as measured by R.B. Cattell's Sixteen Personality Factor Inventory, to compare the national norms of drug addicts/methadone users to the result of the 16 PF of clients of Project Jump Street, Inc., and to determine if significant differences appear among treatment phase I, phase II, and phase III. Twenty-one clients of the program, seven subjects in each of phases I, II, and III, were given the 16 PF by their counselors between September 10 and September 30, 1973. The analysis of the data indicated that while there were no statistically significant differences (p = .05) among the groups, definite trends seemed to be developing among treatment phase I, phase II, and phase III patients. The trends that seemed to be developing were: Increasing sizothymic (A-) response Increasing analytic intelligence response (B-) Increasing ego strength (C+) Decreasing superego strength (C+) Increasing reactivity to threat (H-) Increasing shrewdness (N+) Increasing self-assuredness (0-) Increasing group—dependency (Q2-) In group I significant differences were found as follows in the mean sten scores of the clients when compared to the standard for drug addicts. These factors were: Intelligence(B), Ego Strength(C), Dominance/Submission(E), Superego Strength(C), Praxernia/Autia(M), Artlessness/Shrewdness(N), and Conservatism/Radicalism(Q1). In group I when compared to the standard for methadone users, there were significant differences in the following factors: Intelligence(B), Ego Strength(C), Dominance/Submission(E), Superego Strength (G), Threctia/Parmia (H), Artlessness/Shrewdness (N), and Conservatism/Radicalism (Q1). Group II subjects mean sten scores were compared to the standard for drug addicts and the following factors differed significantly: Intelligence (B), Ego Strength (C), Dominance/Submission (E), and Desurgency/Surgency (F). In group II, when compared to the standard methadone user, significant differences were noted in the following factors: Threctia/ Parmia (H), and Conservatism/Radicalism (Q1). The standard for drug addicts was compared to the mean sten scores for group III. The following factors differed significantly from the standard: Sizothyme/Affectothyme (A), Intelligence (B), Ego Strength (C), Dominance/Submission (E), Alaxia/Protension (L). When group III mean sten scores were compared to the standard for methadone user, there were significant differences in the following factors: Sizothyme/Affectothyme (A), Ego Strength (C), Artlessness/ Shrewdness (N) and Conservatism/Dominance (Q1). These results seem to indicate that there is increasing similarity between the standard for methadone users and the subjects in the study as one approaches group II. Subjects in group I exhibited the greatest amount of variance when compared to the standard for both drug addicts and methadone users, while group III showed only median variance from the standard for drug addicts and methadone users.

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.

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.

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.

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.