<p>Nonsteroidal anti-inflammatory drugs are among the most commonly used prescription and over-the-counter medications, but they often produce significant gastrointestinal ulceration and bleeding, particularly in elderly patients and patients with certain co-morbidities. Novel anti-inflammatory drugs are seldom tested in animal models that mimic the high-risk human users, leading to an underestimate of the true toxicity of these drugs. In the present study we examined the effects of two novel NSAIDs and two commonly used NSAIDs in models in which mucosal defence was expected to be impaired. Naproxen, celecoxib, ATB-346 (a hydrogen sulfide- and naproxen-releasing compound) and NCX 429 (a nitric oxide- and naproxen-releasing compound) were evaluated in healthy, arthritic, obese, hypertensive rats, and in rats of advanced age (19 months) and rats co-administered low-dose aspirin and/or omeprazole. In all models except hypertension, greater gastric and/or intestinal damage was observed when naproxen was administered in these models than in healthy rats. Celecoxib-induced damage was significantly increased when co-administered with low-dose aspirin and/or omeprazole. In contrast, ATB-346 and NCX 429, when tested at doses that were as effective as naproxen and celecoxib in reducing inflammation and inhibiting cyclooxygenase activity, did not produce significant gastric or intestinal damage in any of the models. These results demonstrate that animal models of human co-morbidities display the same increased susceptibility to NSAID-induced gastrointestinal damage as observed in humans. Moreover, two novel NSAIDs that release mediators of mucosal defence (hydrogen sulfide and nitric oxide) do not induce significant gastrointestinal damage in these models of impaired mucosal defence.</p> / Master of Science (MSc)

<p>Colorectal cancer leads to more than 600,000 deaths worldwide per year. An abundance of research has shown that several non-steroidal anti-inflammatory drugs (NSAIDs) can exert chemotherapeutic and chemo-preventative effects in colorectal cancer patients. It is important to note, that use of many different NSAIDs carries a significant risk for cardiovascular and gastrointestinal (GI) complications. A recently developed group of NSAIDs, which release hydrogen sulfide (H₂S), has been shown to have greatly reduced these side effects as compared to conventional NSAIDs. This is likely attributable to the ability of H₂S to increase the resistance of the GI mucosa to injury, as well as to accelerate repair of injury when it occurs. Moreover, H₂S has been shown to be a vasodilator, and therefore may offset some of the hypertensive effects of NSAIDs.</p> <p>We assessed the chemotherapeutic actions of two of these newly developed NSAIDs. ATB-346 is an H₂S-releasing derivative of naproxen and ATB-352 is an H₂S-releasing derivative of ketoprofen. These drugs were tested in the azoxymethane mouse model and in the APC^Min/+mouse model of Colorectal cancer.</p> <p>In the azoxymethane model of colorectal cancer ATB-346 caused a significant reduction in number aberrant crypt foci (ACF), which are pre-neoplastic lesions used as markers of colorectal cancer. The reduction was superior to naproxen at all doses tested. ATB-352 also caused a significant reduction in the number of ACF, however the reduction was not superior to that produced by ketoprofen. In APC^Min/+mice treated with ATB-346 for 14 days (14.5 mg/kg) we observed a complete inhibition of the formation of colonic polyps/tumours and a 97.5% reduction in total polyp score. Shorter treatment with ATB-346 also produced similar reduction in total polyp score. We found that ATB-346-treated mice had lower levels of b-catenin and cmyc without significant changes in APC or p53 levels. <strong></strong></p> <p>These results demonstrate ATB-346 can exert superior chemo-preventative effects in mice models of colon cancer while leading to no gastric or intestinal damage.</p> / Master of Science (MSc)

Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.

Cocaine is a plant-based illicit drug commonly involved in substance use disorder. Although cocaine overdose and cocaine use disorders cause adverse health consequences to individuals and the economic burden on their family and society, there are no FDA (Food and Drug Administration) approved medications for treatment. Recently, it has been recognized that delivery of cocaine hydrolase (CocH) is a promising therapeutic strategy. Human butyrylcholinesterase (hBChE), the primary enzyme involved in cocaine metabolism in human, have advantages over other candidates for the development of CocH. Previous studies in our laboratory have designed and characterized hBChE mutants that have ~4,000-fold improved catalytic efficiency against naturally occurring (-)-cocaine as compared to the wild-type hBChE. Besides the catalytic efficiency, the biological half-life is another essential factor that influences the desired therapeutic value in the long-term treatment of cocaine use disorder. In order to provide prolonged effects to reduce administration frequency in clinical use, efforts have been made to increase the retention time of CocHs in blood circulation by fusing CocHs with other thermostable proteins or their mutants, including human serum albumin (Albu) or the Fc region of the human IgG (Fc). In this dissertation, we demonstrated the clinical potential and the benefits of long-lasting CocHs for cocaine overdose treatment. We used rodent models to show the ability of AlbuCocH1 to block or reverse manifestations of toxic effects of cocaine. In addition, a concomitant LC-MS/MS-based analysis was conducted to investigate the pharmacokinetic profile of a lethal dose of cocaine with the presence of AlbuCocH1. These experimental data demonstrated AlbuCocH1 as an effective cocaine detoxification agent by accelerating the metabolism of cocaine. In order to examine the potential therapeutic value of Fc-fused CocHs in the treatment of cocaine use disorder, we conducted a series of behavioral experiments in rats to evaluate the effectiveness and duration of Fc-fused CocHs in blocking or attenuating cocaine-induced psychostimulant and discriminative stimulus effects. In addition, the intravenous self-administration model was used to investigate the long-term effectiveness of Fc-fused CocHs in blocking or attenuating the reinforcing effects of cocaine. It has been shown that a single dose of E30-6-Fc (3 mg/kg) was able to effectively alter the cocaine dose-response curve and attenuate the reinforcing efficacy of cocaine for at least a month in both male and female rats. In summary, AlbuCocH1 (TV-1380), which failed to meet the primary efficacy endpoint in clinical trials for facilitating abstinence in cocaine-dependent subjects with a weekly dosing schedule (due to the short biological half-life), is more suitable to be developed as a cocaine detoxification agent. On the contrary, the newly designed Fc-fused CocH (e.g. CocH3-Fc, E30-6-Fc) with higher catalytic efficiency and longer biological half-life will be beneficial for long-term abstinence management in cocaine-dependent individuals.

The prevention of mother-to-child transmission is one of the most powerful tools in human immunodeficiency virus type 1 (HIV-1) prevention and has huge potential to improve both maternal and child health. In the absence of any preventative measures, infants born to and breastfed by their HIV-positive mothers have roughly a one-in-three chance of acquiring the infection themselves. HIV can be passed on from mother-to-child during pregnancy, during labor and delivery, and even after during breastfeeding. Intrapartum and neonatal single-dose nevirapine (sd-NVP) is the foundation of preventing mother-to-child transmission in lower resource settings where it has been used alone or as part of combination regimens. Both its simplicity and its long plasma half-life contribute to the success of sd-NVP based therapy. However, sd-NVP frequently results in HIV-1 viral resistance in mothers and children who become HIV infected despite prophylaxis. Sd-NVP leads to the development of non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance, compromising the success of treatment of mother and child with subsequent antiretroviral combinations. Resistance to NNRTIs is particularly worrisome in lower resource settings since many subsequent regimens for maternal and infant antiretroviral therapy include a NNRTI drug.

A novel aryl hydrocarbon receptor nuclear translocator (ARNT)-interacting peptide (Ainpl) was characterized from human liver cDNA library using phage display. Ainpl suppresses hypoxia inducible factor-1a (HIF-1α) signaling pathway through an ARNTdependent manner. HIF-1α is known to be overexpressed in more than 90% of solid tumors, and the inhibition of HIF-1α is proved as an effective approach to suppress tumor growth. ARNT, as the obligatory heterodimeric partner of HIF-1α for downstream gene activation, was used as a bait to screen for Ainpl. Ainpl specifically interacts with the helix-loop-helix (HLH) subdomain of ARNT, but not with HIF-1α. GFP-Ainpl is localized in both cytoplasm and nucleus, and suppresses HIF-1α signaling by two mechanisms: (1) cytoplasmic GFP-Ainp 1 retains ARNT in the cell cytoplasm and (2) nuclear GFP-Ainpl inhibits HIF-1α/ARNT heterodimerization. The suppression of Ainpl on HIF-1α signaling was reversed by introducing ARNT into the cells using transient transfection. We further utilized HIV TAT protein transduction domain to deliver 6His-TAT-Ainpl into three different cancer cell lines (Hep3B, HeLa, MCF-7), and found that 6His-TAT-Ainpl co-localizes with ARNT in the cell nucleus. 6His-TATAinpl can be detected inside the cells after 30 min of transduction, and can reach the maximum level at 2 h. 6His-TAT-Ainp 1 remained detectable in the cells up to 96 h and had a half life of 24 h after transduction. In addition, 6His-TAT-Ainp 1 suppresses HIF-1α downstream genes at both message and protein levels in a dose-dependent manner. Taken together, molecules that target the HIF-1α and ARNT interface can be developed as viable drugs to suppress HIF-1α signaling.

Background: Up to 55% of patients administered ketamine, experience an emergence phenomena (EP) that closely mimics schizophrenia and increases their risk of injury. While genetics accounts for about 50% of severe adverse drug reactions, no studies have investigated genetic association of ketamine-induced EP in healthy patients. Ketamine is metabolized by CYP 2B6 enzymes and CYP 2B^8^ allele significantly alter ketamine metabolism. In addition, ketamine exerts most of its effects by inhibiting the N-methyl-D-aspartate receptor (NMADR), and NMDAR genes (GRIN2B) are associated with learning and memory impairment and schizophrenia. Purpose: To investigate the relationship between CYP2B6*6 and GRIN2B single nucleotide polymorphisms (SNPs) and ketamine-induced emergence phenomena (EP). Methods: This cross-sectional pharmacogenetic study recruited 75 patients having minor orthopedic, hand, foot, anorectal surgeries from two outpatient surgical centers. EP was measured with the Clinician Administered Dissociative State Scale (CADSS). DNA was genotyped using standard Taqman assays and protocols. Genetic association of CYP2B6*6 and GRIN2B (rs1019385 & rs1806191) SNPs and ketamine induced EP occurrence and severity were tested using multivariate logistic and linear regression, adjusting for age, ketamine dose, duration of anesthesia, and time since ketamine administration. Results: Forty-seven patients (63%) received ketamine and were genotyped. Nineteen EP cases were identified (CADSS > 4), leaving 28 non-EP controls. For our population, CADSS has an internal consistency reliability Cronbach’s alpha of 0.82, and could reliably distinguish ketamine from non-ketamine cases. Occurrence and severity of EP were not associated with CYP2B6*6 or GRIN2B (p > 0.1). Models removing genotype and containing age, ketamine dose, duration of v anesthesia, and time since ketamine administration significantly predicted EP occurrence (p = 0.001) and severity (p = 0.007). Presence and severity of EP did not affect patient satisfaction with care. Discussion: Younger age, higher dose and longer duration of anesthesia significantly predicted EP occurrence and severity among our sample. This study provides effect size estimates useful for the design of adequately powered future genetic association studies. The feasibility of recruitment from patients undergoing elective, outpatient surgeries and ease of post-operative EP assessment with CADSS supports our approach. However, the small sample size may have limited about ability to determine significant differences. Conclusion: Fully powered studies are needed to investigate this important phenomena. Determining factors for anesthesia-related EP symptoms may reduce risks and costs associated with this adverse medication effect.

Tumor microenvironmental conditions play a vital role in promoting metastasis and tumor recurrence. Due to inefficient vasculature, cancer cells experience hypoxia, glucose deprivation and low pH even during the early stages of tumor growth. Tumor cells are proposed to adapt to these microenvironmental conditions by acquiring increased migratory and invasion potential and tumor initiating ability. Our research addresses the effect of these biochemical factors of the tumor microenvironment (TME) on motility, epithelial to mesenchymal transition (EMT) and stemness of non-small cell lung cancer (NSCLC). NCI-H292 and NCI-H1650 NSCLC cell lines were used to measure the effect of the above mentioned TME conditions. Apart from acidic pH, low glucose and hypoxia, the effect of high glucose conditions was also measured on H292 and H1650 cell lines. Acidic pH, high and low glucose conditions were observed to have no effect on the motility, EMT and stemness of H1650 cell line. Hence, use of this cell line was discontinued and no further treatment conditions were tested on this cell line. In H292 cell line, acidic pH, low glucose and tumor like conditions combined together (acidic pH + low glucose + hypoxia) [AP+LG+HYP] significantly decreased motility whereas hypoxia significantly increased the motility of H292 cells. High glucose did not affect the motility of H292 cells. Although N-cadherin, a mesenchymal marker, expression was significantly upregulated by acidic pH, high and low glucose conditions, no direct correlation was observed between N-cadherin expression and motility. E-cadherin expression was not affected by acidic pH, high and low glucose conditions. An increase in N-cadherin expression and no change in E-cadherin expression under these conditions might be an indication of partial EMT. Hypoxia and AP+LG+HYP did not alter the expression of E-cadherin and N-cadherin. Although expression of vimentin, another mesenchymal marker, and Sox2, a cancer stem cell marker (CSC), was observed at the mRNA level, no expression of vimentin and Sox2 proteins was observed in H292 cells under any of these treatment conditions. The expression of OCT4, another CSC marker, was also not observed at the protein level in H292 cells. HIF-1α expression was observed in H292 cells under normoxic conditions and was unaffected by hypoxia and AP+LG+HYP. Therefore our research indicates that the effect of these TME conditions might be different on different cancer cell lines or cancer types. Not all cancers may depend on EMT for metastasis. An increase in metastasis under hypoxia may be independent of HIF-1α.

The data presented in this thesis is a comprehensive study on the nature of peptide structure and how subtle and systematic changes in sequence and sidechain affect the basicity, ion stability, and conformation of a peptide. The peptides characterized were acetylated polyalanine di-, tri-, and tetra- peptides containing a proton-accepting probe: lysine and or the non-proteinogenic lysine-homologs: ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid. Peptides were studied in isomeric pairs for which the basic amino acid was placed closest to the N-terminus or the C-terminus of each peptide family (A n Probe vs. ProbeA n ). Using a variety of mass spectrometry based techniques and infrared multiphoton dissociation ion spectroscopy, the isomeric families of polyalanine peptides were characterized. Quantum chemical techniques were employed in parallel to provide theoretical predictions of three-dimensional structure, physical properties (dipole moment, polarizability, and accessible surface area), thermochemical values, and vibrational IR spectra, to gain further understanding of the peptides studied and to push the limits of current theoretical models. Overall it was found that the AnProbe peptide was more basic than their ProbeAn isomer. For the dipeptide systems, the greater basicity of AProbe peptides was due to efficiently charge-solvated ions which formed more compact structures compared to their ProbeA counterpart. For the tri- and tetra- peptide systems, greater basicity of the A 2,3 Probe peptides was likely due to formation of α or 3 10 helix-like structures in the protonated forms., introducing the macrodipolar effect, which cooperatively encouraged helical formation while stabilizing the charged site. On the other hand, ProbeA 2,3 peptides formed charge-solvated coils which do not exhibit any kind of dipole effect, resulting in lower basicity than their A2,3Probe counterpart.

Predicting drug concentrations in the blood and at the site of action is the hottest topic in pharmacokinetics (PK). In vitro-in vivo extrapolation (IVIVE) and physiological based pharmacokinetics (PBPK) models are two major PK prediction strategies. However, both IVIVE and PBPK models are considered as immature methodologies due to their poor predictability. The goal of the research is to investigate the discrepancies within IVIVE and PBPK predictions according to first-principles: convection, diffusion, metabolism, and carrier-mediated transport. In Chapter 2, non-permeability limited hepatic elimination under perfusion steady state is examined. The well-stirred model is re-derived from the convection-dispersion-elimination equation when both dispersion and concentration gradient are ignored and re-named as the zero-gradient model. Pang and Rowland’s lidocaine data are re-analyzed. Their data analysis was based on an unfair comparison of the zero-gradient and parallel- tube models at two different efficiency number ranges. The interference of sensitivity greatly biased the comparison. I also show that both theoretical discussions and experimental results indicate that apparent intrinsic clearance and intrinsic clearance could be affected by blood flow and protein binding. In Chapter 3, I discuss permeability limited hepatic elimination under perfusion steady state. Permeability limited elimination is classified to diffusion dominated, carrier-mediated transport mediated, and mixed effects based on drug passage mechanisms. Each of these three drug passage classes is sub-divided to sink condition and finite volume condition based on the boundary conditions of drug passage. In Chapter 4, the discrepancies within IVIVE for both non-permeability limited and permeability limited drugs are explored. The deficiencies in assay design and data analysis of common in vitro metabolism assays are investigated. The scaling/converting equations for both non-permeability limited and permeability limited drugs are derived. In Chapter 5, I focus on transient PK models. Numerical analysis using finite difference and finite volume methods are introduced into the derivation and discussion of transient PBPK models. In addition, the use of partition coefficient in the non-eliminating tissue/organ models is discussed.