Pharmacy and Pharmaceutical Sciences Commons^™

Microneedle-Assisted Transdermal Delivery Of Naltrexone Species: In Vitro Permeation And In Vivo Pharmacokinetic Studies, Mikolaj Milewski

University of Kentucky Doctoral Dissertations

Naltrexone (NTX) is a drug used primarily in the management of alcohol dependence and opioid dependence. Based on several drawbacks associated with the oral and injectable intramuscular dosage forms of naltrexone currently available on the market, there is substantial interest in delivering naltrexone transdermally. Although naltrexone does not permeate skin at the rate sufficient to reach therapeutic plasma concentrations in humans, novel flux enhancement methods such as microneedles help address this challenge. Earlier work in humans has demonstrated that the use of microneedles achieves plasma concentrations in the lower end of expected therapeutic values. Further flux enhancement is desired to …

Atomic Force Microscopy Method Development For Surface Energy Analysis, Clare Aubrey Medendorp

University of Kentucky Doctoral Dissertations

The vast majority of pharmaceutical drug products are developed, manufactured, and delivered in the solid-state where the active pharmaceutical ingredient (API) is crystalline. With the potential to exist as polymorphs, salts, hydrates, solvates, and cocrystals, each with their own unique associated physicochemical properties, crystals and their forms directly influence bioavailability and manufacturability of the final drug product. Understanding and controlling the crystalline form of the API throughout the drug development process is absolutely critical. Interfacial properties, such as surface energy, define the interactions between two materials in contact. For crystal growth, surface energy between crystal surfaces and liquid environments not …

Isolation And Elucidation Of The Chrysomycin Biosynthetic Gene Cluster And Altering The Glycosylation Patterns Of Tetracenomycins And Mithramycin-Pathway Molecules, Stephen Eric Nybo

University of Kentucky Doctoral Dissertations

Natural products occupy a central role as the majority of currently used antibiotic and anticancer agents. Among these are type-II polyketide synthase (PKS)-derived molecules, or polyketides, which are produced by many representatives of the genus Streptomyces. Some type-II polyketides, such as the tetracyclines and the anthracycline doxorubicin, are currently employed as therapeutics. However, several polyketide molecules exhibit promising biological activity, but due to toxic side effects or solubility concerns, remain undeveloped as drugs.

Gilvocarcin V (GV) (topoisomerase II inhibitor) has a novel mechanism of action: [2+2] cycloaddition to thymine residues by the 8-vinyl side chain and cross-linking of histone …

The Absence Of Abcd2 Reveals A Novel Role For Peroxisomes In The Protection From Metabolic Syndrome, Jingjing Liu

University of Kentucky Doctoral Dissertations

ABCD2 (D2) is a peroxisomal ATP binding cassette (ABC) transporter that is expressed in brain, adrenal and liver. D2 is transcriptionally regulated by key transcriptional factors that control lipid and glucose metabolism. Therefore, we examined its role in adipose tissue. These studies revealed that D2 is highly abundant in adipose tissue and upregulated during adipogenesis. However, D2 deficiency does not affect either adipogenesis or lipid accumulation. An examination of the lipid profile of adipose tissue revealed the accumulation of C20 and C22 fatty acids in D2 deficient (D2‐/‐) mice. When challenged with a diet enriched in erucic acid (C22:1, 10% …

Combinatorial Biosynthetic Derivatization Of The Antitumoral Agent Gilvocarcin V, Micah Douglas Shepherd

University of Kentucky Doctoral Dissertations

Gilvocarcin V (GV), the principal product of Streptomyces griseoflavus Gö 3592 and other Streptomyces spp., is the most prominent member of a distinct class of antitumor antibiotics that share a polyketide derived coumarin-based aromatic core. GV and other members of this class including polycarcin V from Streptomyces polyformus, often referred to as gilvocarcin-like aryl C-glycosides, are particularly interesting because of their potent bactericidal, virucidal and antitumor activities at low concentrations while maintaining low in vivo toxicity. Although the precise molecular mechanism of GV bioactivity is unknown, gilvocarcin V has been shown to undergo a photoactivated [2+2] cycloaddition of …

Evaluation Of The Physicochemical Properties And Stability Of Solid Lipid Nanoparticles Designed For The Delivery Of Dexamethasone To Tumors, Melissa Howard

University of Kentucky Doctoral Dissertations

Pre-clinical and clinical trials suggest that pre-treatment with dexamethasone (Dex) may facilitate enhanced uptake of subsequently administered chemotherapeutic agents. To reduce the side effects associated with systemic administration of Dex, solid lipid nanoparticles (SLNs) containing dexamethasone palmitate (Dex-P) were prepared as a means of achieving tumor-targeted drug delivery. These studies were aimed at evaluating the physicochemical properties and both the physiological and storage stability of the SLNs.

SLNs were prepared using nanotemplate engineering technology. Stearyl alcohol (SA) was used as the lipid phase with Brij^® 78 and Polysorbate 60 as surfactants and PEG6000 monostearate as a long-chain PEGylating agent. …

Effect Of Azithromycin On Macrophage Phenotype During Pulmonary Infections And Cystic Fibrosis, Theodore James Cory

University of Kentucky Doctoral Dissertations

Azithromycin improves clinical outcomes in patients with cystic fibrosis (CF), specifically in patients infected with Pseudomonas aeruginosa. Azithromycin shifts macrophage programming away from a pro-inflammatory classical (M1) phenotype, and towards an anti-inflammatory alternative (M2) phenotype; however, little is known about this mechanism, nor of its impact upon immune response to pulmonary infection. We set out to determine the mechanism by which azithromycin is able to alter macrophage phenotype, and assess the effect of azithromycin induced macrophage polarization on inflammation during pulmonary infections.

Utilizing macrophage cell culture, we found that azithromycin increased IKKβ, a signaling molecule in the NFκB pathway, which …

Nm23-H1 Blocks Cell Motility Independently Of Its Known Enzymatic Activities In A Cohort Of Human Melanoma Cells, Joseph Robert Mccorkle

University of Kentucky Doctoral Dissertations

The metastasis suppressor gene NM23-H1 has been shown to possess three enzymatic activities including nucleoside diphosphate kinase, histidine-dependent protein kinase and 3’-5’ exonuclease activity. While these properties have been demonstrated in vitro using recombinant proteins, the contribution of these activities to suppression of metastatic dissemination is unknown. Site-directed mutagenesis studies were used to identify amino acid residues which are required for proper function of each enzymatic activity associated with H1, providing a platform for studying the importance of each function on an individual basis. To assess the relevance of these activities to melanoma progression, a panel of mutants harboring selective …

Development Of Novel Ahr Antagonists, Hyosung Lee

University of Kentucky Doctoral Dissertations

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic …

Inhibition Of Cholesterol Synthesis By Policosanol, Subhashis Banerjee

University of Kentucky Doctoral Dissertations

Cholesterol is an essential component of the cell, but excessive blood levels are a major risk factor for the development of atherosclerotic plaques that can lead to heart disease and stroke, the foremost cause of premature death in Western societies. Policosanol, a mixture of very long chain alcohols derived from sugarcane, has gained considerable attention among the public as safe and effective means to reduce blood cholesterol levels, a belief based on some early clinical studies. My research investigates one possible mechanism by which policosanol might decrease blood cholesterol levels: the inhibition of cholesterol synthesis in the liver. Previous studies …

Alpha7 Nicotinic Acetylcholine Receptor Regulation In Experimental Neurodegenerative Disease, Christina Margaret Charriez

University of Kentucky Doctoral Dissertations

The α7 nicotinic acetylcholine receptor (nAChR) is involved in learning and memory, synaptic plasticity, neuroprotection, inflammation, and presynaptic regulation of neurotransmitter release. Alzheimer’s disease (AD), a neurodegenerative disease characterized by diminished cognitive abilities, memory loss, and neuropsychiatric disturbances, is associated with a loss of nAChRs. Similarly, traumatic brain injury (TBI) may result in long term neurobehavioral changes exemplified by cognitive dysfunction. Deficits in α7 nAChR expression have previously been shown in experimental TBI and may be related to cognitive impairment experienced in patients following TBI.

The purpose of this dissertation was to investigate changes in α7 nAChR expression in models …

Modulation Of The Alpha-7 Nicotinic Acetylcholine Receptor Following Experimental Rat Brain Injury Improves Cellular And Behavioral Outcomes, Thomas Matt Woodcock

University of Kentucky Doctoral Dissertations

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide, and survivors are often left with cognitive deficits and significant problems with day to day tasks. To date, therapeutic pharmacological treatments of TBI remain elusive despite numerous clinical trials. An improved understanding of the molecular and cellular response to injury may help guide future treatment strategies. One promising marker for brain injury is the translocator protein (TSPO), which is normally expressed at a low level, but is highly expressed following brain damage and is associated with neuroinflammation. The isoquinoline carboxamide PK11195 binds selectively to the TSPO …

Experimental And Molecular Dynamics Simulation Studies Of Partitioning And Transport Across Lipid Bilayer Membranes, Ravindra Wadhumal Tejwani

University of Kentucky Doctoral Dissertations

Most drugs undergo passive transport during absorption and distribution in the body. It is desirable to predict passive permeation of future drug candidates in order to increase the productivity of the drug discovery process. Unlike drug-receptor interactions, there is no receptor map for passive permeability because the process of transport across the lipid bilayer involves multiple mechanisms. This work intends to increase the understanding of permeation of drug-like molecules through lipid bilayers.

Drug molecules in solution typically form various species due to ionization, complexation, etc. Therefore, species specific properties must be obtained to bridge the experiment and simulations. Due to …

Studies On Drug Solubilization Mechanism In Simple Micelle Systems, Shaoxin Feng

University of Kentucky Doctoral Dissertations

Poor aqueous solubilities of drug candidates limit the biopharmaceutical usefulness in either oral or parenteral dosage forms. Lipid assemblies, such as micelles, may provide a means of enhancing solubility. Despite their usefulness, little is known about the means by which micelles accomplish this result. The goal of the current dissertation is to provide the molecular level understanding of the mechanism by which simple micelle systems solubilize drugs. Specifically, the location, orientation and amount of the drug molecules in micelle systems are the focuses of the work.

Three series of model drugs, steroids, benzodiazepines and parabens, in three surfactant systems with …

Insights Into Expression, Cellular Localization, And Regulation Of Supernatant Protein Factor, A Putative Regulator Of Cholesterol Biosynthesis, Elzbieta Ilona Stolarczyk

University of Kentucky Doctoral Dissertations

SPF (Supernatant Protein Factor) is a cytosolic protein that stimulates at least two enzymes in the cholesterol biosynthetic pathway: squalene monooxygenase and HMGCoA reductase. The mechanism of action has not been established but may be related to lipid transfer between intracellular membranes.

There are three human genes for SPF: SEC14L2 (SPF1), SEC14L3 (SPF2) and SEC14L4 (SPF3). The present study differentiates these closely related genes by evaluating their tissue-specific and relative expression levels. SPF1 mRNA was found to be most abundant in liver, mammary gland and stomach. SPF2 showed negligible expression in all tissues tested; SPF3 expression pattern was similar to …

Lipid-Based Paclitaxel And Doxorubicin Nanoparticles To Overcome P-Gp-Mediated Drug Resistance In Solid Tumors, Xiaowei Dong

University of Kentucky Doctoral Dissertations

Multidrug resistance (MDR) is a major obstacle limiting chemotherapeutic efficacy. The purpose of these studies was to investigate the potential application of injectable paclitaxel (PX) and doxorubicin (Dox)-loaded nanoparticles (NPs) engineered from oil-in-water microemulsion precursors for overcoming P-glycoprotein (P-gp)- mediated drug resistance in solid tumors.

An in-vitro study was performed to test whether the oil (stearyl alcohol and cetyl alcohol) used to make lipid nanoparticles could be metabolized. The results showed that the concentrations of the fatty alcohols within nanoparticles, which were quantitatively determined over time by gas chromatography, decreased to only 10-20% of the initial concentration after 15-24 h …

The Pharmacogenomics Of Egfr-Dependent Nsclc: Predicting And Enhancing Response To Targeted Egfr Therapy, Justin M. Balko

University of Kentucky Doctoral Dissertations

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How …

Preparation And Characterization Of Blackberry Extracts And Their Anticancer And Anti-Inflammatory Properties, Jin Dai

University of Kentucky Doctoral Dissertations

Blackberries are rich in polyphenols including anthocyanins. Polyphenols are hypothesized to have biological activities that impact positively on human health. The purpose of these studies was to develop phenolic extracts from selected cultivars of blackberries currently grown in Kentucky as potential Botanical Drug Products for the treatment and prevention of cancer and inflammatory diseases.

An ultrasound-assisted ethanol extraction method was employed to obtain anthocyanin-containing extracts (ACEs) from puree or powder (lyophilized puree) of blackberries. ACEs were analyzed for total anthocyanin and phenolics content, polymeric color, and total antioxidant capacity (TAC). The influence of water content in the extraction system was …

Synthesis And Evaluation Of Prouteolysis Taurgeting Chimeras (Protacs): A Potential Chemical Genetic Approach To Breast Cancer Therapy, Kedra C. Cyrus

University of Kentucky Doctoral Dissertations

The use of small molecules to probe the function of proteins is referred to as chemical genetics. The Proteolysis Targeting Chimera (PROTAC) is a chemical genetic tool that contains the ligand for a target protein of interest and the recognition motif for an E3 ubiquitin ligase attached by a linker. The PROTAC is capable of binding to and recruiting specific target proteins to the intracellular degradation system, the ubiquitin proteasome system (UPS). While the approach has had success it has not been optimized to be used on a broader scale.

Optimization efforts focused on elucidating the ideal linker length between …

Computational Design Of 3-Phosphoinositide Dependent Kinase-1 Inhibitors As Potential Anti-Cancer Agents, Mohamed Diwan Mohideen Abdulhameed

University of Kentucky Doctoral Dissertations

Computational drug design methods have great potential in drug discovery particularly in lead identification and lead optimization. 3-Phosphoinositide dependent kinase-1 (PDK1) is a protein kinase and a well validated anti-cancer target. Inhibitors of PDK1 have the potential to be developed as anti-cancer drugs. In this work, we have applied various novel computational drug design strategies to design and identify new PDK1 inhibitors with potential anti-cancer activity. We have pursued novel structure-based drug design strategies and identified a new binding mode for celecoxib and its derivatives binding with PDK1. This new binding mode provides a valuable basis for rational design of …

Application Of Linear Free Energy Relationships In The Prediction Of Triglyceride/Water Partition Coefficients And Lipid Bilayer Permeability Coefficients Of Small Organic Molecules And Peptides, Yichen Cao

University of Kentucky Doctoral Dissertations

Computational methods such as linear free energy relationships (LFERs) offer a useful high-throughput solution to quickly evaluate drug developability, e.g. membrane permeability, organic solvent/water partition coefficients, and solubility. LFERs typically assume the contribution of structural components/functional groups to the overall properties of a given molecule to be constant and independent. This dissertation describes a series of studies in which linear free energy relationships were developed to predict solvation of small organic molecules in lipid formulations, specifically, triglyceride containing solvents and phospholipid-based liposomes. The formation of intermolecular HBs in triglyceride solvents (homogenous with H-bond accepting ability) and intramolecular HBs within the …

Design, Synthesis, And Pharmacological Evaluation Of A Series Of Novel, Guanidine And Amidine-Containing Neonicotinoid-Like Analogs Of Nicotine: Subtype-Selective Interactions At Neuronal Nicotinic-Acetylcholine Receptor., Aaron Joseph Haubner

University of Kentucky Doctoral Dissertations

The current project examined the ability of a novel series of guandine and amidine-containing nicotine analogs to interact with several native and recombinantlyexpressed mammalian neuronal nicotinic-acetylcholine receptor (nAChR) subtypes. Rational drug design methods and parallel organic synthesis was used to generate a library of guanidine-containing nicotine (NIC) analogs (AH compounds). A smaller series of amidine-containing nicotine analogs (JC compounds) were also synthesized. In total, >150 compounds were examined. Compounds were first assayed for affinity in a high-throughput [³H]epibatidine radioligand-binding screen. Lead compounds were evaluated in subtype-selective binding experiments to probe for affinity at the α₄β_{2 …}

Selective Regulation Of Cardiomyocyte Signaling By Rgl2, Leah M. Allen

University of Kentucky Doctoral Dissertations

A key cardiovascular signaling molecule involved in both physiologic and pathologic regulation of cardiomyocytes is the small molecular weight G-protein, Ras. Differential effects of Ras are mediated by multiple effector molecules, including the RalGEFs which activate Ral. Studies performed in cardiomyocytes have indicated a role for Ral in cardiac hypertrophic signaling and the RalGEF family member, Rgl2, was shown to specifically interact with Ras in the heart. Therefore, I hypothesized that Rgl2 was an important Ras effector that would regulate cardiomyocyte signaling.

To elucidate the potential importance of Rgl2 in regulating cardiomyocyte signaling, a gain-of-function approach was utilized in which …

A Novel Class Of Immunoproteasome Catalytic Subunit Lmp2 Inhibitor And Its Therapeutic Potentials In Cancer, Yik Khuan (Abby) Ho

University of Kentucky Doctoral Dissertations

The immunoproteasome, known to play an important role in MHC class I antigen processing and presentation, have been linked to neurodegenerative diseases and hematological cancers. However, the pathophysiological functions of the immunoproteasome in these diseases are still not very well established. This can be attributed mainly to the lack of appropriate molecular probes that selectively target the immunoproteasome catalytic subunits. Herein, we report the development of a small molecular inhibitor (AM) that selectively targets the major catalytic subunit, LMP2, of the immunoproteasome. We show that the compound covalently modifies the LMP2 subunit with high specificity in human prostate …