Evaluation of dose optimization using the Pencil Beam (PB) and Monte Carlo (MC) algorithms may allow physicists to apply dosimetric offsets to account for inaccuracies of the PB algorithm for lung cancer treatment with Stereotactic Body Radiotherapy (SBRT). 20 cases of Non-Small Cell Lung Cancer (NSCLC) were selected. Treatment plans were created with Brainlab iPlanDose® 4.1.2. The D97 of the Planning Target Volume (PTV) was normalized to 50 Gy on the Average Intensity Projection (AIP) using the fast PB and compared with MC. This exact plan with the same beam Monitor Units (MUs) was recalculated over each respiratory phase. The results show that the PB algorithm has a 2.3-2.4% less overestimation at the maximum exhalation phase than the maximum inhalation phase when compared to MC. Significantly smaller dose difference between PB and MC is also shown in plans for peripheral lesions (7.7 ± 0.7%) versus central lesions (12.7±0.8%)(p< 0.01). / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Mining the human genome for therapeutic target(s) discovery promises novel outcome. Over half of the proteins in the human genome however, remain uncharacterized. These proteins offer a potential for new target(s) discovery for diverse diseases. Additional targets for cancer diagnosis and therapy are urgently needed to help move away from the cytotoxic era to a targeted therapy approach. Bioinformatics and proteomics approaches can be used to characterize novel sequences in the genome database to infer putative function. The hypothesis that the amino acid motifs and proteins domains of the uncharacterized proteins can be used as a starting point to predict putative function of these proteins provided the framework for the research discussed in this dissertation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Choy Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / Table of contents --- p.xi / List of figures --- p.xv / List of tables --- p.xviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe acute respiratory syndrome (SARS) - An overview --- p.1 / Chapter 1.1.1 --- Epidemiology of SARS --- p.1 / Chapter 1.1.2 --- Clinical presentation of SARS --- p.2 / Chapter 1.1.3 --- Diagnostic tests of SARS --- p.5 / Chapter 1.1.4 --- Treatment of SARS --- p.7 / Chapter 1.2 --- Severe acute respiratory syndrome coronavirus (SARS- CoV) --- p.8 / Chapter 1.2.1 --- The etiological agent of SARS --- p.8 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- Genome of SARS-CoV --- p.11 / Chapter 1.3 --- Spike (S) glycoprotein of SARS-CoV --- p.14 / Chapter 1.3.1 --- Functions of SARS-CoV S glycoprotein --- p.15 / Chapter 1.3.2 --- Receptors for S glycoprotein of SARS-CoV --- p.17 / Chapter 1.4 --- 3C-like protease (3CLPro) of SARS-CoV --- p.20 / Chapter 1.4.1 --- Extensive proteolytic processing of SARS-CoV replicase polyproteins --- p.20 / Chapter 1.4.2 --- SARS-CoV 3CLPro --- p.21 / Chapter 1.4.3 --- Substrate specificity of SARS-CoV 3CLPro --- p.22 / Chapter 1.5 --- Combating SARS - Vaccine and drug development --- p.24 / Chapter 1.5.1 --- Vaccine development against SARS --- p.24 / Chapter 1.5.2 --- Drug development against SARS --- p.25 / Chapter 1.6 --- Project objectives of this thesis --- p.27 / Chapter 1.6.1 --- Synthetic Peptide Studies on SARS-CoV S glycoprotein --- p.27 / Chapter 1.6.2 --- Synthetic Peptide Studies on SARS-CoV 3CLPro --- p.28 / Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.30 / Chapter 2.1.1 --- Bioinformatics analyses of SARS-CoV S gly- coprotein --- p.30 / Chapter 2.1.2 --- Peptide design and molecular modeling --- p.32 / Chapter 2.1.3 --- Solid phase peptide synthesis (SPPS) --- p.33 / Chapter 2.1.4 --- Peptide conjugation --- p.35 / Chapter 2.1.5 --- Immunization in rabbits and monkeys --- p.36 / Chapter 2.1.6 --- ELISA analysis --- p.37 / Chapter 2.1.7 --- Immunofluorescent confocal microscopy --- p.39 / Chapter 2.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.40 / Chapter 2.2.1 --- Protein expression and purification --- p.40 / Chapter 2.2.2 --- Solid phase peptide synthesis (SPPS) --- p.41 / Chapter 2.2.3 --- Peptide cleavage assay --- p.44 / Chapter 2.2.4 --- Molecular docking --- p.46 / Chapter 3 --- Results --- p.48 / Chapter 3.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.48 / Chapter 3.1.1 --- General features and structural analyses of the S glycoprotein --- p.48 / Chapter 3.1.2 --- Peptides design and synthesis --- p.53 / Chapter 3.1.3 --- ELISA analysis and immunofluorescent con- focal microscopy --- p.55 / Chapter 3.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.62 / Chapter 3.2.1 --- Substrate specificity of SARS-CoV 3CLPro . . --- p.62 / Chapter 3.2.2 --- Molecular docking of SARS-CoV 3CLPro and peptide substrates --- p.74 / Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.78 / Chapter 4.1.1 --- Synthetic peptides elicited SARS-CoV specific antibodies --- p.78 / Chapter 4.1.2 --- Factors affecting the specificity and antigenic- ity of synthetic peptides --- p.80 / Chapter 4.1.3 --- Next step towards vaccine development --- p.83 / Chapter 4.1.4 --- A synthetic peptide-based approach --- p.84 / Chapter 4.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.86 / Chapter 4.2.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.87 / Chapter 4.2.2 --- Sequence comparison between SARS-CoV 3CLpro cleavage sites --- p.90 / Chapter 4.2.3 --- A rapid and high throughput approach to screen protease substrate specificity --- p.94 / Bibliography --- p.98

<p>This thesis describes the use of a new technology, the Cytosensor^® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor^® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor^® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>

<p>During the last decades, chemotherapy with cytotoxic drugs has played a significant role in cancer therapy. It’s important to develop new anticancer drugs, and drug sensitivity testing in vitro can be used to find the right diagnosis for the newly developed substances.</p><p>The aim of this study was to investigate the cytotoxic activity of the new signal transduction modulators bortezomib, gefitinib and PKC412. The well-established substances cisplatin, cytarabine, doxorubicin and vincristin were investigated for comparison.</p><p>The activity of the cytotoxic drugs was analysed in human tumor samples from patients with ovarian carcinoma (n=16) and lymphoma (n=15) by using the Fluorometric Microculture Cytotoxicity Assay (FMCA). The testing of cellular drug resistance by FMCA was accomplished successfully in 33 out of the 34 samples (97%).</p><p>The results of this study indicated that the activity of cytotoxic drugs in tumor cells obtained from patients with ovarian carcinoma and lymphoma may be detected by the FMCA. It also suggested that bortezomib and gefitinib could represent promising agents for treatment of ovarian carcinoma and that PKC412 might be of less use for patients with this diagnose.</p>

<p>This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [¹¹C]verapamil and [¹¹C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [¹¹C]verapamil, [¹¹C]GR205171 and [¹⁸F]altanserin were compared in various laboratory animal species and in humans.</p><p>A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain.</p><p>The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [¹¹C]verapamil occurred rapidly in the sense that [¹¹C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [¹¹C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [¹¹C]verapamil was P-gp-mediated. The low brain concentrations of [¹¹C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. </p><p>The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates. </p>

3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate (IK9) was recently reported to be a potent inhibitor of cancer cell invasion and angiogenesis. It markedly reduced in vitro invasion of human HT1080 fibrosarcoma cells through collagen-coated porous membranes (Boyden chamber assay) and in vivo tumour growth in athymic nude mice. It was furthermore able to decrease angiogenesis ex vivo in a rat aortic ring assay and in vivo in a choroidal neovascularisation mice model. It nevertheless presents some water solubility and stability problems, which should be taken into account for further investigations. In the first part of the project, we synthesized original IK9 derivatives, modulated at the 3- and 6-positions, by introducing functional groups able to improve water solubility and metabolic stability. Their anti-invasive potency was screened in the Boyden chamber assay and the generated results highlighted some structure-activity relationships. A second part of the project was devoted to the elucidation of the actually unknown mechanism of action of IK9. Anti-invasive or anti-proliferative effects against endothelial cells, main actors of the angiogenic process, were not emphasised. We showed that IK9 acts likely not as an inhibitor of receptor tyrosine kinases (EGFR, PDGFR and VEGFR). The compound generates a weak decrease of mRNA coding for metalloproteinases (MMPs) 2 and 9, and on the other hand a substantial diminution of MMP 2 and 9 secretions by HT1080 fibrosarcoma cells. In conclusion, the consideration of anti-invasive properties together with the worked out solubility and stability profiles highlights several series, notably 6-hydroxycoumarins, 6-hydroxymethylcoumarins and coumarin-3-sulfonamides, whose interest as potential successors to IK9 is undeniable. Le 6-acétoxyméthyl-2-oxo-2H-1-benzopyrane-3-carboxylate de 3-bromophényle (IK9) est un dérivé coumarinique décrit comme inhibiteur puissant de linvasion tumorale et de langiogenèse. Il inhibe linvasion des cellules HT1080 de fibrosarcome humain in vitro à travers une membrane poreuse recouverte dune couche de collagène (test en « chambres de Boyden ») et la croissance tumorale in vivo chez des souris athymiques nues. Il est par ailleurs capable de bloquer langiogenèse, à la fois dans un modèle ex vivo danneaux daorte de rats et dans un modèle in vivo de néovascularisation choroïdale chez la souris. Il présente pour autant une problématique dhydrosolubilité et de stabilité, dont il faudra tenir compte lors dinvestigations futures. Dans une première partie du projet, nous avons synthétisé des dérivés originaux de lIK9, modulés en position 3 et 6 du noyau coumarinique, en introduisant des fonctions susceptibles daugmenter lhydrosolubilité et la stabilité métabolique des molécules obtenues. Leur pouvoir anti-invasif a été évalué dans le test en « chambres de Boyden », ce qui nous a permis de mettre en évidence différentes relations structure-activité. Une deuxième partie du projet fut consacrée à létude du mécanisme daction de lIK9, qui est non identifié jusquà présent. Un effet anti-invasif ou anti-prolifératif envers les cellules endothéliales, actrices principales du processus dangiogenèse, na pu être observé. LIK9 nagit vraisemblablement pas en tant quinhibiteur de plusieurs récepteurs de type tyrosine kinase (EGFR, PDGFR et VEGFR). Le composé engendre une légère baisse de lexpression de lARNm codant pour les métalloprotéases matricielles (MMPs) 2 et 9 mais par contre entraîne une diminution substantielle de la sécrétion des MMPs 2 et 9 par les cellules HT1080 de fibrosarcome humain. En conclusion, la prise en compte simultanée de lactivité anti-invasive, de lhydrosolubilité et de la stabilité met en avant plusieurs séries de dérivés, notamment des 6-hydroxycoumarines, des 6-hydroxyméthylcoumarines et des coumarine-3-sulfonamides, dont lintérêt en tant que successeurs potentiels de lIK9 est indéniable.

This thesis examines the transport of exogenous molecules across the blood-brain barrier (BBB), focusing on active efflux, using positron emission tomography (PET), computer simulation and modelling. P-glycoprotein (P-gp) inhibition was studied using [11C]verapamil and [11C]hydroxyurea was investigated as a new marker for active efflux transport. Simulations were carried out to explore the importance of the efflux transporter location in the BBB. Brain concentrations of [11C]verapamil, [11C]GR205171 and [18F]altanserin were compared in various laboratory animal species and in humans. A central aspect of the studies has been the novel combination of dynamic PET imaging of the brain pharmacokinetics of a labelled drug, administered through an exponential infusion scheme allowing time-resolved consequence analysis of P-gp inhibition, and mathematical modelling of the obtained data. The methods are applicable to drugs under development and can be used not only in rodents but also in higher species, potentially even in humans, to investigate the effects of P-gp or other transporters on drug uptake in the brain. The inhibition of P-gp by cyclosporin A (CsA) and the subsequent change in brain concentrations of [11C]verapamil occurred rapidly in the sense that [11C]verapamil uptake increased rapidly after CsA administration but also in the sense that the increased uptake was rapidly reversible. The P-gp inhibition was best described by an inhibitory indirect effect model in which CsA decreased the transport of [11C]verapamil out of the brain. The model indicated that approximately 90% of the transport of [11C]verapamil was P-gp-mediated. The low brain concentrations of [11C]hydroxyurea appeared to be a result of slow transport across the BBB rather than active efflux. This exemplifies why the extent and the rate of brain uptake should be approached as two separate phenomena. The brain-to-plasma concentration ratios for the three studied radiotracers differed about 10-fold be-tween species, with lower concentrations in rodents than in humans, monkeys and pigs. The increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. The findings demonstrate a need to include the dynamics of efflux inhibition in the experimental design and stress the importance of the choice of species in preclinical studies of new drug candidates.

This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.

A central aim in drug development is to ensure that the new drug is efficacious and safe in the intended patient population. Mathematical models describing the pharmacokinetic-pharmacodynamic (PK-PD) properties of a drug are valuable to increase the knowledge about drug effects and disease and can be used to inform decisions. The aim of this thesis was to develop mechanism-based PK-PD-disease models for important safety and efficacy biomarkers used in anti-diabetic drug development. Population PK, PK-PD and disease models were developed, based on data from clinical studies in subjects with varying degrees of renal function, non-diabetic subjects with insulin resistance and patients with type 2 diabetes mellitus (T2DM), receiving a peroxisome proliferator-activated receptor (PPAR) α/γ agonist, tesaglitazar. The PK model showed that a decreased renal elimination of the metabolite in renally impaired subjects leads to increased levels of metabolite undergoing interconversion and subsequent accumulation of tesaglitazar. Tesaglitazar negatively affects the glomerular filtration rate (GFR), and since renal function affects tesaglitazar exposure, a PK-PD model was developed to simultaneously describe this interrelationship. The model and data showed that all patients had decreases in GFR, which were reversible when discontinuing treatment. The PK-PD model described the interplay between fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c) and haemoglobin in T2DM patients. It provided a mechanistically plausible description of the release and aging of red blood cells (RBC), and the glucose dependent glycosylation of RBC to HbA1c. The PK-PD model for FPG and fasting insulin, incorporating components for β-cell mass, insulin sensitivity and impact of disease and drug treatment, realistically described the complex glucose homeostasis in the heterogeneous patient population. The mechanism-based PK, PK-PD and disease models increase the understanding about T2DM and important biomarkers, and can be used to improve decision making in the development of future anti-diabetic drugs.