Pre-systemic elimination by local enzymatic degradation can play a key role in limiting the bioavailability of intranasally administered drugs. Despite remarkable advancement in the characterization of the nasal biotransformative enzymes, knowledge of the role of the nasal mucosa in limiting bioavailability of therapeutic agents is still inadequate. The aim of this work was to evaluate the expression and substrate biotransformation activity of cytochrome P450 enzymes in the nasal mucosa using bovine olfactory and respiratory explants as in vitro models. Gene expression and localization of major CYP450 isoforms in the nasal mucosa were examined using RT-PCR and immunohistochemistry. The bovine nasal mucosa showed abundant expression of CYP2A6 and 3A4 genes whereas 1A1, 1A2, 2C9, and 2C19 isoforms were expressed at much lower levels. The CYP450 proteins were observed to be present in the epithelial layer and in submucosal glandular cells. The diffusion of melatonin, a CYP1A2 substrate, and the appearance of 6-hydroxymelatonin, its primary metabolite, across bovine olfactory and respiratory explants was measured, and nasal olfactory and respiratory microsomal preparations were used to quantify the kinetic parameters for melatonin 6-hydroxylation. Results indicated that bovine olfactory and respiratory CYP450 isoforms were metabolically active towards melatonin metabolism, and the respiratory mucosa demonstrated the greatest melatonin 6-hydroxylation activity. Numerical simulations were used to probe the effects of the relative magnitudes of the permeability coefficient and enzymatic parameters on net substrate mass transfer across nasal mucosal tissues. The simulations indicated that the concentration gradient of the drug coupled with its permeability coefficient were the most significant factors controlling the transport of drugs across the mucosal tissue. Enzymatic degradation decreased the flux of drugs across the mucosa and had the greatest impact on low permeability compounds. The results from these studies show that the bovine nasal mucosa possesses significant metabolic activity, and the flux of a metabolically labile substrate across the nasal mucosa can be significantly reduced by its enzymatic degradation within the tissue. Use of kinetic modeling to characterize of the extent of biotransformation in the nasal mucosa enables the identification of metabolism-limited bioavailability of intranasally administered drug compounds.

Nearly one-third of all pharmaceutical substances on the market are able to sorb water into their crystal lattices to form hydrates, which can often compromise stability during processing and/or storage[1]. The tendency of a hydrate to lose its water of crystallization during the manufacturing process of tablet compression is of particular concern to formulation scientists. The amount of water freed as a function of increasing compaction pressure can be explained by the mobility of water within the compact. The mobility of water is determined by the size and shape of the crystal lattice, the numbers and strengths of the hydrogen bonds, and the presence of high-energy sites of disorder[2]. Due to their differing crystal structures, theophylline monohydrate (THM), citric acid monohydrate (CAM), theophylline-water-citric acid cocrystal hydrate (CATHP hydrate), and dicalcium phosphate dihydrate (DCPD) make for interesting model systems to examine the dehydration under mechanical load. The thermal dehydration of both powders and tablets was carried out via thermal gravimetric analysis (TGA). By comparing the temperatures required to start removal of water loss from the powder to that of the tablet, the average amount of water of crystallization that is freed by the compaction process may be quantified. The average amount of water freed by the compaction process results from a competition between the mechanically-induced disorder of the crystal structure that increases the molecular mobility of water within the tablets, and the trapping of water within the interparticulate void spaces at high compaction pressures. The compressibilities, compactabilities, and tabletabilities of the materials were calculated as a function of increasing compaction pressure. The consolidation of the powder bed under pressure was modeled by out-of-die Heckel Analysis which demonstrated the ease of deformation of the model compounds. XRD was utilized to show the decrease in overall order of the crystal lattice as a result of compression as well as anisotropy within the tablets. Crystallographic approaches were utilized to demonstrate the compactness of the crystal structure, and how it affects water mobility. Relaxation pulse experiments (T1, T2) utilizing solid-state NMR were used to directly probe the mobilities of the water molecules within the crystal lattice of THM. The results from T1 and T2 relaxation experiments directly measure the change in molecular mobility of water within the tablets as a function of compaction pressure. This provided independent verification of the trends in molecular mobility and average water freed as a function of compaction pressure observed during TGA dehydration. Raman spectroscopy was used to indirectly measure the polarizability and vibrational motions of THM, and these results corroborate those obtained from ssNMR and TGA dehydration experiments. Overall, this work highlights the potential impact that tablet compression can have on API hydrate stability. 1. Hilfiker R (editor). 2006. Polymorphism in the Pharmaceutical Industry. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co, KGaA. 2. Byrn SR, Pfeiffer RR, Stowell JG. 1999. Solid-state chemistry of drugs. SSCI, Inc.

Pseudomonas aeruginosa bacterial biofilms are the leading cause of mortality among cystic fibrosis (CF) patients. Biofilms contain bacteria attached to a surface and encased in a protective matrix. Since bacteria within a biofilm are less susceptible to antibiotics, a new approach is to use dispersion compounds that cause the biofilms to release free-swimming bacteria. Our approach has focused on combining nutrient dispersion compounds with antibiotics to increase eradication of bacteria within biofilms. This approach takes advantage of the enhanced susceptibility of free-swimming bacteria to antibiotics, compared to bacteria within biofilms. Ultimately, this research will guide the development of an aerosol therapy containing both antibiotic and dispersion compounds to treat bacterial biofilm infections. To study the effect of antibiotic and dispersion compound treatments on biofilm eradication, a high-throughput screening assay was used to assess the effect on young Pseudomonas aeruginosa biofilms. In addition, a Lab-Tek chambered coverglass system imaged via confocal microscopy was used to assess the effect on mature Pseudomonas aeruginosa biofilms. Seven antibiotics (amikacin disulfate, tobramycin sulfate, colistin sulfate, colistin methanesulfonate (CMS), polymyxinB sulfate, erythromycin, and ciprofloxacin hydrochloride) were tested alone or in combination with four nutrient dispersion compounds (sodium citrate, succinic acid, xylitol, and glutamic acid) to assess the level of eradication of bacteria within biofilms. For young biofilms, 15 of 24 combinations significantly eliminated more live bacteria within the biofilms (measured in colony forming units per milliliter) compared to antibiotics alone. In the more mature biofilm system, only 3 out of 26 combinations resulted in a higher percentage of live biofilm bacteria being eliminated compared to antibiotics alone, showing the importance of biofilm age in the effectiveness of these potential combination therapies. To aid in confocal microscopic analysis of biofilms, an automated quantification program called STAINIFICATION was developed. This new program can be used to simultaneously investigate connected-biofilm bacteria, unconnected bacteria (dispersed bacteria), the biofilm protective matrix, and a growth surface upon which bacteria are grown in confocal images. The program contains novel algorithms for the assessment of bacterial viability and for the quantification of bacteria grown on uneven surfaces, such as tissue. The utility of the viability assessments were demonstrated with confocal images of Pseudomonas aeruginosa biofilms. The utility of the uneven surface algorithms were demonstrated with confocal images of Staphylococcus aureus biofilms grown on cultured human airway epithelial cells and Neisseria gonorrhoeae biofilms grown on transformed cervical epithelial cells. Finally, a proof-of-concept study demonstrated that dry powder aerosols containing both antibiotic and nutrient dispersion compounds could be developed with properties optimized for efficient deposition in the lungs. A design of experiments study showed that solution concentration was the most significant parameter affecting aerosol yield, particle size, and in vitro deposition profiles. Collectively this work demonstrated that bacterial dispersion from biofilms can enhance antibiotic susceptibility and can be better quantified using the new STAINIFICATION software. Formulation of dispersion compounds and antibiotics into a dry powder aerosol could enable more effective treatment of biofilm infections in the lungs.

Pesticides have been used to control various types of pests, including plants and insects, for thousands of years, however the impact of exposure to these toxic chemicals, with respect to environmental and health consequences, is not fully understood. Two pesticides of interest, molinate and dieldrin, have been shown to cause neurotoxicity in humans, but their mechanisms of toxicity are still unknown. In order to better understand how exposure to these chemicals can cause toxicity, the structure-activity relationship (SAR) was defined to determine how specific changes to the structure of each pesticide affects the toxicity profiles of each of these compounds. Results of this study demonstrated that oxidation of molinate to molinate sulfoxide, and then further to molinate sulfone, a more potent inhibitor of aldehyde dehydrogenase. The sulfone metabolite is capable of covalently modifying the active-site cysteine residue of aldehyde dehydrogenase, accounting for the observed enzyme inhibition. These results indicate that the compound responsible for the toxicity from molinate exposure is not the parent compound, but rather one of the sulfoxidation metabolites. When the SAR of dieldrin was investigated with respect to a Parkinson's disease model, it was determined that the compounds that were previously found to be the least potent insecticides were the most toxic with respect to dopaminergic cells. Each of the compounds tested was observed to disrupt dopamine metabolism in accordance with their toxicity profiles in dopaminergic cells. In combination, these results implicate important structural features responsible for the toxicity with respect to Parkinson's disease. This information is critical for the development of new pesticides, and will be important to increase the selective toxicity for insects while minimizing adverse/off-target effects. This can lead to the development of safer, more effective pesticides that will be essential for future environmental and human health.

In this thesis, the population pharmacokinetics of telapristone and its active metabolite, CDB-4453 was evaluated using nonlinear mixed effects modeling (NONMEM®). A two-compartment (parent) one compartment (metabolite) mixture model with first order absorption and elimination adequately described the pharmacokinetics of telapristone and CDB-4453. For the Phase I/II pharmacokinetic analysis (effect of renal and hepatic impairment), telapristone was rapidly absorbed with an absorption rate constant (Ka) of 1.26 h-1. Moderate renal impairment resulted in a 74% decrease in Ka. Population estimates for oral clearance (CL/F) for the high and low clearance groups were 11.6 L/h and 3.34 L/h, respectively. Twenty-five percent of the subjects were allocated to the high clearance group. Apparent volume of distribution for the central compartment (V2/F) was 37.4 L, apparent inter-compartmental clearance (Q/F) was 21.9 L/h, and apparent peripheral volume of distribution for the parent (V4/F) was 120 L. The ratio of the fraction of telapristone converted to CDB-4453 to the distribution volume of CDB-4453 (Fmetest) was 0.20/L and apparent clearance of the metabolite (CLM/F) was 2.43 L/h. For the pharmacokinetic analysis evaluating the effect of food; food decreased the Ka of telapristone (Ka for the fed and fasted state was 0.467 and 5.06 h-1, respectively). Population estimates of the high and low CL/F groups were 12.0 L/h and 3.15 L/h, respectively. Thirty-one percent of the subjects were allocated to the high clearance group. V2/F, Q/F and V/4 and Fmetest were 52.8 L, 7.53 L/h, 84.8 L and 0.193/L, respectively. CLM/F was 2.10 L/h. An external validation was performed using the final parameter estimates from the pooled pharmacokinetic analysis (effect of renal and hepatic impairment and the effect of food). From this pharmacokinetic analysis, Ka for the fed and fasted state was 0.299 and 2.35 h-1, respectively. Population estimates for the high and low CL/F groups were 11.6 L/h and 3.22 L/h, respectively. The percentage of subjects allocated to the high clearance group was 29%. V2/F, Q/F, V/4 and Fmetest were 52.8 L, 11.6 L/h and 93.8 L and 0.186/L, respectively. CLM/F was 2.23 L/h. The final model did not meet the requirement for adequate predictability using the external validation dataset. However, the external validation dataset only included samples with limited early time points. Because of the limited sampling times, it is difficult to make a conclusion about the overall adequacy of the model. An external validation dataset with more extensive sampling will be needed in order to better assess the predictability final model. This is the first comprehensive review of the pharmacokinetics of telapristone and CDB-4453.

My thesis includes the development of two novel anticancer agents based on natural products; OSW-1 analog (ZJ-201) and truncated Superstolide A analog (ZJ-102). OSW-1 is isolated from the bulbs of Ornithogalum saundersiae. It exhibits an extremely potent anticancer activity against a wide spectrum of cancer cells. Relatively, its anticancer activities are about 10-100 times more potent than many well-known anticancer drugs in clinical use. However, the promise of OSW-1 is dampened by its relatively weak in vivo anticancer activity. We hypothesize that the loss of two ester groups on OSW-1 in mouse causes a discrepancy in its in vivo efficacy. Therefore, replacing both ester groups in the disaccharide portion of OSW-1 with bioisosteric amides should significantly reduce the rate of metabolism and greatly improve its in vivo anticancer activity. This dissertation includes the synthesis of amide analog of OSW-1, ZJ-201. The synthetic route described in this thesis is characterized by its flexibility to synthesize multiple amino analogs of OSW-1. ZJ-201 will be evaluated for its in vitro cytotoxicity, metabolic stability and pharmacokinetic properties. The biological data obtained will enable us to get insights into the SAR of OSW-1 and assist in transforming OSW-1 into a clinically agent. Superstolide A is a highly potent anticancer agent isolated from marine sponge Neosiphonia superstes. In 2013, Jin’s lab reported the design and synthesis of truncated Superstolide A (ZJ-101) in 15 steps with a yield of 6.2%. In vitro cytotoxicity studies showed that ZJ-101 maintains and sometimes exceeds the potent anticancer activity of the parent natural product. As this is the first active analog of Superstolide A reported, there is a need to develop additional analogs of ZJ-101 to probe into the SAR of this anticancer agent. This dissertation includes the synthesis of aromatic analog of truncated Superstolide A, ZJ-102. In vitro cytotoxicity studies showed that ZJ-201 demonstrated poor antiproliferative properties in comparison to ZJ-101. Hence, we can conclude that the cyclohexene ring of ZJ-101 cannot be simplified to an aromatic core as it significantly affects anticancer activity.

The ability to safely delivery efficacious amounts of nucleic acids to cells and tissues remains an important goal for the gene therapy field. Viruses are very efficient at delivering DNA, but safety concerns limit their clinical use. Nonviral vectors are not as efficient at DNA delivery, but have a better safety profile. Limiting the efficaciousness of nonviral vectors are the numerous extra and intracellular barriers that must be overcome for successful DNA delivery in vivo. While single polymers can successfully transfect immortalized cell lines in vitro, multicomponent gene delivery systems are required for delivery in vivo. Key in the development of multicomponent systems is their syntheses. Optimization of a nonviral gene delivery system requires the development of methodologies that incorporate the different components in a controlled fashion, generating homogeneous gene delivery vectors. Such syntheses ensure every polymer has the different components required for successful delivery. The amount of each component and location within the gene delivery system can also be varied systemically, allowing optimization of the vector. The overall scope of this thesis is to develop a chemical method to iteratively couple gene delivery peptides through reducible disulfide bonds. The synthesis of such polypeptides allows the triggered disassembly of a polypeptide polyplexed with DNA upon cellular uptake. To synthesize homogeneous gene delivery polypeptides, a novel iterative reducible ligation strategy was developed, based upon the use of a thiazolidine masked cysteine. Initial studies demonstrated that a thiazolidine could be unmasked to a cysteine in the presence of a disulfide bond without side reaction, though the reported thiazolidine hydrolysis conditions of aqueous methoxyamine were insufficiently robust for high yielding ligations. Discovery of a novel silver trifluoromethanesulfonate hydrolysis led to an efficient process for generating reducible polypeptides, as evidenced in the synthesis of a 4 component polypeptide. Due to the success of the thiazolidine mediated iterative ligation strategy, cysteines were replaced by penicillamines to produce more stable disulfide bonds. The mild thiazolidine hydrolysis and subsequent peptide conjugation reactions led to attempt the iterative ligation strategy on a solid support, eliminating purification steps that lowered the yields in the solution phase methodology. Initial progress at generating gene delivery peptides that could be incorporated into the synthetic strategy included the generation of a tri-orthogonal cysteine protecting scheme that allowed a third cysteine to be derivatized with a targeting ligand or stealthing polymer. Due to the use of terminal cysteines in the iterative ligation strategy, a PEG stealthing polymer could be placed in the center of a polyacridine gene delivery peptide with only a small decrease in the ability to condense and protect DNA during systemic circulation. A convergent synthesis was also developed that was able to synthesize large polypeptides in fewer linear steps. The synthetic methodology of thiazolidine mediated iterative reducible ligation developed in this thesis is important in the gene therapy field as it allows the construction of polypeptides that can be systemically optimized, potentially resulting in highly efficacious nonviral gene delivery.

The dissolution behavior of a drug substance is an important part of its bioavailability. Three solid dissolution mechanisms are recognized: transport control, interface control and mixed-kinetic control. The mixed-kinetic control mechanism is not well studied as the majority of dissolution phenomena in pharmaceutical research are assumed to be transport-controlled. A phenomenological model for mixed-kinetic control was developed in which the interfacial step comprises molecular detachment and re-deposition and is described by chemical kinetic theory. This model encompasses interface control and transport control as limiting cases. Experimental studies on three organic compounds showed that they dissolved by transport control at 37°C, but exhibited certain degrees of interface control at lower temperatures (10°C and 3°C), which, according to the model, indicates that reducing the dissolution temperature slowed down re-deposition more than transport. Using mathematical approaches derived from the model, up to 27% interface control was calculated from the experimental results. The second experimental investigation showed significant degrees of interface control in benzoic acid dissolution in sodium dodecyl sulfate (NaDS) solutions at 25°C. The dissolution behavior was well described by the mixed-kinetic control model and up to 73% interface control was calculated. An extension of the model was proposed to describe a potential micelle-interface interaction mechanism indicated by the model-fitted parameters. The third investigation showed that FD&C Blue #1, a water-soluble dye, inhibited sulfathiazole dissolution in acidic media but not in water. The inhibition was attributed to the blocking of dissolution sites by dye adsorption. A potential pH-dependent adsorption mechanism was proposed in which protonation at sulfathiazole solid surface gives rise to preferential dye adsorption on detachment rates and thus reduced dissolution rates.

Background: In response to high cost and inadequate quality, the healthcare system is in the midst of a transition from paying for volume to paying for value. Billions of dollars could be saved through more effective medication use, and evidence supports the role of the community pharmacist in lowering healthcare cost and improving healthcare quality through medication optimization. Despite this, value-based payment models for community pharmacies are rare, and those that do exist have not been critically evaluated and implementation in a commercially insured population is rare. Objective: The first objective was to design and test a conceptual model of pharmacy value. The second objective was to evaluate variation in the value community pharmacies provide a commercial insurer by assessing the relationship between attributed patients’ healthcare quality and cost. Methods: This study used prescription and medical claims data for 2012 and 2013 from a large commercial insurer in Iowa and South Dakota. Patients were attributed to the pharmacy filling the majority of their prescriptions. Pharmacies’ weekly prescription volume and Sunday prescription filling behavior were used as structural measures of healthcare quality. Percent of days covered (PDC) metrics for beta-blockers, statins, renin-angiotensin system antagonists and non-insulin diabetes agents were used as process metrics. Pharmacies were excluded if the denominator for any PDC metric was less than 15. Outcome metrics consisted of a non-trauma, non-cancer, unplanned hospitalization rate and a non-trauma ED visit rate. Cost impact was categorized into pharmaceutical, medical, and total cost of care. High quality pharmacies with typical or low associated costs or low cost pharmacies with typical to high quality were identified as high value and vice versa for low value. All metrics were risk-adjusted using mixed effect models with a random pharmacy intercept. The ratio between observed and expected quality scores was used for quality scoring. Quality outliers were identified by comparing the 95% CI around pharmacies’ risk-adjusted scores to the all-pharmacy risk-adjusted score mean. A t-test was used to assess variation in pharmacy value. Results: There were 171 pharmacies and 74,581 patients eligible for scoring on all quality metrics. Mixed effects models observed a small but significant impact of pharmacy on process and outcome healthcare quality. No relationship between structures and processes, processes and outcomes was detected. Ten pharmacies were scored as high quality and nine as low quality. Similar numbers were identified for cost outliers, and significant variation in value was detected. Implications/conclusions: Results support the hypothesis that high and low value pharmacies exist. A well-designed value-based payment model could be used to create incentives for pharmacists to enhance care for commercially insured patients, but validation is needed to ensure that incentives are aligned appropriately.

Cancer is a collection of diseases that is characterized by an uncontrolled proliferation of aberrant cells. The conventional treatment strategies including chemotherapy and radiotherapy prove detrimental to healthy cells and are unable to combat primary and secondary metastases. Hence immunotherapy is gaining momentum in cancer clinics and translational research labs as an alternative safer and more efficacious approach to tumor therapy. Vaccines based on antigen alone lack the ability to optimally activate the antigen presenting cells (APCs) including dendritic cells (DCs) to generate significantly greater antigen-specific T cell responses. A typical strategy to overcome this limitation has been the use of adjuvants in order to improve the immunogenicity of the vaccines. A newer class of adjuvants called toll like receptor (TLR) ligands recognize pathogen associated molecular patterns (PAMP) and thus elicit a Th-1 polarized response. The hypothesis of the current research is that co-delivery of antigen and adjuvant in a microparticulate carrier will elicit a strong cell-mediated immune response conferring anti-tumor immunity. Furthermore, we proposed that a combination of TLR ligands when co-delivered with an antigen will be able to mount a synergistic anti-tumor response in comparison to the delivery of antigen alone. There were three primary objectives of the study; 1) Fabrication, process design optimization and characterization of antigen and adjuvant co-loaded microparticles (MP), 2) Study the ability of the MP fabricated in step 1 in eliciting a potent immune response in a murine model by various routes of administration and 3) Study the effect of the various treatment as prophylactic and therapeutic cancer vaccines in a murine tumor model. Objective 1 was achieved by optimizing various process parameters including PLGA type and concentration, surfactant concentration and modification of a previously reported double emulsification solvent evaporation technique. Process optimization lead to the development of the following 5 treatment groups, a) Empty PLGA MP, b) OVA PLGA MP, c) OVA + CpG PLGA MP, d) OVA + poly I: C PLGA MP and e) OVA + CpG + poly I:C tri-component PLGA MP. The optimized microparticles exhibited a mean particle size of 1.5 πm with a smooth spherical surface and a desirable biphasic release pattern of the entrapped components. Results from step 2 indicated the ability of the co-delivery and the tri-component systems to generate several fold higher immunostimulatory response in comparison to the group treated with antigen alone. Of the two routes of administration tested, the intraperitonial (i.p) route gave the strongest response followed by intramuscular (i.m) route. A synergistic antibody response response was observed upon vaccination with the tri-component model. Prophylactic vaccination studies showed that the co-delivery and tri-component system affording the strongest protection against aggressive tumor growth. The therapeutic studies also indicated enhanced tumor protection and 2-fold improvement in survival times when the mice vaccinated with the co-delivery and tri-component MP carriers in comparison to vaccination with microparticles loaded with antigen alone. The carriers also showed strong evidence for the generation of anti-tumor immunogenic memory. In summary, the current study has laid an interesting premise for the use of immunotherapy using a combination of antigen and TLR adjuvants alone or in conjunction with chemotherapy as a treatment strategy for tumor therapy. This research is expected to lead to the utilization of PLGA delivery systems as novel carriers for cancer immunotherapy.