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The pharmacokinetics of ethanol and (-)-trans-isomer of 9-tetrahydrocannabinol (THC), and the pharmacokinetic interaction between them were characterized using statistical models in this thesis. In chapter II, a semi-mechanistic absorption rate dependent hepatic extraction model was developed to characterize ethanol pharmacokinetics. The statistical analysis conducted based on this model indicated no association between ethanol disposition and subject age or sex, and a 23% higher typical Vmax value, a 12.5% lower typical Km value for heavy drinkers compared with moderate drinkers. In chapter III, a parent-metabolite pharmacokinetic model was developed to simultaneously describe the concentration time profile of THC and its active metabolite 11-OH-THC. A parent-metabolite model with 3-compartment pharmacokinetic model for THC and a 2-compartment model for 11-OH-THC was found to best describe the pharmacokinetics of THC and 11-OH-THC simultaneously. In chapter IV, the pharmacokinetic interactions of ethanol on THC, 11-OH-THC and 11-nor-COOH-THC were evaluated using linear mixed effects models. The results suggested that co-administration of ethanol caused an increase in THC and 11-OH-THC systemic exposure, failed to influence the terminal elimination processes of THC and 11-OH-THC, and did not affect the pharmacokinetics of 11-nor-9-COOH-THC.

Fluoroquinolones are broad spectrum antibiotics that have been in use for nearly 50 years. These agents are used to treat a variety of bacterial infections from simple urinary tract infections to tuberculosis. The protein targets of fluoroquinolones are bacterial type II topoisomerases. Fluoroquinolones inhibit the function of these topoisomerases by intercalating in the nick site of the DNA and forming an interaction with helix-4 of the enzyme through a magnesium-water bridge. The binding of a fluoroquinolone stabilizes the DNA-topoisomerase-fluoroquinolone ternary complex. Helix-4 is where some of the most important fluoroquinolone resistance mutations occur. While the fluoroquinolone class of antibiotics has been successful at treating a variety of infections over the past few decades, a number of problems exist. These problems include the inability of many fluoroquinolones to kill non-growing cells, the emergence of fluoroquinolone resistant mutants, and adverse side effects of this antibiotic class. Thus, various aspects of fluoroquinolone structure and activity are explored in this study. The first topic explored is the question of what structural features are necessary for a fluoroquinolone to be able to kill bacteria in the presence and absence of the protein synthesis inhibitor, chloramphenicol (to mimic a dormant, non-growing state of the bacteria). Previous studies have shown that steric bulk at the C-8 position (especially a methoxy group) is necessary to support the ability of a fluoroquinolone to kill non-growing cells. In this study, the N-1 position of a series of C-8 methoxy fluoroquinolones was explored to gain an understanding of what substituents at the N-1 position of C-8 methoxy fluoroquinolones support the ability to rapidly kill bacteria in the presence of a protein synthesis inhibitor. In a second study the N-1 position is further explored, but with different goals. A recent crystal structure of a fluoroquinolone bound in the ternary complex with topoisomerase IV and DNA has revealed that the N-1 position of the fluoroquinolone is near in space to the catalytic tyrosine residue. It was reasoned that new interactions can be made with active site tyrosine residue through the N-1 position of the fluoroquinolone core. A number of N-1 fluoroquinolone derivatives were designed, synthesized, and evaluated for their ability to inhibit the DNA supercoiling activity of DNA gyrase, as well as the poisoning ability of the fluoroquinolones. The advantages of targeting the catalytic tyrosine residue are that this amino acid cannot be mutated without loss of enzyme function, and that by forming a new binding contact to the enzyme, activity can be maintained against helix-4 mutants. Finally, in a step toward the goal of mitigating the tendon related side effects of fluoroquinolones (thought to be due to Ca2+ coordination), the metal binding domain of the fluoroquinolone was altered. These fluoroquinolones were tested for their ability to inhibit and poison DNA gyrase. From the studies described, we have learned that the N-1 position is very sensitive to modification, that novel binding contacts to bacterial topoisomerases can be made through the N-1 position, and that modifying the metal binding domain of fluoroquinolones can lead to retention of activity against DNA gyrase. These accomplishments all push the fluoroquinolone field ahead by introducing a novel binding interaction to optimize (with the goal of creating a fluoroquinolone that is active against current fluoroquinolone resistant mutants) and by showing that fluoroquinolone activity can be retained even when the metal binding domain is altered, thus moving us closer to the goal of reducing tendon-related side effects.

Sulfonamide- and sulfone-induced hypersensitivity reactions are thought to be mediated through bioactivation of parent drug molecule(s) to their respective reactive metabolite(s). In order to explain the cutaneous drug reactions caused by sulfonamides and sulfone, a mechanism can be proposed by which the bioactivation of these drugs in keratinocytes of the skin forms reactive hydroxylamine metabolites that can covalently bind to cellular proteins, which in turn act as antigens leading to the cascade of immune reactions resulting in a cutaneous drug reaction. In order to probe the proposed mechanism, we determined the enzymes responsible for the bioactivation of these parent drugs to their hydroxylamine metabolites in cultured human keratinocytes. It was found that flavin containing monooxygenases and peroxidases play an important role in the bioactivation of these drugs in keratinocytes. We also confirmed the presence of these enzymes in keratinocytes. Interestingly, though cytochrome P450s are important in the oxidation of parent arylamine xenobiotics to their hydroxylamine metabolites in the liver, they do not appear to play a significant role in the bioactivation of these drugs in keratinocytes. The hydroxylamine metabolites of sulfamethoxazole and dapsone can undergo autooxidation, generating reactive free radicals. Our studies showed that both of these metabolites elevate oxidative stress in keratinocytes by forming reactive oxygen species. Though the cytotoxicity induced by these metabolites is not correlated with the extent of oxidative stress, the generation of reactive oxygen species may be important finding as these species can act as danger signals that activate antigen presenting cells in the skin. As a possible explanation for the idiosyncratic nature of these reactions, folate deficiency was studied as a potential risk factor. However, the results of these studies suggested that deficiency of folic acid in keratinocytes does not predispose such cells to the toxicity associated with the parent drugs or their metabolites. Unexplored is the potential role of such deficiency on the immune response itself.

Cationic antimicrobial peptides (CAPs) are an important component of the innate immune system and are instrumental in the elimination of bacteria, viruses, protozoa, yeast, fungi and cancerous cells from the body. CAPs are comprised of less than 100 amino acids and have a net positive charge due to a multitude of basic residues in their primary sequences. CAPs exert their antimicrobial activity primarily through the formation of pores in microbial membranes, but also play important immunostimulatory roles in the body. Glycosaminoglycans (GAGs) are negatively charged, polydisperse linear polysaccharides found at cellular surfaces. Although many protein-binding interactions of the GAG family, including heparin and heparan sulfate, have been well-characterized, it is not known to what extent endogenous GAGs affect the innate immune system. In the studies here the modulatory activities of GAGs and other polyanionic polysaccharides (PPSs) on CAPs were probed. Initial studies focused on interactions between a short peptide derived from bovine lactoferricin and GAGs. GAGs and other PPSs were then tested for their ability to modulate the antimicrobial activities of a number of CAPs against Gram-positive and -negative organisms. GAGs were also tested for the ability to modulate CAPs binding to bacterial lipopolysaccharide. CAP affinities for the GAGs were determined from lipopolysaccharide competition binding assays. Finally GAGs were evaluated for the ability to protect CAPs from proteolytic degradation. The modulatory activities of GAGs and other PPSs are largely dependent upon all components of the test system and, to a lesser extent, the charge of the molecule.

Fluoroquinolones are broad-spectrum antibacterial agents based on the structure of nalidixic acid. For nearly five decades it has been known that fluoroquinolones inhibit bacterial growth by blocking the enzymatic action of type II topoisomerases such as DNA gyrase and topoisomerase IV. Only recently has it been discovered that some fluoroquinolones are capable of a mechanism that results in fragmented DNA and leads to rapid bacterial cell death. This mechanism is not well understood. Presented here are studies towards understanding the structure activity relationship (SAR) of fluoroquinolones, specifically to determine what leads to the novel mechanism termed "rapid lethality." This work is based on the hypothesis that structurally unique fluoroquinolones interact with the DNA-topoisomerase complex in a unique manner that ultimately leads to rapid cell death. The first approach to understand SAR for killing was to evaluate the effect of a ring fusion between N-1 and C-8 of the fluoroquinolone core. Known lethal fluoroquinolones are substituted by N-1 cyclopropyl and C-8 methoxy, but some clinically important fluoroquinolones contain a 2-methylmopholino moiety between these two positions. Novel fluoroquinolones were synthesized and clinically available agents were obtained to create a panel of drug molecules with one of six C-7 substituents and either the morpholine ring system or N-1 cyclopropyl and C-8 methoxy. Bacteriostatic and bactericidal activities of these compounds were determined. Bactericidal studies were conducted both in the presence and absence of chloramphenicol, a protein synthesis inhibitor used to simulate non-growing bacteria. Lethality in the presence of chloramphenicol is also important when considering co-administration of fluoroquinolones with other antibiotic classes. In a second study, fluoroquinolones were synthesized with a C-2 thioalkyl substitution. Substitutions at the C-2 position are severely lacking in clinical fluoroquinolones, with only prulifloxacin, a newly developed antibiotic, being substituted by an N-1 to C-2 thiazetidine ring structure. Analogs of ciprofloxacin and moxifloxacin were synthesized such that the N-1, C-2, and C-8 positions were substituted with cyclopropyl, thioethyl/thioisopropyl, and methoxy groups, respectively. The compounds were then evaluated for antibiotic activity against three different bacterial strains to evaluate the contribution of the C-2 thioalkyl substituent to antibacterial activity. In a third study, quinazoline-2,4-diones, a new antibiotic class structurally and mechanistically similar to fluoroquinolones, were modified at the C-4 position in an effort to understand the binding interaction between these compounds and the target enzyme. Importantly, the quinazoline-2,4-diones typically retain activity against bacterial cells known to be resistant to fluoroquinolones and are less likely to select for resistant mutants. In this study, the C-4 carbonyl was replaced with either a thiocarbonyl or a hydroxylimine and the new compounds, bearing C-7 substituents common to potent antibiotic fluoroquinolones and quinazolines, were evaluated for activity against bacterial cells. Despite the findings of recently published X-ray crystallography, it was determined that one of the greatest determinants in antibiotic activity of fluoroquinolones is the C-7 substituent. Additionally, there is increasing evidence that the C-2 carbonyl of quinazoline-2,4-diones affords the increase in activity against resistant mutants by creating a unique binding interaction. Collectively, the conclusions reached here add to our understanding of the structure activity relationship of the fluoroquinolone antibiotic class for rapidly killing bacterial cells and overcoming resistant mutants.

Alkaline degradation of Carbaryl in the presence of CTAB micelles has been reported as the most efficient method; however, the factors accounting for it are not yet clear. The main objective of this work was to study some of the factors affecting the alkaline degradation of Carbaryl in the presence of cetyl trimethylammonium bromide (CTAB). Three specific aims were researched in order to address the main objective. Solubility studies, UV-vis, fluorescence, and 1D-HNMR and 2D-HNMR spectroscopies were used to research the solubilization of carbaryl in CTAB micelles. Solubility studies showed that carbaryl partitions into CTAB micelles with a binding constant of 553 ± 8 M-1, and each mole of micellized surfactant incorporates about 0.336 moles of carbaryl. Spectroscopy studies showed that carbaryl does not interact electrostatically with micelles but does through van der Waals interactions. 1D-HNMR and 2D-HNMR indicated solubilization in the Stern layer, oriented with its hydrophilic moiety towards the Goüy-Chapman layer and the hydrophobic moiety towards the core of the micelle. Kinetic studies as a function of the surfactant concentration along with micellar kinetic models were used to calculate micellar rate constants (k’M) for each of four different cationic surfactants: cetyl trimethylammonium hydroxide (CTAOH), cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTACl), and cetyl pyridinium chloride (CPCl), and compared to the corresponding rate constants (k’W) in water; the results in all cases showed k’M / k’W > 1. This fact led to the conclusion that additional factors beyond solubilization of substrates are playing a role. Solubility studies revealed the following binding constant order and solubilization capacity order: CPCl > CTAOH ≈ CTAB > CTACl, CPCl > CTAOH ≈ CTAC > CTAB, indicating that for CPCl, Coulombic interactions, such as charge-transfer complexes, may be favoring the concentration effects, while for other surfactants, such as CTAOH, the [–OH] as the micelle counterion increases Carbaryl’s concentration in the Stern layer compared to its bulk concentration. In contrast, large, weakly-hydrated polarizable ions such as Br– displace hydrophilic ions, providing less enhancement. Kinetic experiments as a function of the surfactant head’s charge led to the conclusion that cationic and zwitterionic surfactants have a catalytic effect of the alkaline hydrolysis of carbaryl, while nonionic and anionic surfactants have inhibitory effects: kobs (cationic) > kobs (zwitterionic) > kobs(nonionic) > kobs (anionic). A similar order for solubility parameters (Ks and SC) was observed from equilibrium solubility studies. Experiments as a function of the polarity of the medium in the presence of both polar and nonpolar solvents showed that the hydrolysis rate is inversely proportional to the medium polarity. Ionic strength experiments showed that the hydrolysis rate is inversely proportional to the ion concentration.

Fluoroquinolones, a class of type-II topoisomerase inhibitors, have successfully been used as antibiotics for the last several decades, beginning with the use of nalidixic acid in urinary tract infections. This led to the broad-spectrum activity of ciprofloxacin in the 1980s. Unfortunately, use of fluoroquinolones has led to the emergence of resistant bacteria. Recently, this has generated new bacteria such as multidrug-resistant and extensive-drug-resistant strains of M. tuberculosis that are also fluoroquinolone-resistant. Infections caused by these bacterial strains are widespread, with high mortality rate in immune-compromised populations such as the elderly, infants, and in AIDS or HIV-positive patients. Fluoroquinolone resistance is acquired through amino acid substitutions of key fluoroquinolone-binding residues of the type-II bacterial topoisomerases DNA Gyrase and Topoisomerase IV, the enzyme targets of fluoroquinolones. Amino acid substitutions that result in fluoroquinolone resistance are located on Helix-4 of these enzymes, which is the site of a magnesium (Mg)-water bridge that is a crucial binding interaction for fluoroquinolones. When certain substitutions to Helix-4 occur, the Mg-water bridge is compromised and no longer available to anchor fluoroquinolones into a ternary complex composed of topoisomerase, fluoroquinolone, and DNA. This results in drug resistance. Herein are described attempts to generate fluoroquinolones that are capable of overcoming this mechanism of resistance. In the first study, attempts were made to generate a series of novel tricyclic fluoroquinolones and diones designed to exploit intercalative or pi-stacking binding interactions with the bacterial DNA in the ternary complex in order to lessen the importance of the Mg-water bridge interaction. Despite numerous attempts, no complete synthetic pathway to these core structures was ever discovered. The second study investigated the utility of a C7-aminomethylpyrrolidine group on the fluoroquinolone structure. This was done in order to explore the mechanistic reasons why previously generated fluoroquinolones possessing this C7-aminomethylpyrrolidine group maintained activity against common Helix-4 mutants. A panel of fluoroquinolones with C7-aminomethylpyrrolidine groups and diverse core structures was synthesized and docking studies with the original C7-aminomethylpyrrolidine fluoroquinolone and other fluoroquinolones were performed. Target compounds were synthesized and evaluated for inhibition/poisoning purified enzyme and for the ability to inhibit growth with wild-type and fluoroquinolone-resistant cells. In a third study, fluoroquinolones possessing structural variations of the C7-aminomethylpyrrolidine were designed and synthesized to explore structural requirements of the aminomethylpyrrolidine group binding and overcoming fluoroquinolone-resistance caused by alterations of Helix-4. This led to further exploration of the binding space around the C7-position of the fluoroquinolones. In both the second and third studies, the new fluoroquinolones were evaluated for the ability to specifically target bacterial topoisomerases over human topoisomerase. The results of these studies have contributed new knowledge to the binding requirements of fluoroquinolones that maintain potency against fluoroquinolone-resistant type-II topoisomerases, and represent a step towards methodology to overcome bacteria resistant to fluoroquinolones.

Pyronaridine/Artesunate (PA) 3:1 fixed dose combination is a novel artemisinin-based combination therapy (ACT) in development for the treatment of acute uncomplicated Plasmodium falciparum or Plasmodium vivax malaria. An understanding of both pharmacokinetics and pharmacodynamics of pyronaridine is of importance in order to achieve optimal therapeutic outcome. In this thesis, population pharmacokinetic models for pyronaridine in healthy subjects, and adult and pediatric malaria patients were developed. Pyronaridine pharmacokinetics in both adult and pediatric populations were best described by a two compartment model with first order absorption and elimination from the central compartment. A presence of malaria infection and body weight were the significant covariates that explained pyronaridine pharmacokinetic variability in the adult population. For the pediatric population, age was the only significant covariate that explained pyronaridine pharmacokinetic variability. Monte Carlo simulations were also performed to address differences in pyronaridine exposures among these populations and to explore the exposures of pyronaridine among recommended dosage regimens for pediatric and adult malaria patients. Healthy adults had a higher exposure to pyronaridine as compared to adult malaria patients. For the pediatric population, younger children had a higher exposure to pyronaridine as compared to older children. The overall range of pyronaridine exposures among dosing groups for adult and pediatric malaria patients were relatively similar. The cut-off values of pyronaridine pharmacokinetic parameters associated with successful treatment outcome were also determined by means of receiver operating characteristic (ROC) curve. These cut-off values can be used to optimize the outcome of malaria treatment. Additionally, Cox proportional hazard model was conducted to determine the relationship between several covariates and time to the occurrence of re-infection or recrudescence. The models showed that as the levels of predicted pyronaridine concentrations on day 7 increased, the risks of acquiring re-infection or recrudescence decreased. Finally, pharmacokinetic drug-drug interaction of pyronaridine and ritonavir was assessed based on the overlap pathway for metabolism of both drugs and the high rates of HIV and malaria co-infection. There was an effect of ritonavir on pyronaridine pharmacokinetics. However, the results were not considered clinically relevant. An increase in ritonavir exposure was observed in the presence of fixed dose PA.

OSW-1 is a natural saponin and its anticancer activities are 10- to 100-fold more potent than many well-known anticancer agents in clinical use. Its cytotoxicity profile suggests that it may have a unique mode of action that is different from other well-known anticancer agents. However, its mechanism still remains as a mystery after years of study, and no paper has ever been published in this area. Extensive in vitro and in vivo testing has been conducted and toxicology experiments have also been carried out by our collaborator Prof. Huang's laboratory at MD Anderson Cancer Center. In order to identify the pharmacophore and mechanism of OSW-1 and increase its in vivo activity and selectivity, amino analogues are synthesized for the SAR study employing the chemistry developed in our lab. Superstolide A (1) is a highly potent anti-tumor reagent that was isolated from deep water marine sponge in 1996. The potent anticancer activity, molecular complexity (11 chiral centers) and scarcity in natural resources make this molecule an attractive synthetic target. Currently I am working on the model study for the construction of the 16-membered macrolactone present in Superstolide A. Specifically I am focusing on the investigation of three crucial carbon-carbon bond-forming reactions in our synthetic strategy including Julia olefination, Suzuki coupling and Horner-Emmons olefination.