Accurate prediction of target activity of a drug and rational design of dosing regimen requires knowledge of concentration-time course of the drug at the target. In vitro in vivo correlation (IVIVC) successfully predicts activity and pharmacokinetics of some drugs but is unsuccessful with many others due to poor permeability, transporter activity and use of plasma drug concentrations for determination of PK parameters. According to the free drug hypothesis, at steady state, the unbound drug concentration on either side of a biomembrane is equal. In this case, unbound plasma drug concentration acts as a good surrogate for unbound cell concentrations. However, presence of transporters coupled with poor membrane permeability result in violation of the free drug hypothesis. This results in failure of IVIVC and subsequent discrepancies in the prediction of target activity of pharmacokinetic predictions. Since it is the unbound drug that is capable of exerting the pharmacodynamic effect and available for intracellular metabolizing and transport machinery, knowledge of the unbound concentration inside the cell is very important. Experimental measurement of intracellular unbound concentration is very difficult due to the small size of the cell and complex cellular disposition resulting from activity of metabolizing enzymes, transporters, target binding and organelle binding within the cell. The present study, therefore, aims at predicting the intracellular unbound concentrations using modeling and simulation approach. Liver perfusion experiments were conducted in male Sprague Dawley rats with uptake transporter substrates atorvastatin and bosentan, in presence and absence of inhibitors of active uptake and metabolism, to study tissue distribution of these drugs in presence of uptake transport and metabolism. The outflow perfusate data thus obtained were used as input for the explicit membrane model for liver to predict the unbound intracellular concentrations of atorvastatin and bosentan. Similarly, in vivo pharmacokinetic experiments were also conducted in rats in presence and absence of inhibitors of active uptake and metabolism. The data obtained were used as input for hybrid compartmental models to predict unbound concentrations of these drugs upon intravenous dosing. Modeling exercises were also conducted to study the differential impact of inhibition of active uptake on plasma versus unbound cell concentrations. The effect of uptake transport on the induction potential of bosentan was studied in sandwich cultured rat hepatocytes and in in vivo studies in rats. Inhibition of active uptake in the liver perfusion studies increased the outflow perfusate concentrations, decreased the amount recovered in the bile for atorvastatin and bosentan, and decreased the liver concentrations for atorvastatin. The liver concentrations for bosentan with inhibition of active uptake were not different than the control group. Inhibition of active uptake in the in vivo studies also decreased the systemic clearance of atorvastatin and bosentan. Inhibition of metabolism decreased the systemic clearance of bosentan. It was observed that the perpetrators for metabolism and transport used for this project were not specific for the pathway of interest. Active uptake appeared to be of major significance for disposition of atorvastatin. The model predicted unbound concentrations of atorvastatin at the end of 50 min perfusion were about 7-fold higher in presence of active uptake than in absence of active uptake. On the other hand, inhibition of metabolism resulted in 1.26 fold increase in unbound atorvastatin concentrations inside the cell. Modeling the in vivo data indicated that atorvastatin disposition was not affected until 90% inhibition of active uptake clearance was achieved. However, any further inhibition of active uptake clearance had a largely increased the exposure of this drug. The predicted unbound intracellular bosentan concentrations in presence of active uptake were only marginally higher than in the absence of active uptake, possibly due to inhibition of apical efflux of this drug by the uptake inhibitor, rifampin, used in this study. The modeling exercise showed that in the in vivo studies, BOS disposition was sensitive to intrinsic uptake clearance until 99% inhibition was achieved. However, any further inhibition resulted in minimal change in the exposure of this drug. The differential sensitivity of atorvastatin and bosentan exposure for active uptake clearance was thought to be due to the different diffusional clearance for these drugs. For both atorvastatin and bosentan, simulations indicated that any extent of inhibition of the active uptake clearance did not affect the cell exposure of these drugs. In vitro induction of bosentan could not be characterized in sandwich cultured rat hepatocytes. Bosentan appeared to be a weak inducer of cyp3a mediated metabolism in rats. In summary, the impact of uptake transport and metabolism on the systemic and intracellular disposition of atorvastatin and bosentan was studied. Liver perfusion and in vivo pharmacokinetic studies along with explicit membrane models were successfully used to predict unbound cell concentrations of atorvastatin and bosentan. / Pharmaceutical Sciences

The objective of this study was to design, develop and evaluate an amorphous solid dispersion (ASD)-loaded controlled release (CR) matrix system for poorly water-soluble drug in order to enhance its solubility and dissolution rate while maintaining the stability of the supersaturated state during and after dissolution. Two types of polymeric carriers, namely hydroxypropyl methylcellulose acetate succinate (HPMCAS) and copovidone S-630, were used in spary-dried dispersion (SDD) formulations. Weak acid drugs glipizide and gliclazide which tend to have limited solubility and dissolution rate were chosen as poorly soluble model compounds representing Class-II drugs according to the FDA adopted Biopharmaceutics Classification Scheme. The unique features of the hydrating CR matrix system consisting of amorphous drug dispersion that provides supersaturation and probable mechanisms to inhibit precipitation are investigated. SDD-loaded CR matrix tablets prepared in this study were based on use of automated spray dryer and compaction simulator with optimized process parameters in order to control the critical quality attributes (CQAs) of final formulations. A 3×4 full factorial design was conducted to understand the effect of drug loading level and polymer type on the performance of SDD products for both glipizide and gliclazide. Drug loading level and succinoyl content % of HPMCAS were identified as two critical factors for meeting goals set to achieve maximum concentration under non-sink condition referred to as Cmax. Compliance to Cmax limit is assured by selecting drug loading level and type of HPMCAS within design space. Spray-dried dispersions (SDDs) of glipizide and gliclazide were prepared using HPMCAS (H, M and L grades) and copovidone as amorphous matrix forming polymer in order to improve the solubility and dissolution rate of the drug. The SDDs appeared as a single amorphous phase with up to 60% drug loading level as revealed by X-ray powder diffraction (XRPD), modulated differential scanning calorimetry (mDSC) and scanning electron microscopy (SEM). Supersaturated micro-dissolution testing of SDD powders in fasted state simulated intestinal fluid showed prolonged supersaturation state (up to 180 minutes) with significant solubility increase relative to crystalline drug under same conditions. Moreover, plot of relative dissolution AUCs (AUC (SDD) /AUC (crystalline)) versus stable supersaturated concentration ratio (C180/Cmax) was provided as an appropriate formulation strategy for selecting SDDs with improved solubility and supersaturation stability. Selected SDDs were formulated into matrix tablet with rate-controlling hydrophilic polymer, hydroxypropylmethylcellulose (HPMC) and other excipients. Dissolution data based on standard USP paddle method indicated that SDD-loaded CR tablets provide stable supersaturated concentration within the hydrated matrix with increased rate and extent of drug dissolution. Co-existence of HPMCAS and HPMC within the hydrating matrix showed strong suppression of drug crystallization and allowed achievement of zero-order and slow-first order release kinetics. In conclusion, results of this study indicate that use of HPMCAS as drug carrier in spray-drying process is able to produce homogeneous single phase SDDs which are stable and promising for inclusion into CR systems in development of CR dosage forms of poorly water-soluble drugs. / Pharmaceutical Sciences

Cancer is the second most common cause of death and a major cause of mortality in the world. The high mortality associated with cancer is due to the fact that usually it is discovered too late, when it is metastasized to different organs and is very hard to cure. Finding more efficient, convenient and selective ways for early diagnosis and eradication of pre-malignant or malignant tumors of small dimensions is a task of utmost importance. The development of many malignant tumors was associated with hypoxia and the over-expression of specific membrane-bound carbonic anhydrase (CA) isozymes CA IX and CA XII. Malignant tissue of relatively small dimensions can grow fast and can invade the surrounding tissues by reverting to anaerobic metabolism and by acidifying the extracellular milieu around the tumor, increasing its invasiveness. Our goal is to detect and treat malignant hypoxic tumors using selective inhibitors of CA IX and CA XII and the objective of this thesis was to develop selective and efficient carbonic anhydrase inhibitors (CAIs) for the tumoral CA IX and CA XII that will leave unaltered the normal tissues. Two new sets of membrane-impermeant carbonic anhydrase inhibitors are proposed to be developed based on pyridinium positively-charged moieties attached to know CAI pharmacophores. Our guiding hypothesis was that modulation of carbonic anhydrase potency and tissue penetrability is possible to be achieved via fine tuning of pyridinium substitution. The use of appropriate substitutents on the pyridinium ring allowed the creation of CAI with special optical properties (e.g. fluorescence). The rationale for the research summarized in this thesis was that a CAI selective for membrane isozymes CA IX and CA XII over-expressed in cancer with controlled tissue penetrability can open new avenues in cancer early detection and treatment that will complement and/or potentiate present technologies and therapies. / Pharmaceutical Sciences

Pharmacologic blockade of 5-Lipoxygenase (5-LO) through its inhibitor, zileuton (Zyflo®), has been shown to reduce both gamma secretase-catalyzed misprocessing of amyloid precursor protein and over-phosphorylation of tau protein (two hallmarks of Alzheimer’s disease (AD)) in transgenic mice (3xTg, Tg2576). However, zileuton suffers from low potency, liver toxicity, gastrointestinal side effects, and a suboptimal metabolism profile that hamper its development as a viable disease-modifying treatment for AD patients, who are usually older. Prodrugs of zileuton that deliver therapeutic concentrations of the parent molecule to the brain at low plasma concentrations could overcome these problems. In addition, other 5-LO inhibitors (ABT-761, BWA4C) with similar structure but greater potency might also be amenable to facilitate the discovery of the best prodrug for AD. A prodrug is a biologically inactive compound that is metabolized in the target tissue to release the parent drug. Prodrug strategies for Central Nervous System (CNS) delivery include masking polar groups with lipophilic moieties that promote brain penetration, Chemical Delivery Systems (CDS) that trap prodrugs in the brain and incorporating brain nutrients which engage Blood Brain Barrier (BBB) nutrient transporters. We have pursued all three strategies to enhance CNS delivery of the prototype hydroxyurea 5-LO inhibitor analog zileuton. To accomplish this task we synthesized and characterized 48 5-LO prodrugs for zileuton that falls into all three prodrug categories, and several lipophilic and CDS prodrugs for the other two 5-LO inhibitors (22 for ABT-761 and 22 for BWA4C,). The prodrugs were tested in a battery of in vitro assays that included solubility, plasma/simulated gastrointestinal fluid/microsomal stability, cell toxicity, and 5-LO inhibition assays. The promising compounds then went to the second tier of screening by equilibrium dialysis with plasma/brain homogenate/microsomes, and in vivo pilot pharmacokinetics study and full pharmacokinetics studies. As a consequence of this work, we identified six zileuton prodrugs (RF14, RF15, RF75, RF77, RF87, and RF88) with reasonable solubility, stability, safety, protein/lipid binding, and no 5-LO inhibitory activities and advanced them to the in vivo pilot pharmacokinetics studies. The most lipophilic prodrug RF58 was also tested in vivo as a comparator to RF14 and RF15 despite its fast metabolism in plasma. However, none of them demonstrated better CNS delivery of parent drug compared to administration of the parent itself. For the lipophilic prodrugs, two carbamate prodrugs (the racemic mixture of RF14 and RF15, RF58) were tested in the in vivo pilot study. The racemic mixture of RF14 and RF15 (RF14/15) with slightly increased lipophilicity (CLogP = 2.81 compared to 2.48 of zileuton) didn’t demonstrate a better brain penetration (Brain/Plasma = 0.06 in RF14/RF15 compared to 0.5 for zileuton). In addition, the carbamate linker was relatively stable in the brain, which resulted in a low zileuton brain-to-plasma ratio (0.03). The very lipophilic RF58 (CLogP = 6.51) demonstrated a large brain-to-plasma ratio (Brain/Plasma = 21.7), however, it suffered from rapid metabolism and extremely slow conversion to zileuton. The conclusions from this category of prodrug were that an increase in CLogP could increase the brain penetration, but the carbamate linker was too stable in the brain to ensure reasonable conversion to zileuton. RF14/15 had less brain penetration possibly because its rotatable bonds were doubled compared with zileuton (rotatable bond = 3). The large increase of entropy offseted the small increase of CLogP. For the CDS prodrugs, distal ester prodrugs were unstable in the in vitro screening, while two distal amide prodrugs with trigonelline and dimethoxy-dihydroquinoline promoieties RF75 and RF77, respectively, showed reasonable in vitro data and advanced to pilot in vivo pharmacokinetics studies. Both of them demonstrated a better zileuton brain-to-plasma ratio (0.726 for RF75, 1.98 for RF77 compared to zileuton 0.5), which indicated a CNS-targeted effect. However, RF75 suffered from rapid peripheral elimination and RF77 suffered from low brain penetration (Brain/Plasma = 0.008), which led to a very low zileuton concentration in the brain and thus both prodrugs did not advance to full pharmacokinetics study. The low level of RF75 was probably due to its fast oxidization to the charged pyridinium intermediate which then suffered from rapid elimination. Similarly, a rapid oxidization would also cause RF77 to show low brain penetration (although p-glycoprotein efflux cannot be ruled out since it was not measured). The conclusions from this approach are that CDS is a good CNS targeted delivery system for zileuton and the distal amide linker is tolerable in this system although the brain conversion (approximately 10%) is slower than the plasma conversion (approximately 30%). The dihydroquinoline CDS system was more stable than the trigonelline CDS system. The low brain penetration of dihydroquinoline system should be investigated and additional substituted dihydrquinoline CDS analogs should be synthesized to continue this investigation. For the transporter-mediated prodrugs, esters were not stable while one glucose prodrugs RF87 with a glycosidic bond and one lysine prodrugs RF88 with carbamate bond were advanced to in vivo pilot pharmacokinetics study. Similar with RF75, although RF87 demonstrated better zileuton brain-to-plasma ratio (0.79), it suffered from rapid peripheral metabolism that prevented its further development. RF88 demonstrated limited brain penetration (Brain/Plasma = 0.013) which indicates either that RF88 was a weak substrate for LAT1 or that this prodrug was more a LAT1 inhibitor than a substrate. However, encouraged by its high plasma concentration at 30 min, it was possible that at a later time the brain concentration of zileuton could be higher. So the full pharmacokinetics studies were performed. However, this prodrug did not provide better zileuton exposure than zileuton itself throughout a 6 hour time period. Although the current prodrugs did not provide better brain zileuton exposure compared to administering the parent drug itself, the project could be further investigated with the parent drugs ABT-761 and BWA4C. In addition, analogs that are less stable in plasma than the ones advanced in the screening triage, CDS promoieties with different substitution patterns to modulate oxidative potential; and transporter-mediated promoieties containing different amino acids and sugars could be further investigated to pursue the discovery of better prodrugs of 5-LO inhibitors for Alzheimer’s disease. The key to success in future efforts will be the identification of a reliable in vitro screening assay to measure the ability of the prodrugs to be converted to parent drug in the brain. / Pharmaceutical Sciences

Time-dependent inactivation (TDI) of CYPs is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to over-predict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human liver microsomes. Inhibitors evaluated include troleandomycin (TAO), erythromycin (ERY), verapamil (VER), Paroxetine (PAR), itraconazole (ITZ) and diltiazem (DTZ) along with primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (MA). Complexities incorporated in the models included multiple binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The different factors affecting TDI kinetics were evaluated such as lipid partitioning, inhibitor depletion, presence of transporters. The inactivation parameters obtained from numerical method were incorporated into static in-vitro – in-vivo correlation (IVIVC) models to predict clinical DDIs. For 123 clinically observed DDIs, using a hepatic CYP3A synthesis rate constant of 0.000146 min-1, the average fold difference between observed and predicted DDIs was 2.97 for the standard replot method and 1.66 for the numerical method. Similar results were obtained using a synthesis rate constant of 0.00032 min-1. These results suggest that numerical methods can successfully model complex in-vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach. Chapter one presents the detailed introduction along with the hypothesis and significance of the project. Chapter 2 includes the development of the bioanalytical method for quantitation of various compounds which includes inactivators and their primary metabolites. Chapter 3 entails the discussion on in-vivo studies in rats involving TDI mediated DDI studies. Chapter 4 discusses the in-vitro studies and use of the numerical method for evaluation of TDI kinetics. Chapter 5 and chapter 6 provides discussion on the impact of inhibitor depletion and partitioning of TDI kinetics and how these two could lead to misinterpretation of TDI results. Chapter 6 also provides a discussion on how transporters could affect TDI results mainly from hepatocyte studies. Chapter 7 involves prediction of TDI mediated DDI using static modeling. Chapter 8 is a case study on bosentan involving induction mediated DDI. / Pharmaceutical Sciences

Hypoxia is a common feature of many solid tumors, including colorectal, breast and ovarian carcinoma. Under hypoxia, tumor cells escape p53-mediated apoptotic cell death and instead adopt a HIF-mediated pro-survival and pro-metastatic pathway. CA IX is a membrane bound carbonic anhydrase isozyme over-expressed in hypoxic tumor cells as a HIF-mediated adaptive response to tumor hypoxia. CA IX-mediated intracellular pH homeostasis and extracellular pH drop have been implicated in survival, invasion, metastasis, and resistance of hypoxic tumor cells to weakly basic chemotherapeutic drugs, e.g., doxorubicin. Inhibition of CA IX by small molecule inhibitors as well as monoclonal antibodies (mAbs) has been shown to be an effective approach to regress the tumor growth. Given its high expression level and the functional importance in hypoxic tumors, inhibition of CA IX catalytic activity by small molecule CA IX inhibitors has the potential to rescue, potentiate, or even synergize with the efficacy of doxorubicin against CA IX expressing hypoxic tumors. Building upon this hypothesis, in this dissertation, we focused our efforts towards evaluating combinations of CA IX inhibitors together with doxorubicin and map for synergistic cytotoxicity. In Chapter 2, we established the cellular models we used throughout this dissertation: colorectal cancer cell model HT-29, triple negative breast cancer cell model MDA-MB-231 and ovarian cancer cell model SKOV-3, previously known to express CA IX. In Chapter 3, we explored and established a relationship between CA IX expression and the efficacy of weakly basic drug doxorubicin against tumor cells taking a set of 8 high grade serous ovarian carcinoma cell models (representing ovarian cancer of different origin and subtypes). In Chapter 4, we screened a series of ureido sulfonamide CA IX inhibitors to select leads to use in combination with doxorubicin against the same cancer cell lines. We used a plethora of in vitro and in silico screening techniques to shortlist the best performing compounds for efficient tumor killing. Subsequently, in Chapter 5, we developed and validated LC-MS/MS methods for simultaneous quantitation of doxorubicin and ureido sulfonamide CAIs and successfully applied them to identify and quantitate these drugs/drug combinations in SKOV-3 cell lysates and external cell growth media. In Chapter 6, the best performing CAIs selected in Chapter 4 were initially tested in combination with doxorubicin 2D under normoxia and hypoxia. Cell viability upon treatment with drug combinations and the combination index values were calculated from the cell viability assay data to map for synergism between doxorubicin and CAIs. The 2D experiments allowed us to further select CAIs to be tested in combination with doxorubicin in 3D against tumor spheroids generated from the same cancer cells. As expected, we were able to identify CAI and doxorubicin combinations that synergized to kill the cancer cells in 3D tumor spheroids. Towards the end of this chapter, using SKOV-3 as the model cell line, we established the mechanism of synergism by LC-MS/MS as well as by live cell image analysis. In Chapter 7, we optimized a PEG-PCL based polymeric drug delivery system (DDS) for co-delivery of doxorubicin and CAIs. Thorough pre-formulation and formulation studies allowed us to identify critical formulation and process parameters and optimize them to maximize drug loading and encapsulation efficiency. The optimized method was also tested for its reproducibility and for the stability of the final formulations. Stability of the final formulations was the final criteria used to further screen the compounds and combinations in the polymeric DDSs. A release study was conducted towards the end of this chapter to discern the release kinetics of the drug pairs co-loaded in the polymeric micelles. In Chapter 8, lead formulations were tested for their efficacy against the cell models displaying the highest and lowest CA IX expression, namely SKOV-3 and MDA-MB-231 cell lines. Several control experiments were also conducted in parallel and free CAIs, free doxorubicin, micelle loaded with CAIs, micelle loaded with doxorubicin, liposome loaded with doxorubicin and liposome loaded with doxorubicin in presence of CAIs were included to test on the same tumor models. A thorough comparative analysis of these different delivery modalities was also made. Finally, a correlation between efficacy and drug release kinetics of co-loaded micelles were identified and discussed. In conclusion, the synergistic cytotoxicity of ureido sulfonamide CA IX inhibitors were thoroughly investigated and synergistic dose combinations were identified. A PEG-PCL-based drug delivery system was optimized to co-load and deliver synergistic dose combinations in vitro in 2D and 3D cell models. / Pharmaceutical Sciences

Background: Primary cilia are cellular organelles project from the cell surface of mammalian cell and play important roles in vertebrate development, organogenesis, health, and others genetic diseases. Primary cilium functions as a mechano-sensor and chemo-sensor. Defect in primary cilia causes the progression of polycystic kidney disease (PKD) which further leads to the inflammatory responses. We, therefore, investigated the role of Toll-like receptors 4 and 9 (TLR) in primary cilia towards PKD. Purpose: The main purpose of the proposed study is to identify and target the immune reactive proteins i.e. TLRs in the primary cilia. By targeting those primary cilia immune reactive proteins using suitable agonist and antagonists to study the control of cystic formation and their progression mechanisms. Methods: To target the ciliary immune TLR proteins (TLR4 and TLR9), we did immunostaining to evaluate their localization on primary cilia. Cilia lengths were measured and compared using differential interference contrast (DIC) and fluorescent imaging techniques. The in vitro3D cyst progression was monitored by adding agonists lipopolysaccharide (LPS) and oligodeoxynucleotides (ODN) and antagonist 4-hydroxy chloroquine (HCQ). Results: From our results we found that the TLR antagonist HCQ increases ciliary length in treated scrambled control, Pkd2knockout (KO) and TLR4KOcells as an immune response, whereas opposite results were observed with TLR9KO. However, the selected agonists for TLRs (LPS/ODN) increases cilia length in TLR9KO cells and decreases scrambled control, Pkd2KO and TLR4KO. In our 3D cyst cultures, we used agonists and antagonist for both the TLRs and observed that the cyst formations and progressions were inversely related to the cilia lengths. From these observations, we speculated that the new ciliary TLR proteins have a role in cystic progression. In conclusion, we found that the TLRs agonists/antagonist can modulate cilia length and TLRs role in inflammatory actions. The primary cilium already has central roles throughout cell biology, but here we propose, for the first time, that the cilium and the regulation of its structural importance in inflammation of PKD.

Excerpt:To the Editor: Recently, the American Journal of Pharmaceutical Education published a research brief entitled, “Spirituality and Religiosity of Pharmacy Students” by Purnell and colleagues.1 The authors describe their recent survey assessing the presence and impact of religiosity/spirituality on various facets of student life and students’ opinions. In three separate instances, the authors advocate for greater support to be provided by pharmacy schools for student religiosity/spirituality.

<p>Heavy water moderated thermal nuclear fission reactors have a greater inherent neutron economy than light water or graphite moderated reactor designs. Consequently, such units, operating on a variety of fuel cycles, may play an ever-increasing role in meeting future global energy demands. This thesis explores, analytically, the operational advantages and challenges associated with the use of a low enriched uranium (LEU) fuel cycle in advanced reactors based on the CANDU heavy water moderated, pressure tube design concept. The flexibility afforded through the use of LEU fuel is applied to enhance the operational and safety characteristics of reactors utilizing this fuel cycle. An investigation of factors influencing coolant void reactivity is conducted. Design modifications are introduced to reduce the coolant void reactivity, while maintaining the continued capability of high power operation. An enrichment and element radius graded fuel bundle design is developed with a central graphite core, an inner ring of 14 fuel elements, and an outer ring of 21 fuel elements. Fuel and lattice design perturbations are investigated to examine the effect of lattice pitch variations, capability of radioisotope production, the use of burnable poisons, and light water coolant. Xenon override requirements with LEU fuel are addressed. The efficacy of using modified two group (M-2) neutron diffusion theory for LEU fuel management studies is investigated. A modelling strategy is developed for the simulation of reactivity devices and fuel lattice properties using the M-2 methodology with a fixed energy cut-off. Detailed fuel management studies are conducted to examine the operational intricacies of LEU fuelling. Improved checkerboard type fuelling strategies are developed. Finally, the CANDU - Spectral Shift Advanced Thermal Reactor (CANDU-SSATR) is introduced and characterized. This multi-spectrum high burnup advanced reactor design utilizing simplified fuel management strategies holds great promise for the future.</p> / Doctor of Philosophy (PhD)

<p> Inappropriate expression of nicotinic acetylcholine receptors in the central nervous system is associated with nicotine addiction, Alzheimer&rsquo;s disease, Parkinson&rsquo;s disease and other disorders. Modulators (drugs) have the potential to restore circuit properties that arise from inappropriate expression of nicotinic receptor&rsquo;s. Compounds that interact with allosteric sites have a distinct advantage over agonists and partial agonists, in that, they retain normal activation patterns by allowing binding of the endogenous ligand. Positive allosteric modulators boost the receptors ability to respond to agonist. Studies of these modulators constitute a first step toward the identification and development of better compounds that minimize signaling errors at cholinergic synapses. We have used single molecule methods to investigate the action of a novel positive allosteric modulator, desformylflustrabromine (dFBr), on nicotinic receptors. Our studies were focused on the &alpha;4&beta;2 subtype of nicotinic receptors in the brain. These receptors exist in two forms with low sensitivity (&alpha;4₂&beta;2₃) or, alternatively, high sensitivity (&alpha;4₂&beta;2₃) to agonist. Our experiments allowed us to develop detailed gating models for high and low sensitivity receptors, as well as gain new insights regarding the mechanisms that underlie potentiation by allosteric modulators. We found that dFBr potentiates low sensitivity receptors by destabilizing desensitized states of the receptor. In contrast, potentiation of high sensitivity receptors arises from a synchronization of openings following an application of agonist due to an increase in the opening rate. Based on our results we now have a better understanding of the advantages of dFBr on high and low sensitivity receptors.</p>