The 5-HT7¬ receptor is the most recently discovered 5-HT receptor subtype. 5-HT7 is a GPCR that exhibits a regulatory role in many biological functions in both the central nervous system (CNS) and the periphery. Recent literature has demonstrated a connection between the 5-HT7 receptor and Inflammatory Bowel Disease (IBD) progression. IBD is a devastating disease that affects 1.4 million Americans. Patients suffer from life altering symptoms as a result of severe, chronic inflammation of the gastrointestinal tract. Current treatments mitigate symptoms with no effect on disease progression. Targeting the 5-HT7 receptor as a novel treatment option is a viable medicinal chemistry project that could result in a therapy capable of providing relief to IBD patients. A novel series of butyrolactones were discovered during a prior thesis project completed by Dr. Rong Gao at Temple University’s School of Pharmacy. Broad screening indicated that many of the compounds within this series were potent binders of the 5-HT7 receptor. These results led to the initiation of a medicinal chemistry program aimed at the development of this series with the intent to identify novel 5-HT7 receptor antagonists that are suitable for pre-clinical and clinical evaluation for the treatment of IBD. Medicinal chemistry strategies were utilized in order to optimize each structural aspect of the butyrolactone pharmacophore. This required the preparation of several small series of compounds wherein one structural feature was systematically changed while the remaining features were held constant. The particular properties that were studied for optimization included 5-HT7 affinity, subtype selectivity, liver microsomes stability (mouse and human), and the topological polar surface area (to minimize CNS penetration). Implementing these strategies led to the identification of potent 5-HT7¬ antagonists, some of which exhibited excellent subtype selectivity and improved mouse liver microsome stability. Two analogs, 170073 and 230168, were chosen for further study. Both analogs exhibited adequate in vivo pharmacokinetic profiles capable of supporting efficacy in an in vivo setting. 170073 distributed rapidly and extensively into brain tissue, while 230168 moderately distributed into brain tissue. Moving forward, reducing CNS penetration will become a top priority. These two compounds were examined in the DSS induced mouse model of IBD and both exhibited efficacy. Specifically in the acute DSS model of colitis, 170073 and 230168 significantly lowered the disease activity index, mitigated histological damage and reduced the production of proinflammatory cytokines. In addition, 170073 demonstrated efficacy in the chronic DSS model of colitis. 230168 has yet to be tested in the chronic model. The results of this dissertation support the validity of this project and the use of 5-HT7¬ ¬¬antagonists as a potential novel treatment option for IBD. / Pharmaceutical Sciences

HIV- associated neurocognitive disorder (HAND), also known as HIV encephalopathy and AIDS dementia, is one of the critical complications of HIV infection that causes severe morbidity and even shortens survival. This complication is challenging to treat because most of the antiretroviral therapeutic (ART) agents cannot achieve the desired therapeutic levels in the central nervous system (CNS) because they cannot efficiently cross the blood-brain barrier (BBB). The goal of this study is to develop a new transferrin conjugated nanoemulsion system (Tf-NE) for antiretroviral medication delivery and evaluate its potential to cross the BBB. Nanoemulsions were prepared based on the solvent evaporation sonication method using lipids and phospholipids. To achieve brain delivery, holo-transferrin was conjugated to DSPE-PEG (2000)-Maleimide by NHS ester crosslinking reaction. Darunavir (DRV) was encapsulated into Tf-NE as an antiretroviral agent. Size, polydispersity index, and dispersion stability were characterized using dynamic light scattering (DLS) system. Morphology of the Tf-NE was investigated using transmission electron microscopy (TEM). Differential scanning calorimetry was performed to study the extent of drug solubilization inside the nanoemulsion system. To evaluate the in vitro toxicity of Tf-NE-DRV, MTT assay was performed by using the human brain endothelial capillary hCMEC/D3 cells and 293T cells. Cell uptake and drug transport assays were also conducted to investigate the in vitro activity of Tf-NE-DRV. In vitro efficacy studies, ex vivo imaging studies and in vivo biodistribution studies were performed to investigate the brain targeting function of Tf-NE-DRV. Tf-NE-DRN was prepared 100-130nm in diameter with polydispersity index smaller than 0.3. This nanoemulsion system was stable in serum-enriched medium and PBS at 37 ℃ for 5 days. All the therapeutic compounds were well-dispersed inside the oil core of Tf-NE-DRV. Within the therapeutic concentration range, Tf-NE-DRV did not cause a significant reduction in the cell viability, indicating low toxicity of the formulation. Considerable uptake of Tf-NE-DRV into the BBB model of hCMEC/D3 cells was observed. Tf-NE-DRV can maintain the same therapeutic function as the free drug form of darunavir in vitro. Imaging and biodistribution results revealed the formulation was superior to the free drug and able to transport the drug across the BBB in vivo to reach the therapeutic level. We successfully developed a biocompatible nanoemulsion system that can effectively penetrate the BBB. This Tf-NE-DRV system shows the clinical potential to deliver antiretroviral agents into the CNS system to achieve improved treatment of HAND. / Pharmaceutical Sciences

Osteomyelitis is a bone infection disease that is caused by microbes. One of the reason that a successful antimicrobial therapy has not been achieved in bone related infection is due to the physiological and structural limitations and multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Alendronate, a type of bisphosphonate, is a commonly used drug to treat osteoporosis that can strongly chelate with the calcium ions in bone mineral (hydroxyapatite), could be utilized as an active targeting moiety in a drug delivery system to bone tissues. Since nanomedicine can provide a robust drug delivery platform, with the properties of encapsulating molecules of different hydrophilicity, tunable drug release profile, and potential of differential targeting cells and tissues, we proposed a lipid-polymer nanoparticle system, Bone-Targeting Nanoparticle (BTN), with surface modified with covalently bonded alendronate. In this study, BTN encapsulates linezolid, which has dose-related adverse effect that prevent long duration usage. According to our current results, BTN demonstrates three distinguished traits that potentially improves the therapeutic effect of linezolid towards MRSA induced osteomyelitis: a) a hydrophobic polymeric core that can encapsulate a high amount of linezolid; b) alendronate as a targeting moiety that can guide BTN to bone tissue and accumulate near the site of infection; and c) a PEGylated lipid interface that can enhance the drug release profile and provide increased serum stability relative to standard delivery methods. / Pharmaceutical Sciences

With the growing use of physiologically-based pharmacokinetic (PBPK) modeling to predict the pharmacokinetics of a drug, accurate prediction of the tissue: plasma water partition coefficients (Kp,m) has become increasingly important. In these predictions, drug-lipid interactions have been traditionally described using the octanol: water partition coefficient (logP) and the vegetable: oil: water partition coefficient (logPvo). However, the logP does not fully represent all of the drug interactions with phospholipids, while the logPvo is calculated from the logP and not determined experimentally. Partitioning into microsomes has been used as a potential surrogate for phospholipid partitioning in our previous steady-state volume of distribution prediction method. Microsomal partitioning is able to act as a total phospholipid partitioning term, representing both acidic and neutral phospholipid interactions. Partitioning into adipocytes potentially can provide an alternative surrogate for d / Pharmaceutical Sciences

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