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Malaria is a mosquito borne infectious disease caused by a unicellular Apicomplexan parasite of the Plasmodia genus. The emergence and subsequent spread of drug resistance in the highly virulent Plasmodium falciparum parasite has been a major setback in eradicating malaria, which affects an estimated 216 million individuals and causes 445,000 deaths annually worldwide. Chloroquine (CQ) was once used as the first-line antimalarial drug treatment, until CQ-resistant parasites emerged in endemic regions including Africa, Southeast Asia, and South America. More recently, parasites have developed resistance to the current first line drug piperaquine (PPQ), used in combination with dihydroartemisinin (DHA) in Southeast Asia. Plasmodium falciparum chloroquine resistance transporter (PfCRT), a member of the drug/metabolite transporter (DMT) superfamily, is a 49-kDa integral transmembrane protein localized in the digestive vacuole (DV) of the pathogenic parasite. Mutations in PfCRT have been identified as the core determinants of Plasmodium falciparum resistance to CQ and PPQ by mediating the efflux of these antimalarial drugs. All CQ resistance-conferring PfCRT isoforms share the K76T mutation, which is widely used as a molecular marker for CQ resistance. Despite the significance in the impact of drug-resistant malaria, a detailed understanding of PfCRT physiological function and the molecular basis of PfCRT-mediated drug resistance have been hampered by a lack of high-resolution structural information. This dissertation describes the first structure of PfCRT and reveals the interaction of drugs with the purified and reconstituted protein. We determined the structure of the 49-kDa PfCRT 7G8, a clinically relevant CQ-resistant isoform found in South America, to 3.2 Å resolution by single-particle cryo-electron microscopy (cryo-EM), in complex with a specific antigen-binding fragment (Fab) to overcome current size limitations in cryo-EM. Our PfCRT structure displays an inward-open conformation, consists of 10 transmembrane (TM) helices with an inverted topology, and has unique elements including two juxtamembrane helices and a highly conserved cysteine-rich loop between TM helix 7 and 8. The architecture of PfCRT is similar to other members of the DMT superfamily. TM helices 1-4 and 6-9 in PfCRT form a central cavity which is a potential binding site for both CQ and PPQ. A striking feature is that virtually all the CQ resistance mutations, identified from decades of investigation into PfCRT variants that have evolved independently across the malaria-endemic world, map around this central, negatively-charged cavity. Distinct mutations that have been proposed to cause high-level PPQ resistance in parasites, which cause a loss of CQ resistance, form a planar ring that also lines this cavity. Functional experiments with various purified PfCRT isoforms or mutants provide evidence that drug resistance is possibly due to pH- and membrane potential-dependent drug transport. We also show that PfCRT CQ-resistant isoforms bind and transport arginine, suggesting that positively charged amino acids may be putative transport substrates for CQ-resistant PfCRT. This work provides a structural and functional framework to understand the mechanism of PfCRT-mediated drug resistance in the malaria parasite.

Abstract not available

Wang, Shu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 119-139). / Abstracts in English and Chinese. / Table of contents --- p.i / Abstract --- p.v / 摘要 --- p.viii / Acknowledgements --- p.x / List of tables --- p.xi / List of figures --- p.xiii / List of abbreviations --- p.xvi / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Overview of nasal drug delivery --- p.2 / Chapter 1.1.1 --- Structure and permeability of the nasal mucosa --- p.3 / Chapter 1.1.2 --- Pathways of drug permeation across nasal mucosa --- p.6 / Chapter 1.1.3 --- Models for nasal drug permeation studies --- p.7 / Chapter 1.1.3.1 --- In vitro models --- p.7 / Chapter 1.1.3.2 --- In situ models --- p.13 / Chapter 1.1.3.3 --- In vivo animal models --- p.16 / Chapter 1.1.4 --- Factors affecting drug absorption across nasal mucosa --- p.19 / Chapter 1.1.4.1 --- Biological factors --- p.20 / Chapter 1.1.4.2 --- Physicochemical properties of drugs --- p.25 / Chapter 1.1.4.3 --- Formulation factors --- p.29 / Chapter 1.1.5 --- Profile of a suitable drug candidate for nasal delivery --- p.33 / Chapter 1.2 --- Physicochemical properties and human pharmacokinetics of the four drug candidates --- p.35 / Chapter 1.2.1 --- Rizatriptan --- p.35 / Chapter 1.2.2 --- Meloxicam --- p.37 / Chapter 1.2.3 --- Lomoxicam --- p.39 / Chapter 1.2.4 --- Nebivolol --- p.40 / Chapter 1.3 --- Scope of the current study --- p.44 / Chapter Chapter Two. --- Preliminary validation of Calu-3 cell line model as an in vitro model for nasal drug permeation screening --- p.45 / Chapter 2.1 --- Introduction --- p.45 / Chapter 2.2 --- Materials --- p.46 / Chapter 2.2.1 --- Chemicals --- p.46 / Chapter 2.2.2 --- Materials for cell culture --- p.46 / Chapter 2.2.3 --- Instruments --- p.47 / Chapter 2.3 --- Methods --- p.47 / Chapter 2.3.1 --- Cell culture --- p.47 / Chapter 2.3.2 --- Cytotoxicity studies by MTS/PES assay --- p.48 / Chapter 2.3.2.1 --- Optimization of MTS/PES assay for the initial cell seeding density and the incubation time --- p.49 / Chapter 2.3.2.2 --- Cytotoxicity studies of non-physiological pH and osmolarity on Calu-3 cells by MTS/PES assay --- p.49 / Chapter 2.3.3 --- Integrity of Calu-3 cell monolayers --- p.50 / Chapter 2.3.3.1 --- Transepithelial electrical resistance (TEER) --- p.50 / Chapter 2.3.3.2 --- Permeabilities of marker compounds --- p.51 / Chapter 2.3.3.3 --- Effect of osmolarity on the Calu-3 cell monolayers --- p.53 / Chapter 2.3.4 --- Inter-passage variation --- p.53 / Chapter 2.3.5 --- Statistical analysis --- p.54 / Chapter 2.4 --- Results and discussions --- p.54 / Chapter 2.4.1 --- Cell culture --- p.54 / Chapter 2.4.2 --- Cytotoxicity studies by MTS/PES assay --- p.55 / Chapter 2.4.2.1 --- Optimization of MTS/PES assay for the initial cell seeding density and the incubation time --- p.55 / Chapter 2.4.2.2 --- Cytotoxicity studies of non-physiological pH and osmolarity on Calu-3 cells by MTS/PES assay --- p.57 / Chapter 2.4.3 --- Integrity of Calu-3 cell monolayers --- p.58 / Chapter 2.4.3.1 --- Transepithelial electrical resistance (TEER) --- p.59 / Chapter 2.4.3.2 --- Permeabilities of marker compounds --- p.60 / Chapter 2.4.3.3 --- Effect of osmolarity on the Calu-3 cell monolayer --- p.63 / Chapter 2.4.4 --- Inter-passage variation --- p.65 / Chapter 2.5 --- Conclusion --- p.66 / Chapter Chapter Three. --- Permeation studies of selected drug candidates using the Calu-3 cell line model --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.2 --- Materials --- p.69 / Chapter 3.2.1 --- Chemicals --- p.69 / Chapter 3.2.2 --- Materials for cell culture --- p.69 / Chapter 3.2.3 --- Instruments --- p.69 / Chapter 3.3 --- Methods --- p.70 / Chapter 3.3.1 --- HPLC assay development and validation for the drug candidates --- p.70 / Chapter 3.3.2 --- Stabilities of the drug candidates in loading solutions at different pHs --- p.71 / Chapter 3.3.3 --- Cell culture --- p.71 / Chapter 3.3.4 --- Cytotoxic effects of the drug candidates on Calu-3 cells by MTS/PES assay --- p.71 / Chapter 3.3.5 --- Permeation studies of drug candidates using Calu-3 cell line model --- p.72 / Chapter 3.3.5.1 --- Effect of concentration on the permeabilities of drug candidates across Calu-3 cell line model --- p.72 / Chapter 3.3.5.2 --- Effect of pH on the permeabilities of drug candidates across Calu-3 cell line model --- p.73 / Chapter 3.3.5.3 --- Effect of osmolarity on the permeabilities of drug candidates across Calu-3 cell line model --- p.73 / Chapter 3.3.6 --- Permeation studies of drug candidates in artificial membrane model at different pHs --- p.73 / Chapter 3.3.7 --- Correlation of the permeabilities of drug candidates between Calu-3 cell line model and artificial membrane model --- p.74 / Chapter 3.3.8 --- Statistical analysis --- p.75 / Chapter 3.4 --- Results and discussions --- p.75 / Chapter 3.4.1 --- HPLC methods for the drug candidates --- p.75 / Chapter 3.4.2 --- Stabilities of the drug candidates in loading solutions at different pHs --- p.75 / Chapter 3.4.3 --- Cytotoxic effects of the drug candidates on Calu-3 cells by MTS/PES assay --- p.76 / Chapter 3.4.4 --- Permeation studies of drug candidates in Calu-3 cell line model --- p.81 / Chapter 3.4.4.1 --- Effect of concentration on the permeabilities of drug candidates across Calu-3 cell line model --- p.81 / Chapter 3.4.4.2 --- Effect of pH on the permeabilities of drug candidates across Calu-3 cell line model --- p.84 / Chapter 3.4.4.3 --- Effect of osmolarity on the permeabilities of drug candidates across Calu-3 cell line model --- p.87 / Chapter 3.4.5 --- Permeation studies of drug candidates in artificial membrane model at different pHs --- p.88 / Chapter 3.4.6 --- Correlation of the permeabilities of drug candidates between Calu-3 cell line model and the artificial membrane model --- p.92 / Chapter 3.5 --- Selection of drug candidate for further in vivo studies --- p.93 / Chapter 3.6 --- Conclusion --- p.93 / Chapter Chapter Four. --- In vivo absorption studies of the most promising drug candidate --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials --- p.96 / Chapter 4.2.1 --- Chemicals --- p.96 / Chapter 4.2.2 --- Instruments --- p.96 / Chapter 4.3 --- Methods --- p.97 / Chapter 4.3.1 --- HPLC conditions --- p.97 / Chapter 4.3.2 --- Preparation of standard solutions --- p.97 / Chapter 4.3.3 --- Calibration curves --- p.98 / Chapter 4.3.4 --- Sample preparations --- p.98 / Chapter 4.3.5 --- Validation of the assay method --- p.98 / Chapter 4.3.5.1 --- Specificity --- p.98 / Chapter 4.3.5.2 --- Precision and accuracy --- p.99 / Chapter 4.3.5.3 --- Recovery --- p.99 / Chapter 4.3.5.4 --- Sensitivity --- p.99 / Chapter 4.3.5.5 --- Stability --- p.99 / Chapter 4.3.6 --- Animals --- p.100 / Chapter 4.3.7 --- Drug administration --- p.102 / Chapter 4.3.8 --- Data analysis --- p.102 / Chapter 4.4 --- Results and discussions --- p.103 / Chapter 4.4.1 --- Validation of the assay method --- p.103 / Chapter 4.4.1.1 --- Specificity --- p.103 / Chapter 4.4.1.2 --- "Precision, accuracy and linearity" --- p.105 / Chapter 4.4.1.3 --- Recovery --- p.106 / Chapter 4.4.1.4 --- Sensitivity --- p.107 / Chapter 4.4.1.5 --- Stability --- p.108 / Chapter 4.4.2 --- "In vivo absorption studies through the nasal, intravenous and oral routes in rat model" --- p.108 / Chapter 4.4.3 --- Preliminary correlation between permeabilities of compounds in Calu-3 cell line model and their nasal bioavailabilities in animal models --- p.111 / Chapter 4.5 --- Conclusion --- p.113 / Chapter Chapter Five. --- Overall conclusion --- p.114 / Chapter Chapter Six. --- Future studies --- p.117 / References --- p.119

Molecular capsules consist of two or more molecules that bind through either covalent or noncovalent interactions to form a structure with an internal void capable of containing guest molecules. These capsules can be used in catalysis/biocatalysis, in drug transport and delivery, in supramolecular arrays, and to stabilize reactive intermediates. Cavitands and calix[4]arenes are two types of macrocycles that have been used to form molecular capsules. Cavitands are used to form capsules called carceplexes, hemicarceplexes, and hemicarcerands through covalent bonds when two molecules are bridged together rim to rim. Calix[4]arene derivatives self-assemble reversibly through noncovalent interactions such as hydrogen bonding and ionic bonding to form capsules. Capsules formed form cavitands and calix[4]arenes have been shown to encapsulate a variety of guest molecules in nonpolar solvents. In order for the capsules to be used for biological applications, the capsules need to encapsulate guest molecules in water. There are only a few examples of capsules that encapsulate guests in polar solvents. Calix[4]arenes derivatives substituted with charged substituents on the upper rim and propyl groups on the lower rim were synthesized. These derivatives dimerize through ionic interactions in polar solvents forming both heterodimers and homodimers. These dimers will be used to encapsulate various guest molecules. Although the ionic propoxycalix[4]arene monomers are water-soluble, the heterodimers are not. This is due to the shielding of the charges upon assembly leaving only the propyl groups on the lower rim exposed to the polar solvent. To increase dimer solubility in water, calix[4]arene derivatives are being synthesized with hydroxy ethyl groups instead of the propyl groups on the lower rim. When the charged hydroxyethoxycalix[4]arene derivatives dimerize, the alcohols will be exposed to the polar solvent instead of the propyl groups increasing the water-solubility of the capsules.

Indiana University-Purdue University Indianapolis (IUPUI) / Drug-drug interactions (DDIs) refer to situations where one drug affects the pharmacokinetics or pharmacodynamics of another. DDIs represent a major cause of morbidity and mortality. A common adverse drug reaction (ADR) that can result from, or be exacerbated by DDIs is drug-induced myopathy. Identifying DDIs and understanding their underlying mechanisms is key to the prevention of undesirable effects of DDIs and to efforts to optimize therapeutic outcomes. This dissertation is dedicated to identification of clinically important myopathic DDIs and to elucidation of their underlying mechanisms. Using data mined from the published cytochrome P450 (CYP) drug interaction literature, 13,197 drug pairs were predicted to potentially interact by pairing a substrate and an inhibitor of a major CYP isoform in humans. Prescribing data for these drug pairs and their associations with myopathy were then examined in a large electronic medical record database. The analyses identified fifteen drug pairs as DDIs significantly associated with an increased risk of myopathy. These significant myopathic DDIs involved clinically important drugs including alprazolam, chloroquine, duloxetine, hydroxychloroquine, loratadine, omeprazole, promethazine, quetiapine, risperidone, ropinirole, trazodone and simvastatin. Data from in vitro experiments indicated that the interaction between quetiapine and chloroquine (risk ratio, RR, 2.17, p-value 5.29E-05) may result from the inhibitory effects of quetiapine on chloroquine metabolism by cytochrome P450s (CYPs). The in vitro data also suggested that the interaction between simvastatin and loratadine (RR 1.6, p-value 4.75E-07) may result from synergistic toxicity of simvastatin and desloratadine, the major metabolite of loratadine, to muscle cells, and from the inhibitory effect of simvastatin acid, the active metabolite of simvastatin, on the hepatic uptake of desloratadine via OATP1B1/1B3. Our data not only identified unknown myopathic DDIs of clinical consequence, but also shed light on their underlying pharmacokinetic and pharmacodynamic mechanisms. More importantly, our approach exemplified a new strategy for identification and investigation of DDIs, one that combined literature mining using bioinformatic algorithms, ADR detection using a pharmacoepidemiologic design, and mechanistic studies employing in vitro experimental models.