• A Computational Study Of Novel Kynurenine 3 Monooxygenase Inhibitors

      Hughes, Tamera Dionne
      Alzheimer’s Disease (AD) is a chronic, neurodegenerative condition that gradually affects an individual’s memory leading to dementia and ultimately death. � Acetylcholinesterase inhibitors have been the mainstay of treatment for AD, but they are only able to control cognitive deficits. Such strategies only temporarily delay the symptoms and do not stop or reverse the progression of the disease. � While AD was initially thought to be the result of plaque accumulation in the brain, this is being reexamined. The kynurenine pathway (KP) has been discovered to play a major role in many neurodegenerative diseases, including AD. � The KP represents a major route for the catabolism of tryptophan (TRP) and accounts for most of the metabolism of TRP that is not committed to protein synthesis. Of particular interest in the KP is the KMO enzyme, which produces toxic metabolites. � This accumulation leads to AD. It is believed that inhibiting KMO will lead to a decrease in the production of these toxic products downstream attenuating or improving the effects of AD. The objective of this study is to identify novel KMO inhibitors using computer aided drug design approaches. � To accomplish this objective the following specific aims will be pursued: Aim 1: To determine the most thermodynamically stable conformations of each potentially novel potent inhibitors of KMO The molecular geometry for stable conformations must be calculated through energy-based methods in order to predict the corresponding molecular properties. � Knowing the molecular geometry is critical for understanding structure-function relationships, as well as the design of novel drugs. Molecular mechanics will be used to explore the conformational space of potential KMO inhibitors. � Each minimum energy structure will be characterized using quantum mechanics with frequency calculations. Based on ab initio and DFT full geometry optimization all stable conformations within an energy cut-off will be identified. � In addition, conformational flexibility will be explored by plotting specific dihedral angles vs. the corresponding energy to generate a conformational energy profile. � We hypothesize that generating a set of diverse conformations will yield the bioactive conformation that will further assist in discovery of a novel KMO inhibitor. Aim 2: To obtain a solvent equilibrated homology model of the substrate–free human KMO Understanding the conformational flexibility and tertiary structure of KMO is critical for structure-based drug design and interpreting structure-function relationship, which will be used in the design of novel therapeutics for disease. � Various KMO inhibitor-bound KMO models will be generated in order to obtain accurate atomic descriptions of the inhibitor-specific catalytic site of KMO. For KMO, different homology models of human KMO will be constructed using the x-ray crystal structures of the substrate free and bound form of yKMOs. � Aim 3: To better understand the specific molecular interactions between the KMO enzyme and bound inhibitors via docking and molecular dynamics simulations Docking and molecular dynamics simulations (MD) will be carried out to establish the specific molecular interactions that exist between the KMO enzyme and its bound inhibitors. � Molecular modeling methodologies available through YASARA will be utilized. � The chemical properties can be mapped to help identify the chemical space that contributes to inhibition. These docking algorithms will generate a score that attempt to distinguish between those potential inhibitors that bind strongly in the KMO binding pocket from those that bind weakly. � Also, MD simulations will be carried out on compounds that are identified through docking using YASARA.
    • Application Of Population Pharmacokinetic Modeling And Simulation In Anti-infective Therapy

      ABSTRACT VINEET GOTI APPLICATION OF POPULATION PHARMACOKINETIC MODELING AND SIMULATION IN ANTI-INFECTIVE THERAPY Under the direction of AYYAPPA CHATURVEDULA, PH.D. AND REBECCA BURNS, PHARM.D, PH.D. The application quantitative approaches to drug research have gained acceptance over the last few decades. One such quantitative approach is population pharmacokinetic (PopPK) modeling and simulation. PopPK modeling and simulation in this dissertation has been applied to therapeutic problems and evaluate the performance of below the limit of quantitation (BQL) data handling approaches in nonlinear mixed effect models. A PopPK model is usually developed by iteratively fitting several models to clinical data and by so doing reduces the pharmacokinetic behavior of a drug to a few PopPK parameters. The PopPK approach has the ability to quantify the variability in the pharmacokinetics of the drug. This enables the application of Monte-Carlo simulation procedures to test several what if situations. In this dissertation, the PopPK modeling and simulation approach is applied to answer therapeutic and methodological problems by using the gold standard software NONMEM®. Therapeutic problems involved: 1) the evaluation of the risk of HIV transmission to an uninfected member on Truvada (fixed dose combination of emtricitabine/tenofovir) in serodiscordant relationship 2) optimization of vancomycin dosing nomogram in Emory University hospitals. PopPK models were developed for tenofovir and vancomycin using clinical data obtained from the subject population of interest. Simulations conducted using the tenofovir PopPK model revealed that protective concentrations of tenofovir were achieved at majority of the times of perceived risk of HIV exposure. The nomogram of vancomycin was optimized by iterative simulations using the developed PopPK model. The optimized nomogram incorporated a loading dose and reduced the maintenance dose. This resulted in more subjects consistently falling in the therapeutic concentration range. The methodological problem involved the evaluation of two newly suggested below the quantification limit (BQL) data handling methods: fractional conditional single imputation (FCSI) and conditional multiple imputation (CMI). These two methods were contrasted with the well accepted M3 method in terms of efficiency in estimating PopPK parameters. It was found that both CMI and FCSI were inferior to the M3 approach. The M3 method, for the most part, gave PopPK parameters with acceptable bias and precision.
    • Applications Of Mechanistic Modeling And Simulations In Compound And Dosage Forms Selections / By Eric Akwasi Mintah

      Mintah, Eric Akwasi
      Physiologically based pharmacokinetic (PBPK) modeling and simulation techniques have been adopted in the pharmaceutical industry to aid in compound selection and dosage form development in recent years. This is a result of easier access to computers and advanced knowledge of species physiology. The mechanistic modeling approach utilizes the compound’s physiochemical properties, formulation related factors, route of administration and species physiology in order to predict the concentration-time profile in plasma and tissues. In this dissertation, different predictive and mechanistic models (ADMET®, ACAT®, OCCAT® and metabolite tracking approaches in Gastroplus®) were applied to simulate the concentration time profiles of various compounds. We applied mechanistic modeling techniques to predict the concentration-time profiles of curcumin and its analogs in order to identify potential drug candidates for future preclinical and clinical studies. An in silico based absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction tool in Gastroplus® (version 8.5, Simulations Plus, Inc., Lancaster, CA, USA) was utilized. For this purpose, we performed model qualifications by comparing the simulated pharmacokinetics data of pure curcumin and compared to the observed data from literature. Curcumin analogues and other compounds that showed higher potential for oral absorption were selected for further study. In our second project, we evaluated the predictability of the new oral cavity compartmental absorption and transit (OCCAT®) model by utilizing commercial buccal and sublingual (fentanyl, buprenorphine, nicotine, miconazole, rizaptriptan and testosterone) formulations. The new OCCATTM model was able to simulate the PK parameters/profiles of published multiple doses of buccal and sublingual drugs administered to healthy and patient population. Varying degrees of bias was observed for all the simulated PK parameter values as compared to the published parameter values for the compounds tested based on the computed % predictability error. Although, the new OCCAT model can be used to support the formulation development and regulatory decisions, its applicability and the predictability for specific drug needs to be adequately qualified. In another project, we conducted mechanistic analysis to track the metabolites of tenofovir disoproxil fumarate (TDF) using PBPK modeling. The main goal of the project was to track the pharmacokinetics of a prodrug, tenofovir disoproxil fumarate (TDF) and its parent drug, tenofovir. Finally, the mechanistic modeling approach was utilized to simulate the disposition, including potential metabolic pathways of 4-benzylpiperidine based on its predicted physiochemical properties.
    • Association Of Genetic Variation And Lipid-related Concentrations In African American Men With And Without Type 2 Diabetes

      Samedy, Lesly-Anne Carline
      Abnormal levels of lipids and cholesterol in the blood lead to atherosclerosis, which can then cause cardiovascular disease (CVD). Specifically, decreased high-density lipoprotein (HDL) and increased low-density lipoproteins (LDL) increase the risk of CVD such as myocardial infarction and ischemic stroke. These lipid abnormalities occur more frequently in individuals with Type 2 diabetes mellitus (T2DM). In addition, uncontrolled diabetes causes damage to the blood vessels, in turn making them more prone to damage from atherosclerosis, putting these individuals at especially high risk for the development of CVD. African Americans with T2DM are at an even greater risk of CVD as a result of a combination of risk factors, among which dyslipidemia plays a prominent role. However, their lipid profile is often different from Caucasians with T2DM. Genetic variations in ADIPOQ, APOE, CETP, NOX3, PCSK9, and PON1 are thought to lead to altered levels of circulating LDL and HDL and may potentially explain the variability seen in lipid levels. Given the increased risk of CVD in African Americans with T2DM, it is important to gain an understanding of the underlying physiology and genetics. The goal of this study is to understand the genetic association with lipid levels in AA males with and without T2DM. Our long-term goal is to identify polymorphisms that can serve as biomarkers in the prediction, development and management of CV events in patients with both T2DM and dyslipidemia. To address each aim and analyze any potential relationships and/or interactions, we employed a candidate gene case-control approach where we compared genotype frequencies of lipid related candidate genes in unrelated African American males, 30 years of age of older, not currently treated with lipid lowering agents, with T2DM and healthy nondiabetic controls. Our data suggest that alterations in HDL and LDL particle levels are potentially due to genetic variation as well as the presence of T2DM. The CETP Taq1B B2-allele was associated with higher levels of HDL 2b and HDL L, but a lower level of HDL 3a and HDL 3b. In addition, the CETP Taq1B B2-allele was associated with decreases in small subclasses of LDL: LDL IVb, LDL IVa & LDL IIIb, where small LDL subclasses are considered more atherogenic compared to larger LDL. PON1 L55M variant allele was associated with decreases in HDL S as well as significant interactions between genotype and diabetic status on HDL 3a. The APOE E4 allele was associated with higher levels of LDL subclass: LDL 3a, LDL 2b, LDL 2a, LDL M and LDL L. With respect to PCSK9, the presence of diabetes abrogates the effects of the A443, potentiating a more atherosclerotic profile. In the absence of T2DM, levels of HDL 3a and 3b were reduced and HDL 2b elevated with the variant allele compared to the wildtype. However, we observed that, alternatively, diabetic variant allele carriers had elevated levels HDL 3a, and 3b and reduced levels of HDL 2b. The ADIPOQ variant allele was associated with lower levels of IDL 2, where lower levels of IDL potentially indicate a decrease in CVD risk. African Americans suffer disproportionately from CVD but only an estimated 50% of CVD incidence can be explained by traditional risk factors. Inclusion of genetics as well as lipoprotein subclass information may be beneficial. We observed ADIPOQ variant allele carriers had significantly lower levels of LDL 1 and IDL 2 and APOE variant allele carriers had significantly lower levels of LDL 1, subclasses considered less atherogenic than other LDL subclasses. CETP Taq1B and PON1 L55M variant allele carriers were significantly associated with HDL subclasses, where we observed increases in larger, more efficient HDL and decreases in smaller HDL. In addition, a significant interaction between genotype and diabetic status was observed, only for CETP Taq1B, PON1 L55M and PCSK9 A443T genotype. Targeting these particular genes, as well as many others known and unknown, may serve as promising interventions for all dyslipidemic individuals. Understanding the influence that co-morbidities may have in the presence of genetic variations will facilitate the design of innovative strategies for the prevention of morbid cardiovascular events via early detection of CVD risk.
    • Design And Evaluation Of Novel Topical, Transdermal And Microneedle Formulations

      Sivaraman, Arunprasad
      Arunprasad Sivaraman Design and evaluation of novel topical, transdermal and microneedle formulations (Under the direction of AJAY K. BANGA, Ph.D) Purpose: The broad aim of this project was to formulate passive transdermal patches, topical gel and microneedle and evaluate their physical properties and delivery efficiency via skin. Methods: Polyvinyl alcohol polymer transdermal patch was prepared in combination with DURO-TAKTM 387-2516 acrylate adhesive using oxybutynin and characterized for coating efficiency, matrix structure, rheology, tack, shear, peel adhesion and tested for in vitro permeation using dermatomed human skin for 72 hours. A semi-synthetic opioid transdermal patch was prepared using a formulation blend of oleic acid, BIO-PSA 7 - 4301 silicone adhesive and an amphiphilic solvent and characterized for coating efficiency, matrix structure, thickness, tack, peel adhesion and tested for in vitro permeation using dermatomed human skin for 72 hours. In situ forming hydrogel microneedles were investigated using a non-ionic triblock thermosensitive copolymer with methotrexate. The microneedles were characterized for sol-gel transition, geometry, depth of micropores, histology, rheology, methylene blue staining and evaluated for transdermal delivery in full thickness porcine ear skin and dermatomed human skin. A topical diclofenac gel was prepared using polyvinyl alcohol polymer and characterized for matrix arrangement, pH, adhesion, spreading efficiency, crystallization, rheology, skin irritation and evaluated for in vitro drug distribution and permeation with dermatomed human skin. Results and Conclusions: The polyvinyl alcohol transdermal patch showed an emulsion matrix formation with an average in vitro cumulative permeation and flux of 343.80 ± 74.36 μg/cm2 and 4.79 ± 1.3 μg/cm2/h respectively with no skin irritation. The semi-synthetic opioid transdermal patch delivered an average drug cumulative permeation and flux of 25.98 ± 0.19 μg/cm2 and 0.28 ± 0.2 μg/cm2/h respectively with no drug crystallization. The in situ formed hydrogel microneedles delivered an average cumulative drug amount of 32.2 ± 15.76 μg/sq.cm and 114.54 ± 40.89 μg/sq.cm for porcine ear skin from 0.2% w/w and 0.4% w/w methotrexate formulations. The topical diclofenac gel delivered an average cumulative drug amount of 22.85 ± 9.41 μg/cm2 and distribution of 10.30 ± 9.09 μg/cm2 for 24 h with no skin irritation. In conclusion, transdermal patches, topical gel and microneedles were formulated and characterized for their physical properties and evaluated for skin delivery.
    • Development and Characterization of a Novel Immunotherapy for Treatment of Breast Cancer by Combining Particulate Cancer Vaccines with Immune-Modulators

      Mulla, Nihal S; College of Pharmacy
      In the last half of the century, advances in the field of cancer therapy including chemotherapy, hormonal therapy and targeted therapy have been responsible for improvements in breast cancer related mortality. Although such advances have benefited all cancer types, there are considerable challenges faced by researchers striving to realize the goal of complete tumor remission. Immunotherapy is a great alternative as it has minimum side effects and several advantages over traditional cancer therapies. The aim of this project is to develop a novel immunotherapy for treatment of cancer by combining cancer vaccines with various immune-modulators. We take advantage of micron-sized particles to deliver vaccine along with other immune modifiers to target immune cells and to initiate immune response against breast cancer antigens. These particles were evaluated for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. The efficacy of breast cancer vaccine microparticles was tested in a murine breast cancer model. The immunized animals showed significantly lower tumor growth compared to the naive animals that did not receive any treatment. The delay in tumor growth in vaccinated animals was due to a strong immune response generated against tumor- associated antigens encapsulated within the microparticles. We observed a significant increase in the CD4+ T cell population. The suppression mechanism employed by regulatory T cells are thought to contribute significantly to the failure of current therapies that rely on potentiation of anti-tumor responses. We evaluated the therapeutic efficacy of vaccine microparticles after depleting the immunosuppressive regulatory T cells. We observed a significant improvement in the efficacy of vaccine microparticles by depleting regulatory T cells. The tumor inhibitory effect of vaccine microparticles was due to depletion of regulatory T cells by cyclophosphamide. We observed a significant increase in the CD8+ T cell population. The final aim of my research project was to enhance the immunogenicity of cancer vaccines using adjuvants. Based on our findings we conclude that Alum, Addavax (like MF59) R848 and CpG significantly enhanced the immunogenicity of breast cancer associated antigens.
    • Development And Characterization Of Polymeric Formulations And Microneedles For Dermal Drug Delivery

      Kim, Yujin
      Skin provides an attractive route for drug delivery, as transdermal delivery systems can ensure noninvasive and sustained delivery of medication across the skin into systemic circulation. Although the skin is a convenient site for drug administration, it only permits passive penetration of permeants with certain physicochemical properties. As a result, different strategies or devices have been developed to enhance the drug permeation into and across skin. The first aim of this study was to evaluate the efficacy of multiple dosing of trolamine salicylate (TS) as a topical analgesic on in vitro skin permeation. Also, the effect of sonophoresis on topical delivery of analgesic was evaluated. Results suggested that the multiple dosing (17.4 ± 4.1 μg/sq.cm) delivered a significantly higher amount of drug than the single dose (6.5 ± 0.6 μg/sq.cm). Also, the use of ultrasound enhances topical absorption of TS into and across skin. In the second aim, we focused on developing a new formulation to reduce the frequency of application of topical analgesics. A Poly lactic-co-glycolic acid (PLGA) based bio-adhesive polymeric solutions were prepared successfully and formed a thin film upon application in situ. A significantly higher amount of TS was delivered from a formulation containing 20% PLGA (45 ± 4 µg/sq.cm) as compared to PLGA-free counterpart (0.6 ± 0.2 µg/sq.cm). In the third aim, hyaluronic acid (HA) microneedles (MNs) were fabricated with and without magnesium ascorbyl phosphate (MAP) to facilitate the delivery of the active agent into and across the skin. The results showed that successful fabrication of HA MN, and the application of the current MN technology enhanced the delivery of MAP into skin (96.8±3.9 µg/sq.cm) compared to passive delivery (44.9±16.3 µg/sq.cm). In the fourth aim, the feasibility of transdermal delivery of heparin, a hydrophilic macromolecule, through laser-microporated skin was investigated. There was no passive permeation of heparin across the skin. However, permeation of heparin from the laser-treated group delivered 13.4 ± 0.62 µg/sq.cm after 24 h. Furthermore, heparin was not delivered to either the epidermis or dermis passively, whereas the laser-treatment group enabled delivery of heparin to the epidermis as well as the dermis.