Aliskiren (ALS) is a direct renin inhibitor with low bioavailability and high drug cost. The goal of this study was to increase the bioavailability of ALS through nanoformulation. The optimized formulation was then evaluated in spontaneously hypertensive rats (SHRs). We developed an ALS poly(lactic-co-glycolic) acid nanoparticle (ALS-NP) through the emulsion–diffusion–evaporation method with various solvents, stabilizer concentrations, and centrifugation speeds. SHRs were orally dosed with 30 mg/kg ALS or dose equivalent ALS-NP. Several parameters were assayed in plasma and/or urine at baseline and 24 h post-dose, while pharmacokinetic analysis included serial sampling. The optimum formulation was found with ethyl acetate, a 1.00% w/v didodecyldimethylammonium bromide concentration, and a 10,000 r/min (15,554 g) centrifugation speed. A 168% relative bioavailability was observed as a result of ALS-NP administration along with significant, as determined by Student’s t-test, increases in the maximum ALS plasma concentration (p = 0.0189) and the area under the plasma concentration–time curve from 0 to infinity (p = 0.0095). Conversely, a reduction was found in oral volume of distribution (p = 0.0009) and oral clearance (p = 0.0298). Blood urea nitrogen increased significantly after dosing in both groups (p < 0.0001 and p < 0.0001); however, no statistical difference was found between endpoint levels (p > 0.05) following one-way analysis of variance (ANOVA). Kidney injury molecule-1 increased following ALS dosing (p = 0.0486), while ALS-NP showed a decrease (p = 0.027) which was also significantly decreased compared to ALS-Final (p = 0.0008) when examined using two-way ANOVA. Urinary potassium excretion decreased significantly, as shown by two-way ANOVA, only in the ALS group (p = 0.0274) which was also significantly reduced compared to ALS-NP-Final (p = 0.016). Using the current formulation and at the dosage tested, ALS-NP showed a more favorable pharmacokinetic profile and positive kidney changes compared to ALS in regard to select outcomes. Thus, NP formulation may further improve ALS renoprotection in addition to increasing bioavailabilty.

No description available.

Phospholipids (PLs) are a component of cell membranes, biological fluids and tissues. These compounds are problematic for the bioanalytical chemist, especially when PLs are not the analytes of interest. PL interference with bioanalysis highly impacts reverse-phase chromatographic methods coupled with mass spectrometric detection. Phospholipids are strongly retained on hydrophobic columns, and can cause significant ionization suppression in the mass spectrometer, as they out-compete analyte molecules for ionization. Strategies for improving analyte detection in the presence of PLs are reviewed, including in-analysis modifications and sample preparation strategies. Removal of interfering PLs prior to analysis seems to be most effective at moderating the matrix effects from these endogenous cellular components, and has the potential to simplify chromatography and improve column lifetime. Products targeted at PL removal for sample pre-treatment, as well as products that combine multiple modes of sample preparation (i.e. Hybrid SPE), show significant promise in mediating the effect on PL interference in bioanalysis.

Drug makers have developed numerous techniques to influence treatment choices. Almost no information exists regarding the pharmaceutical industry's influence on rheumatologists and how these pressures could affect patient care. This phenomenological research, conducted within the framework of social exchange theory, explored the lived experiences of rheumatologists regarding their interactions with agents of the pharmaceutical industry. A researcher-designed interview protocol was used to gather feedback from 10 rheumatologists regarding how interactions with agents of the pharmaceutical industry made them feel. Using horizonalization, meaningful statements made by rheumatologists were condensed into specific themes and patterns, which provided a composite summary of their experiences with agents of the pharmaceutical industry. The experiences of rheumatologists' interactions with drug manufacturing personnel provided insights about medication access and patient financial assistance. Other key themes from rheumatologists' feedback included relationships, respectfulness, value appraisal and credibility, and authority and oversight. Rheumatologists' preferences and animosities towards the pharmaceutical industry revealed potential opportunities to both improve and curtail specific activities. Such opportunities would allow rheumatologists and the pharmaceutical industry to increase equitable exchanges and facilitate the appropriate application of medical care for the greater society.

Although tamoxifen has been successfully utilized in the treatment and prevention of estrogen-dependent breast cancer for decades, its use is limited by its low incidence of endometrial cancer. The carcinogenic effects of tamoxifen are complex and may involve a combination of estrogen receptor-mediated hormonal effects as well as the metabolic activation of tamoxifen to reactive electrophiles that are genotoxic. Moreover, a significant population of patients develop clinical resistance to tamoxifen, which leads to breast cancer recurrence and a decrease in patient survival. Therefore, the goal of the current study was to examine the interactions of major metabolites of tamoxifen with the human cytosolic sulfotransferases hSULT2A1, hSULT1E1, and hSULT1A1*1. Changes in the catalytic activity of hSULT2A1 by tamoxifen metabolites may inhibit the formation of the genotoxic Α-sulfooxy tamoxifen intermediate catalyzed by this enzyme. Moreover, tamoxifen metabolites might interfere in the inactivation of hydroxysteroids catalyzed by hSULT2A1 as a part of the variable responses to tamoxifen therapy. Endoxifen was the most potent inhibitor of the hSULT2A1, which suggests that this metabolite may inhibit the role of hSULT2A1 in the metabolic pathway for genotoxicity that is seen with tamoxifen.N-desmethyltamoxifen (N-desTAM) was a substrate for the hSULT2A1, and the product of this reaction, N-desmethyltamoxifen sulfamate (N-desTAM-S), displayed greater inhibition of the enzyme than its unconjugated precursor. Thus, endoxifen, N-desTAM, and N-desTAM-S might serve protective roles in some tissues as they may inhibit the role of hSULT2A1 in the genotoxicity of tamoxifen. Metabolites of tamoxifen were then examined as inhibitors of hSULT1E1 and hSULT1A1*1 due to the roles of these enzymes in the inactivation of estrogens. Each of the metabolites studied were weak inhibitors of hSULT1E1; thus, endoxifen is not likely to promote increased estrogen signaling in breast tissue when administered as an independent breast cancer therapeutic agent in ongoing clinical trials. However, 4-hydroxytamoxifen (4-OHTAM) was a very potent inhibitor of hSULT1A1*1 when examined with estradiol as substrate. This suggests the potential for 4-OHTAM to interfere in estrogen metabolism in tissues where hSULT1A1*1 is expressed and hSULT1E1 is not. This information will be useful when interpreting the clinical trials of endoxifen and will aid in the design of related molecules

Anemia of prematurity is characterized by a progressive decline in hemoglobin level during the first month of life. Unlike term newborns, preterm infants become anemic and often require red blood cell transfusions. Various factors contribute to the development of this anemia. These include short infant red blood cell (RBC) lifespan, decline in erythropoiesis rate after birth, and blood losses caused by repeated phlebotomies. The objectives of this work were to develop novel models to evaluate fetal and neonatal erythropoiesis, and to study in vivo adult and neonatal RBC survival in low birth weight preterm anemic infants. The model developed to evaluate fetal erythropoiesis was based on the in utero growth of the fetus over time. Neonatal erythropoiesis rate was estimated using a hemoglobin (Hb) mass-balance based method that has the advantage of not relying on specific structural pharmacodynamic model assumptions to describe the Hb production, but instead utilizes simple mass balance principles and nonparametric regression analysis to quantify the amount of Hb produced and the Hb production rate during the first month of life. To study RBC survival, two separate models, one describing the elimination of neonatal RBCs produced under non-steady state conditions, and the second describing the elimination of adult RBCs produced under steady state conditions were developed and applied to the RBC survival data obtained from low birth weight anemic preterm infants. The proposed mathematical models and its implementation provides a flexible framework to study both in utero non-steady state (non-SS) fetal erythropoiesis and neonatal erythropoiesis in newborn infants.

Nasal sprays used for the treatment of cold and allergy symptoms use same device and formulation in children and adults. Owing to the obvious differences in nasal cavity dimensions between adults and children, the performance of nasal sprays products in children needs to be critically assessed. In an effort to evaluate the deposition patterns of nasal sprays administered to children, a nasal cast based on MRI images obtained from a 12-year-old child’s nasal cavity was developed using 3D printing technology. Glycerin-water mixtures providing sprays with a range of plume angles (26° - 62°), along with three additional commercial nasal sprays, were investigated by actuating the device into the cast under controlled conditions. Following spray administration, the cast was disassembled and subjected to image analysis followed by quantification of formulation deposition in each section of the cast using both chemical and image analysis. The results showed that nasal sprays impacted entirely in the anterior region of the 12-year-old child’s nasal cavity and limited amount of spray entered the turbinate region – the effect site for most topical drugs. Additional experiments were conducted to measure the deposition patterns of nasal sprays in the presence of a mucus layer on the surface of the nasal cast. In an effort to make the nasal deposition studies more relevant to human nasal conditions, the mucus coated nasal cast was tilted in order to induce a physical movement of the mucus layer from the anterior region to the nasopharynx. The presence of mucus did not result in a significant increase in the turbinate region deposition but tilting of the mucus coated nasal cast resulted in 20% - 40% deposition in the turbinate region, improving the posterior region deposition of sprays. Even with the enhanced posterior movement nearly 60% of the spray formulations remained in the anterior region, a site with poor absorption characteristics. The computational fluid dynamic simulations evaluated the impact of multiple parameters including plume angle, droplet diameter and administration conditions on the deposition of nasal sprays in the 12-year-old child’s nasal cavity. The simulations showed significant anterior deposition for all plume angles (10° – 50°) and droplet diameters (30 µm – 400 µm) tested, similar to the observations from the in vitro experiments. An additional parameter, the direction of nasal spray actuation in the nasal cavity, was identified as a critical factor improving the turbinate region deposition of sprays in the 12-year-old child’s nasal cavity in spite of the narrow nasal valve region.

Diseases of the liver have a large impact on human health. Genetic disorders, metabolic disorders, alcoholism, cancer, or infections can all impair liver function. If serious enough, a liver transplant may be necessary, a major surgical procedure which requires life-long immune suppression and relies on the availability of donor livers. Gene therapy is being intensively studied as a potential method to treat many disorders, including disorders of the liver. While viral gene therapy has seen some success, possible side effects make it risky, so nonviral gene delivery vectors are being developed. Unfortunately, these nonviral vectors do not yet have the efficiency of the viral vectors. Nonviral gene delivery vectors face many chges in vivo. The vectors must protect DNA from nucleases while it moves through the bloodstream, they must avoid nonspecific uptake, they must be enter the correct cells, and must enter the nucleus before the DNA can be expressed. If any step of this process fails, there will be very little, if any, expression, and it may be impossible to determine what went wrong. One impediment to nonviral gene delivery research is the transition from in vitro studies to in vivo studies. The cancer derived cell lines most often used for in vitro transfections are rapidly dividing, which makes nuclear entry much easier than in the whole animal. While primary cells would be a more accurate model of the in vivo environment, the number of cells that can be obtained from tissues is small, and primary cells usually cannot be cultured for long. This limits the number of experiments that can be done with each preparation of cells. To overcome this, we have miniaturized transfection assays, including the transfection of mouse primary hepatocytes with luciferase in 384 well plates. Because fewer cells are needed, more experiments can be performed with each liver preparation. Another issue introduced by the differences between in vitro and in vivo research is circulatory stability. In vitro, large particles with strong positive charges are desired, because they sink down onto the cells and are attracted to the negatively charged cellular membranes. However, in vivo these particles will aggregate serum proteins and become lodged in narrow capillary beds in the lungs or other organs, often causing toxicity. While this behavior can usually be overcome through PEGylation, improving a particle's circulatory half-life will still improve its chances of finding the correct target. Scavenger receptors found on liver nonparenchymal cells are very efficient at removing negatively charged particles from the bloodstream. We have shown that dosing large amounts of PEGylated polyacridine DNA polyplex can saturate the scavenger receptors and improve circulatory half-life. We have also shown that large doses of PEGylated peptide with or without acridine groups can inhibit scavenger receptor uptake through the formation of peptide-protein nanoparticles. By inhibiting scavenger receptor uptake, DNA can be successfully hydrodynamically stimulated at times up to 12 hours post-delivery, demonstrating a longer circulatory half-life and suggesting a mechanism to explain how delayed hydrodynamic stimulation can achieve full level gene expression in the liver after the DNA has had time to circulate throughout the whole animal. Once a nonviral vector finds its target cell, it must still enter the cell through endocytosis and then escape the endosome before it becomes digested in the lysosome. Before the DNA cargo can be expressed, it must enter the nucleus. Nuclear entry in nondividing cells is a major barrier to efficient gene delivery. One method to over come this barrier is to avoid the need for nuclear entry altogether by delivering mRNA instead of DNA. mRNA can produce protein in the cytoplasm by finding a ribosome and initiating translation. However, it is even less stable in the bloodstream than DNA. We have produced an mRNA construct capable of high-level expression in the liver through hydrodynamic delivery. The PEGylated polyacridine peptides used to protect DNA were applied to mRNA and shown to enhance expression, allowing a 1μg dose of mRNA peptide polyplex to produce higher expression than an equal dose of DNA. The peptides were also shown to provide some protection against nuclease digestion in serum. This suggests that efficient, if transient, protein expression can be achieved through peptide protected mRNA delivery. However, DNA delivery is still desired for longer term expression, and the nuclear entry of DNA is still a problem. In an effort to help facilitate nuclear entry, the membrane disrupting enzyme phospholipase A2 was modified in several ways. The enzyme was conjugated with DNA binding peptides, nuclear localization peptides, and hepatocyte targeting oligosaccharides. Additionally, mutant forms of the enzyme were prepared in bacterial expression systems to achieve site-specific conjugation. Unfortunately, none of these efforts produced a useful tool for nuclear entry. The research presented in this thesis represents some progress toward the goal of nonviral gene delivery to the liver. Hopefully, some of this work will be useful in the development of new treatments and therapies to improve human health.

Immunotherapy has been widely investigated in cancer, infectious diseases and allergies for prevention or amelioration of disease progression. In the case of vaccines, the key cellular target in stimulating an effective and appropriate immune response is the professional antigen presenting cell or dendritic cell (DC). Cancer vaccines are primarily aimed at the activation of a tumor-specific cytotoxic T lymphocyte (CTL) response whilst vaccines to allergies are aimed at reducing IgE responses. Such vaccines normally involve the administration tumor-associated antigens (TAAs) for cancer, or antigens (Ags) derived from infectious microbes and allergens in the case of allergies. Ags, whether derived from tumor or allergen, can be combined with adjuvants, that include immunostimulatory molecules recognized by the pathogen associated receptors expressed by DCs and can trigger the activation/maturation of DCs. Co-delivery of an appropriate adjuvant with an Ag can stimulate DCs to subsequently promote a robust Ag-specific CTL response which may favor anti-tumor immunity. Cancer vaccines have been widely investigated in the clinics as a complementary therapy to surgery, radiation and chemotherapy. Activation of CTLs against tumor cells that express TAAs could lead to the complete eradication of a cancer and prevent its reoccurrence. In this study I developed microparticles using a polyanhydride polymer prepared from 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) that has shown inherent adjuvant properties. I prepared 50:50 CPTEG:CPH microparticles encapsulating a model tumor Ag, ovalbumin (OVA), and synthetic oligonucleotide containing an unmethylated CpG motif, CpG, as an adjuvant. CpG has shown significant potential as an adjuvant for TAA-based vaccines leading to significant anti-tumor immune activity. I have shown that mice vaccinated with OVA-encapsulated 50:50 CPTEG:CPH microparticles developed OVA-specific CTL responses. These mice showed enhanced survival compared to the control treatment groups when challenged with OVA expressing tumor cells . In a more novel in-situ cancer vaccine, TAAs from dying tumor cells (caused by certain chemotherapeutic drugs) can be used as the source of Ags delivered to DCs. The presence of an adjuvant with dying cancer cells can assist in appropriate maturation of DCs so as to promote the generation of an effective tumor/TAA-specific CTL response against released TAAs. In this work I developed a therapeutic in situ tumor vaccine encapsulating a chemotherapeutic drug and CpG. Doxorubicin (Dox) is a widely used chemotherapeutic drug that induces tumor cells to undergo an immunogenic form of apoptosis. Sustained release of Dox in solid tumors of mice can cause the release of a variety of TAAs which can be presented by DCs and, in the presence of CpG, stimulate a strong anti-tumor CTL response. I prepared formulations of poly(lactic-co-glycolic acid) (PLGA) particles loaded with Dox and CpG which demonstrated sustained release of their cargo. I show that among various formulations of Dox and CpG, co-delivery of Dox and CpG in the same PLGA particles in-vivo showed the highest reduction in tumor growth and longest survival when compared to treatment groups of PLGA particles delivering Dox and CpG either alone or in combination. PLGA particles have also been investigated as a prophylactic vaccine delivery system that generates a robust Ag-specific CTL response. This system has been employed for the development of vaccines against various infectious diseases and allergies. However, there has been conflicting opinions regarding the optimum size of PLGA particles required to stimulate an active CTL response. Thus, I developed different sizes of PLGA particles encapsulating OVA and CpG to study the relationship of particle size with the magnitude of OVA-specific CTL responses. I showed that the degree of particle uptake and activation of DCs increased with decreasing size of PLGA particles. I also showed that immunization of mice with 300 nm sized particles demonstrated a higher proportion of OVA-specific CTLs and increased the secretion of IgG2a antibody responses. I also evaluated the efficacy of these particles with a clinically relevant Ag, Dermatophagoides pteronyssinus-2 (Der p2). Mice vaccinated with different sizes of PLGA particles loaded with CpG and coated with Der p2 displayed different magnitudes and types of immune activation against Der p2. The small sized particles decreased the airway hyperresponsiveness associated with allergy-induced asthma. The presence of CpG in the PLGA particle vaccines also reduced the airway hyperresponsiveness. This thesis research has contributed to the identification and development of a delivery system for Dox in combination with CpG which gives sustained release of these molecules within tumors and show the longest survival in tumor bearing mice. This study also optimized the size of PLGA particles for the delivery of vaccine to produce a robust Ag specific immune response for development of vaccination against intracellular diseases, cancer and allergy.

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.