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

Dysregulation of PI3K-AKT-mTOR pathway has been reported in various pathologies, such as cancer and insulin resistance. The proline-rich AKT substrate of 40-kDa (PRAS40), also known as AKT substrate 1 (AKT1S1), lies at the crossroads of these cascades and inhibits the activity of the mTOR complex 1 (mTORC1) kinase. Firstly, our findings showed that disruption of PRAS40, a substrate of AKT and component of mTORC1, alters glucose homeostasis and prevent hyperglycemia in the streptozotocin (STZ)-induced diabetes mouse model. PRAS40 ablation resulted in a mild lowering of blood glucose levels and glycated hemoglobin (HbA1C), a lowered insulin requirement, and improved glucose tolerance in untreated PRAS40 gene knockout (PRAS40 (-/-)) as compared to wild-type (PRAS40 (+/+)) mice. PRAS40 deletion significantly attenuates hyperglycemia in STZ-induced PRAS40 (-/-) mice through increased hepatic AKT and mTORC1 signaling, a lowered serum insulin requirement, and altered hepatic GLUT4 levels. Furthermore, we investigated the role of PRAS40 in possible feedback mechanisms, and altered AKT/PRAS40/mTOR signaling in the pathogenesis of tumor progression. For this we probed new datasets extracted from Oncomine, a cancer microarray database containing datasets derived from patient samples, to further understand the role of PRAS40 (AKT1S1). These data strongly supports the previous findings that PRAS40 may serve as a potential therapeutic target for various cancers. Elevated levels of HER2 and PRAS40 are found in some human breast cancers. To directly test the importance of these genetic events in mammary tumorigenesis, we assessed whether disruption of PRAS40 could alter mammary tumor occurrence in HER2 overexpressing mice. HER2 overexpressing mice expressing the activated rat Erbb2 (c-neu) oncogene under the direction of the MMTV promoter was bred with Cre-recombined homozygous (PRAS40-/-) mice. We examined mammary tumor development in the presence (PRAS40+/+) or absence (PRAS40-/-) of PRAS40 using this double transgenic mouse mammary tumor model. Loss of PRAS40 resulted in a delayed mammary tumor onset, improved tumor-free survival, and reduced mammary pre-cancerous lesions in PRAS40-/- versus PRAS40+/+ HER2 overexpressing mice. These results suggest that PRAS40 knockdown could be an attractive target and adjuvant therapy in HER2-positive breast cancers.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African Americans, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 years causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the characterization of AG10 , a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10 , combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life of therapeutic peptides. The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of a model peptide Gonadotropin Releasing Hormone (GnRH) and its analog GnRH-A without compromising their potency. Apart from GnRH, we have used other peptides to study their proteolytic stability in vitro . Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo . We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.

There is significant interest in the field of neuroscience to gain a better understanding of how neurons die in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have used the neuronal cell line NG115-401L with unique calcium signaling characteristics to test the hypothesis that improving calcium loading into the endoplasmic reticulum (ER) to increase ER calcium levels acts as a possible neuroprotective response. We approached this problem using both pharmacological and genetic approaches targeting the central mediator of calcium uptake in the ER localized sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) enzyme. The pharmacological studies involved use of the ginger root compound 6-gingerol, which to date is the best documented agent for activating SERCA enzymes in heart and skeletal muscle. However, in our experiments, gingerol did not appear to activate NG115-401L SERCA pumps; indeed, the compound produced a response more like that of a SERCA inhibitor inducing a rapid ER calcium depletion. In addition, gingerol stimulated robust calcium influx responses, an unexpected result given the NG115-401L neural cell line is uniquely deficient in calcium influx pathways. Our genetic approach involved expressing the stromal interaction molecule 1 (STIM1) protein in the NG115-401L cell, which is also an ER localized protein that serves as a pivotal calcium influx channel regulator. NG115-40lL neurons present a native deficiency of STIM1 expression in a background phenotype with well characterized perturbations in ER calcium regulation and control of calcium influx pathways. Thus, STIM1 may be predicted to increase ER calcium levels, conferring protection against neuron cell death due to ER calcium store defects. STIM1 expression reconstituted the corrupted calcium influx pathway in NG115-401L neurons, which conferred neuroprotective responses to ER calcium perturbation, mitochondrial oxidative stress and subsequent cell death. Our results argue for unique and undiscovered regulatory effects of gingerol on the ER calcium circulation system, and suggest that the expression of STIM1 in these neurons protects against ER stress and oxidative stress via reconstruction of cellular calcium homeostasis.

Five projects are presented here that use Photoelectron Photoion Coincidence (PEPICO) Spectroscopy to determine high-accuracy thermochemical data on small and intermediate size molecules and radicals that are relevant in modeling combustion and atmospheric processes. Some of the experiments were carried out on the laboratory-based Threshold PEPICO (TPEPICO) apparatus which has the advantage of having a low-temperature inlet system and unlimited measurement time, while most of the projects involved the use of the Imaging PEPICO (iPEPICO) apparatus at the Swiss Light Source, which is capable to determine ionic dissociation energies to sub-kJ/mol accuracy. The iPEPICO on the synchrotron beamline was also useful where measurements required energies in excess of 14 eV. The modeling framework of PEPICO is based on the RRKM statistical theory of dissociation kinetics and statistical energy distributions and models complex dissociation pathways to extract both kinetics and thermochemical data from the experiment. In the first project, we measured the onsets of Br- and I-loss reactions for C 2 H 5 Br and C 2 H 5 I using TPEPICO, respectively. The heats of formations of the two molecules are related through the ethyl cation, which was used in their determination. The second project involved measuring Cl-loss from four S i O j Cl k compounds viz. SCl 2 , S 2 Cl 2 , SOCl 2 , and SO 2 Cl 2 to obtain reliable thermochemistry. The second Cl-loss from S 2 Cl 2 + and SOCl 2 + helped us conclude that assuming three-dimensional translational degrees of freedom yields a more reliable statistical model of product-energy distributions. The third project investigated methanol and its isotopologues to explore the dissociation pathways through the H/D-losses. The 0 K appearance energies were used to determine the accurate heat of formation of CH 2 OH and the proton affinity of formaldehyde. The fourth project explores the dissociation pathways of cis -1-bromopropene, trans -1-bromopropene, 2-bromopropene, 3-bromopropene and bromocyclopropane to find that except for 2-bromopropene, all other isomers dissociate into the allyl cation. To derive accurate thermochemical information on the neutral precursors, a mixed theoretical and experimental thermochemical network was used to determine their 0 K heats of formation. The last project involves measurements on dimethyl disulfide (DMDS) and dimethyl diselenide, which are the simplest models that can be used to study disulfide and diselenide linkages. There are several discrepancies in the thermochemistry of DMDS, whereas ours is the first experimental attempt to study the ionic thermochemistry of dimethyl diselenide experimentally.

The low redox potential of guanines (G 1.29 V vs. NHE) compared to other nucleobases, makes them potentially susceptible to attack by exogenous and endogenous damaging species. This property of guanine has also been utilized for the development of several anticancer agents including the well-known platinum complexes, cisplatin and carboplatin. The two closely related nickel complexes, NiCR and NiCR-2H, exhibit significant differences in cytotoxicity towards MCF-7 cancer cells. In the first part of this work, we explain this difference using biochemical and biophysical approaches to study their interactions with duplex DNA. The nickel complexes were found to selectively oxidize guanines in bulged DNA structures in the presence of oxidant and notably NiCR-2H oxidizes guanines more efficiently than NiCR. According to 1 H NMR studies, NiCR-2H binds strongly to the N7 position of dGMP compared to NiCR and could be an important oxidation product of NiCR under physiological conditions. The second part of this work focuses on the secondary DNA structures known as G-quadruplex formed in the guanine rich telomeric region. G-quadruplex is formed by stacking of G-quartets (a coplanar cyclic array of four Gs) on top of each other. Its formation is known to inhibit the activity of the reverse transcriptase telomerase that is overexpressed in 80-90% cancer cells. The guanines in telomeric DNA are readily oxidized due to their low redox potential and the major oxidation product is 7, 8-dihydro-8-oxoguanine (OxodG). OxodG (0.58 V vs. NHE) can further be oxidized in the presence of one electron oxidants and the resulting product forms adducts with endogenous nucleophiles such as spermine. In light of these findings, we hereby designed and synthesized novel bifunctional perylene derivatives that can selectively bind to the telomeric DNA via G-quadruplex formation and subsequently react with OxodG in close proximity. These compounds have strong binding affinity towards G-quadruplex and can significantly stabilize the OxodG containing G-quadruplex motif by end stacking on the upper G-quartet. The effect of these compounds on telomerase activity and cytotoxicity towards Hep3B cancer cells was also evaluated.

Administration of drugs sublingually allows direct absorption into the systemic circulation which results in quick onset of action and a higher bioavailability as a consequence of by-passing first pass metabolism. Absorption of drugs across sublingual mucosa is typically determined by means of in vitro permeation studies using excised sublingual tissue during early phases of drug development. Although in vitro set up has been designed to mimic in vivo system yet the results of in vitro studies often deviate from in vivo results. Therefore, it is not known if the in vitro studies can be used as surrogate for in vivo studies in a predictable manner. To understand the relationship between in vitro and in vivo system for sublingual drug delivery, the first objective of this dissertation research was to investigate difference/similarities between in vitro and in vivo system by performing parallel in vitro and in vivo studies and establish a correlation. Five model drugs possessing diverse physicochemical properties and New Zealand White rabbits were used for these studies. Comparison of time course of absorption revealed a significant difference in time lag between in vivo (less than 5 min) and in vitro (30-120 min) systems. However, the derived absorption parameter permeability coefficient was similar in in vitro and in vivo system for caffeine: (2.10±0.22)×10 –5 , (2.06±0.47)×10 –5 ; Naproxen: (1.91±0.44)×10 –5 , (2.34±0.26)×10 –5 ; Propranolol: (2.93±0.52)×10 –5 , (3.51±0.75)×10 –5 ; Verapamil: (3.95±0.29)×10 –5 , (4.75±0.81)×10 –5 and Atenolol: (2.01±0.68)×10 –6 , (2.95±0.32)×10 –6 cm/s, respectively (p>0.05). The discrepancy between in vitro and in vivo system was hypothesized in this study to be due to the difference in thickness and role of extensive microcirculation in the two systems. Histological evaluation revealed the presence of rich vasculature 10-20 μm below the epithelium which is responsible for quick removal of drug permeating the epithelium (100-150 μm) of sublingual mucosa and reaching systemic circulation in an in vivo system. In contrast, in in vitro system the permeated drug can only be detected after crossing the excised sublingual tissue of 250±50 μm thickness. A mathematical model based on the monolayer (epithelium) and bilayer (epithelium+connective tissue) nature of the membrane representing in vivo and in vitro system, respectively demonstrated the nature of membrane to be responsible for difference in time lag but similar permeability coefficient. To be able to predict in vivo result using in vitro data, the second objective of this dissertation research was to develop a predictive pharmacokinetic model based on the established in vitro in vivo correlation (IVIVC) of sublingual absorption parameters across two systems. Predicted plasma concentration-time profiles of propranolol, verapamil, naproxen, atenolol and caffeine were found to be in good agreement with the experimental profile with the coefficient of determination of 0.85, 0.80, 0.97, 0.98 and 0.88, respectively. The applicability of the model was further evaluated by predicting in vivo performance of Zolpidem and Propranolol following sublingual administration in human beings and comparing area under the plasma concentration-time curve. Percent prediction error was 12.02% and less than 10% (4.69, 6.69, 5.02 for 1, 1.75 and 3 mg dose, respectively) for Propranolol and Zolpidem, respectively. The final objective of this dissertation was to extend the established IVIVC to other suitable animal models such as pig for assessing sublingual absorption. Histological evaluation revealed the similarity in the structure of sublingual mucosa of pig and New Zealand White rabbit. Similar transport characteristics (p>0.05) of model drugs across sublingual mucosae of two species were observed indicating the possibility of using them interchangeably. In conclusion, a rational attempt was made in this dissertation research to identify the root cause of the discrepancy between in vitro and in vivo system and establish a correlation correcting the discrepancies. The established IVIVC and predictive pharmacokinetic model will help in rationale design and development of new sublingual formulations and will be a valuable tool in the preclinical phase of early drug development stage.

As a promising pH-triggerable molecular switch, trans -2-aminocyclohexanol (TACH) has a variety of applications. By introducing two hydrocarbon tails, multiple TACH-based lipids (flipids) have been designed and studied that are able to perform a drastic conformational flip upon protonation, loosening the stacking of hydrocarbon tails in lipid bilayers. Liposomes constructed from such flipids (fliposomes) can be disrupted by this acid-triggered conformational flip to cause a rapid release of a cargo specifically in areas of increased acidity (such as inflammation or ischemic tissues, solid tumor, and endosome pathway). A library of flipids has been built based on structural modifications of both amino headgroups and hydrophobic tails. A series of fliposomes have been constructed and their colloidal stability, capacity and pH-dependent leakage were investigated. A good correlation between the conformational switch of flipids studied by 1 H-NMR and the fliposomes' leakage indicated that the former is a cause for the latter. The obtained results showed that all the properties of fliposomes can be manipulated by selection of the amino headgroups structure and basicity, and the length and shape of hydrophobic tails, by using mixtures of different flipids or fliposomes, and by changing the content of flipids while constructing fliposomes. As a result, we prepared the pH-triggerable fliposomes with extraordinary characteristics: high stability in storage combined with instant release of their cargo in response to a weakly acidic medium. Fliposomes encapsulating the anticancer drug methotrexate (MTX) were applied to HeLa cells and demonstrated much higher cytotoxicity than the free drug and negative controls, indicating that they could conduct more efficient cellular delivery of MTX. The MTX-loaded fliposomes inhibited tumor growth in B16F1-melanoma-bearing nude mice compared to the control group, suggesting the anticancer activity of MTX delivered by pH-triggerable fliposomes in vivo. The results of research demonstrated the potential of fliposomes to serve as a viable drug delivery system.

NF-κB is a transcription factor protein complex that plays an important role in some cancers and inflammatory responses. It can enhance the proliferation rate, reduce apoptosis, as well as create more blood flow to ensure the survival of cancer. Thus blocking the NF-κB pathway has potential therapeutic benefit. We designed a series of compounds based on quinazoline scaffold pharmacophore model which may have high binding affinity with p50 subunit of NF-κB. The compound series with phenyl substitution at position 2 of quinazoline proved to be more effective at inhibiting NF-κB function both theoretically and experimentally. These compounds also reduce the proliferation of numerous tumor cell lines and the mean GI50 for representative compound 2a is 2.88μM on NCI 60 cell lines. Compound 2a can induce significant apoptosis at the concentration of 1μM. The exploration of the mechanism of action of these compounds found that 2a does not inhibit kinases upstream of NF-κB and does not inhibit p65 translocation from the cytosol to the nucleus as 2b does. However 2a inhibits NF-κB dependent Luciferase expression as well as NF-κB target genes better than 2b. This may suggest that 2a inhibits the NF-κB pathway by directly blocking gene transcription, while 2b acts at cytoplasmic stage.