Orofacial pain is associated with various pathologies such as headache, dental pain and ophthalmic pain. The trigeminal system innervates a large section of the head, including the nasal and oral cavities, the cornea and facial skin, and is responsible for the transmission of pain signals from the orofacial regions to the brain. These investigations were undertaken to study the effect of intranasal delivery of analgesics on orofacial pain using an operant testing method in mice. Doses of either lidocaine HCl or butorphanol tartrate were administered to mice, and the analgesic effectiveness was measured using a thermal operant behavior test involving a facial heat stimulus. Two parameters were measured in the operant assay: the number of licks and the duration of facial contact. Pain response was measured at two different temperatures: 37 ºC and 49 ºC. The magnitude of analgesic response was also compared between intranasal and intraperitoneal drug administration at 49 ºC. Mice showed a significant decrease in the number of licks and duration of facial contact for both treatment and control groups as the temperature was increased from 37 ºC to 49 ºC. A significant difference in the duration of facial contact was observed following either lidocaine or butorphanol by nasal administration. One group of animals receiving intranasal lidocaine did exhibit an increase in the duration of facial contact compared to the control. Two doses of butorphanol were tested and increases in the duration of facial contact were observed at both levels, but no significant difference was observed in the number of licks recorded. No convincing differences were observed in the mice behaviors for intranasal or intraperitoneal dosing of lidocaine or butorphanol. This suggests that nasal administration of these two analgesics at the doses tested did not provide superior pain relief compared to systemic delivery of the agents.

Calcineurin inhibitors (CNIs) are the cornerstone of immunosuppressive therapy following transplantation; however, immunosuppressive drug regimens consist of multiple medications with narrow therapeutic indices and substantial inter-patient variability. Despite intensive therapeutic monitoring, considerable time can elapse before the desired therapeutic concentration is achieved, which increases the risk of graft rejection or drug-related toxicities. In addition, maintaining therapeutic concentrations of CNIs does not prevent the development of toxicities, such as nephrotoxicity. Pharmacogenomics can greatly benefit solid organ transplant recipients through individualized drug therapy; tacrolimus is a widely used CNI and a substrate of cytochrome P450 3A (CYP3A) metabolizing enzymes and the efflux transporter p-glycoprotein (PGP) encoded by the ATP-binding cassette subfamily B member 1(ABCB1) gene. This dissertation describes work conducted in order to examine the effect of genetic variability in the above mentioned genes on the pharmacokinetics of tacrolimus and their contribution to a predisposition to adverse events or drug interactions in the transplant population. Our retrospective study investigating the effect of genetic polymorphisms on the risk of CNI-induced renal dysfunction identified a time-sensitive effect for the CYP3A5 expressor genotype, which predicts increased renal tubular CYP3A5 expression, in modifying the risk for renal dysfunction in liver transplant patients. This dissertation also examines the hypothesis that local tissue levels of tacrolimus and/or its major metabolite may be an improved indicator of nephrotoxicity, and through development of a robust and sensitive liquid chromatography/ mass spectrometry (LC/MS) analytical method to co-determine tacrolimus and its major metabolite, 13-O-demethyl tacrolimus (13-ODMT), in rat kidney tissues, we identified a possible relationship between tacrolimus dose and the extent of metabolite accumulation in the kidneys of rats receiving tacrolimus intra-peritoneally, paving the way for examining this relationship in kidney transplant recipients with calcineurin inhibitor-induced nephrotoxicity (CNIT). Overall, my research aims to identify biomarkers that might assist in early prediction of optimal tacrolimus starting and maintenance doses. Importantly, these studies provide the foundation for prospectively identifying patients at higher risk for adverse effects or drug interactions, with the ultimate goal of improving treatment outcome and quality of life for the transplant recipient receiving tacrolimus.

Non-viral gene therapy offers the potential to deliver nucleic acids producing therapeutic proteins to treat genetic diseases without the limitations observed with viral vectors. Before the therapeutic potential of non-viral gene delivery can be realized, several barriers to efficient gene delivery must be overcome. One delivery barrier of interest is the enhancement of endosomal escape to prevent vehicle and DNA degradation within the lysosome. However, to properly investigate the generation of analogues designed to enhance endosomal escape, one must also develop a gene delivery vector capable of addressing the deficiencies of traditional cationic polymer vectors. The overall scope of this thesis project is to address the deficiencies and concerns encountered with traditional non-viral vectors. This has led to the hypothesis involving the development of novel systems based on polyintercalation afforded by incorporation of multiple acridine moieties within a modular polyacridine peptide. Initial studies focused on proof of principle experiments in vitro to assess the polyacridine peptides viability as a gene delivery vector by tethering the fusogenic peptide melittin to polyacridine. Polyacridine-melittin allowed us to conduct SAR (Structure Activity Relationship) studies relating to the sequence and structure of the polyacridine peptides using biophysical measurements and luciferase expression levels in cell culture to dictate peptide design. This data led to the discovery of (Acr-Arg)4-Cys as the optimal in vitro polyacridine-peptide scaffold. (Acr-Arg)4-Cys was chosen as the lead polyacridine peptide for further development for in vivo mouse studies following PEGylation of the C-terminal cysteine. Polyplexes formulated with the (Acr-Arg)4-PEG peptide demonstrated the ability to produce efficient in vivo gene transfer after delayed hydrodynamic (HD) stimulation. Further in vivo polyacridine peptide SAR studies resulted in identification of (Acr-Lys)6-Cys as a PEGylated analogue that offered superior delivery capability by moderating stimulated gene expression comparable to HD pGl3 after a 1 hr delay between formulation dose and hydrodynamic stimulation. The properties of (Acr-Lys)6-Cys allowed the in vivo study of multi-component complexes composed of polyacridine PEG, N-glycan targeting ligand, and fusogenic peptide to overcome the delivery barriers, most notably endosomal escape and nuclear localization. Multi-component complexes were formulated with 25 μg of pGL3 and liver gene expression was evaluated by bioluminescence imaging (BLI). Multi-component complexes containing polyacridine-PEG, N-glycan targeting ligand, and/or the charge neutral fusogen PC-4 produced detectable luciferase expression. Alternatively, multi-component complexes formed with the cationic fusogen melittin or anionic fusogen JTS-1 were unable to produce a BLI response, suggesting that multi-component complexes are intolerant of excessive charge. Upon further optimization, polyacridine peptides hold great therapeutic potential due to their modular design and unique nucleic acid binding properties to produce delivery vehicles capable of enabling efficient gene transfer in vivo.

A human-derived, mammary epithelial cell culture model would allow drug transport in the mammary epithelium to be studied in greater detail while minimizing risks to mothers and nursing infants. MCF10A and primary human mammary epithelial cells (HMECs) were investigated for their utility as human, cell-based model systems for drug transport studies. Polarized monolayers are essential for transcellular flux studies of drug transporter function, and their formation was measured by transepithelial electrical resistance, immunofluorescence microscopy and vectoral flux studies. Both cell types failed to form adequately polarized monolayers despite various modifications to the cells or culture conditions. Transporter-mediated drug uptake and efflux in MCF10A cells was measured using flow cytometry, a technique which enables the measurement of intracellular drug concentrations. The fluorescent drug, mitoxantrone, was used to assess active efflux transport by the ABC transporters ABCG2 (BCRP) and ABCB1 (MDR1). After accounting for the inter-day variability with a linear mixed effects model, inhibitor effects on intracellular drug concentrations were evident. Specific inhibition of MDR1 using verapamil increased mitoxantrone accumulation as expected; however, BCRP-specific inhibition with fumitremorgin C decreased accumulation. Flow cytometry studies on mitoxantrone uptake suggested that it is a substrate for an unidentified active uptake transporter. PEPT1 and PEPT2 transporter functionality in MCF10A cells was evaluated using a fluorescently labeled dipeptide (A-K-AMCA). A-K-AMCA uptake showed an active component which was inhibited by a general metabolic inhibitor, the dipeptide Gly-Gln, and the peptidomimetic cefadroxil, indicating the involvement of a peptide transporter in A-K-AMCA uptake. Drug transporter expression levels in MCF10A cells and HMECs were measured using RT-PCR. Transporter expression levels, which were similar in the MCF10A cells and the HMECs, were compared with expression levels in lactating and non-lactating mammary epithelial cells. Low expression of BCRP, MDR1 and PEPT1 was seen in MCF10A cells, yet the effects of these transporters could still be observed in functional flow cytometry transport assays. Flow cytometry studies MCF10A cells may useful as a mammary drug transport model for transporters which have similar expression levels to lactating mammary epithelial cells.

The primary objective of the research was to investigate the pharmacodynamics of digoxin in dogs. Initially an assay specific for digoxin in the presence of its major metabolites, viz., digoxigenin, digoxigenin mono-digitoxoside, digoxigenin bis-digitoxoside and dihydrodigoxin was developed using HPLC-RIA. Methodology for non-invasive measurement of left ventricular ejection time (LVET) and other systolic time intervals (STI) in beagle dogs were developed. This involved surgery for exteriorization of the carotid artery in the dogs and subsequent measurements of LVET and STI after recovery. STI, heart rate (HR) and digoxin levels were monitored in normal beagle dogs administered 0.05 mg/kg or 0.025 mg/kg i.v., infused uniformly over a 5 min. period. The STI did not lend itself to pharmacodynamic modelling. The LVET, QS2 and P-R interval were found to be inversely, but linearly, related to the heart rate. Therefore, the bradycardic response to digoxin was extensively investigated in beagle dogs. Pharmacodynamic models evaluated for modelling the bradycardic response to digoxin were: the pharmacokinetic model with a direct linear link, the linear model, the physiologic-pharmacokinetic model with direct linear link and the effect compartment model. The physiologic pharmacokinetic model was simulated using SPICE2 which uses network thermodynamics to simulate biological systems. Criteria for the selection of appropriate models were established. Using the established criteria, the effect compartment model was demonstrated to be the best model. The implications and applications of pharmacodynamic models in general and specifically of the pharmacodynamic model for the bradycardic response to the digoxin are discussed.

The feasibility of producing inhalable microparticles of sugars and protein-sugar molecular mixtures using spray drying was investigated as an alternative to conventional micronization techniques. Four sugars; lactose (L), trehalose (T), sucrose (S) and mannitol (M) were spray dried using a commercial bench-top spray dryer and their physicochemical stability, with respect to particle size, moisture uptake and crystallinity changes, investigated after storage at 23%, 52%, 75% and 93% relative humidity (RH) and 25 °C for 30 days. Two crystalline size fractions (“coarse” = 125-212 μm and “fine” = 44-74 μm) of each sugar, were also characterized, as possible replacements for lactose as carriers for admixture with drugs in dry powder inhalers (DPIs). Sieve fractions of lactose, trehalose and mannitol failed to show significant moisture uptake at RHs ≤ 93% and 25 °C indicating their thermodynamic stability under most realistic storage conditions. While sucrose failed to show moisture uptake at ≤ 75% RH, it dissolved in sorbed moisture at 93% RH. Spray dried sugars were collected successfully in particle sizes suitable for inhalation. Spray dried lactose, trehalose and sucrose were amorphous and remained in this state after storage at 23% RH. At higher RHs, however, they recrystallized completely in ≤ 30 days. Spray dried mannitol was completely crystalline after collection from the spray dryer. It did not show moisture uptake or physical state changes at all RHs. A fine particle collection apparatus incorporating a nebulizer and a wire-in-tube type electrostatic precipitator (EP), built and characterized for particle collection efficiency, was used to review protein activity following spray drying with or without the four sugars as stabilizers. Bacterial (BAP) and bovine intestinal alkaline phosphatase (BIAP) were used as model proteins. Sugar free BAP solutions (apparent protein concentration ~120 μg/ml) lost 23% of initial enzyme specific activity after spray drying at ~63 °C and collection in the EP. Protection offered by the sugars to BAP during drying, was however statistically indistinguishable from the sugar-free protein solution (dried from the same protein concentration solution). When BIAP was dried from sugar free solutions (apparent protein concentration ~1 mg/ml), it lost 31% of its initial specific activity; activity which could be completely recovered when BIAP was co-dried with L, T or S (ANOVA, p < 0.05). However, M which crystallized during spray drying failed to protect the enzyme from this loss of activity. These results implied that the physical state of sugar (amorphous or crystalline) in the final dried product may be an important determinant for offering protection to proteins during spray drying and storage. Even so, multiple factors could potentially influence the selection of a sugar to form inhalable microparticles with a protein. These factors are described and discussed in this thesis, whether or not they appeared to be important with respect to the drying and stability of particular proteins selected for experimental investigation.

An original HPLC assay was developed for pyrimethamine (PYR) in plasma, RBCs, and buffer for the purpose of studying its plasma protein binding and RBC partitioning. Equilibrium dialysis (ED) was used to study protein binding. Isotonic phosphate buffer used in ED did not prevent small volume shifts. The pH of the plasma affected the protein binding of PYR although it was not significant for the comparison of binding at pH 7.4 vs. 8.0. PYR at 1000 ng/ml averaged 93.1% bound to plasma proteins. Binding to pure human albumin was 86.5% at lower levels of albumin and PYR (350 ng/ml). There was a significant difference (p<.03) in the plasma binding at two levels in the therapeutic range, with more free at higher levels. There was also concentration dependent binding at higher concentrations; the drug did not follow to law of mass action when binding increased at higher concentrations. This is a solubility phenomenon. Linear regression at the effect at albumin concentration on plasma binding yielded the equation percent free = -0.467(albumin g/L) + 23.5. The binding to pure albumin was only slightly above that predicted by this equation (83.1%). The first and second stoichiometric binding constants are K1 = 2.83 x 104 and K2 = 1.74 x 104 M-1 from nonlinear regression at data. There was no binding to normal levels at α1-acid glycoprotein. PYR is preferentially bound to plasma proteins in comparison to RBCss. The mean RBC/plasma ratio was 0.42 (10.2% CV, n=5). when plasma was removed and pH 7.4 isotonic buffer substituted, mean RBC/buffer ratio was 5.2 (11.8% CV, n=2). Mean percent bound to hemolysate was 42.5% (19% CV, n=10). Binding to hemoglobin did not account for all the RBC uptake. Therefore, PYR binds to RBC membranes.

The kinetics and mechanisms of macromclecular absorption from the airways of the rat lung were studied in vivo and in vitro, following identical solute administration methods. The isolated perfused rat lung (IPRL) was used as an in vitro model, and the disposition of 7.4 and 4.3 kDa fluorophore-labeled polyhydroxyethylaspartamide (F-PHEA) and other small and macromolecular reference solutes was investigated across doses and in the presence of a variety of biochemical inhibitors. Absorption profiles of F-PHEA, and the solute's lung distribution at different times after administration, were statistically identical in vivo and in vitro, showing that the IPRL possessed a viable mucociliary escalator and that macromolecular solute absorption rates from the IPRL were predictive of those in vivo. A kinetic model of the IPRL incorporating mucociliary clearance alongside active and passive absorption was developed and validated. This model was employed successfully to analyze each solute's absorption components from the rat lung following simultaneous, aeross-dose, nonlinear regression analysis of airway-to-perfusate absorption data. 7.4 and 4.3 kDa F-PHEA absorption from the pulmonary lung compartment occurred actively via the polyaspartamide transporter with values for Vmax,P and Km,p of 4.37/3.60 µg/min and 56.6/76.8 µg, respectively. In contrast, the magnitude of F-PHEA's passive absorption component, ka,p, was inversely related to its molecular weight, consistent with the absorption of a small proportion of each solute dose passing by restricted diffusive transport through tight junctions in the pulmonary epithelium. The active component of 7.4 kDa F-PHEA absorption, which enhanced airway-to-perfusate transfer at low doses, was significantly inhibited in the IPRL at lowered temperature (25 °C; 68.4 %) and in the presence of 1.0 mM 2,4-dinitrophenol (53.3 %), 100 µM ouabain (75.8 %), 30.0 µM monensin (66.0 %) and 30.0 µM noeodazole (68.4 %), This suggested that polyaspartamide-transporter was dependent on ATP-derived energy, and probably employed an intracellular vesicular transcytotic mechanism. The airway-to-perfusate transfer of 376 Da fluorescein and 4.4 kDa fluorophore-labeled dextran had no active (dose-dependent) absorption component. However, each of these solutes showed values for solute's molecular weight-dependent passive absorption and solute-independent mucociliary clearance, when compared to those for F-PHEA.

The goal of non-viral gene delivery is to treat illnesses stemming from gene deficiencies or overexpression without the use of viruses, which can cause severe immunogenic response. Many barriers face the delivery of DNA both in vivo and in vitro and must be overcome by the development of a complex multi-component carrier designed to address each challenge. While it is intuitive to develop a carrier in vitro, the requirements for in vivo gene delivery differ greatly, and often a non-viral carrier optimized in vitro will fail in the bloodstream in vivo due to high surface charge, which encourages blood protein binding, or dissociation of the polyplex leaving the DNA vulnerable to nucleases. It is evident that development of a non-viral gene delivery vector for use in vivo requires an easily amended platform to develop the carrier and a reproducible, calibrated assay to determine the expression of polyplexed DNA in vivo. Polyacridine peptides conjugated to polyethylene glycol (PEG) are a unique and characterizable set of carrier molecules that can be modified by peptide synthesis and various PEGylation strategies. Through the use of bioluminescence imaging and hydrodynamic stimulation (HS), a physical method that provides high levels of expression with small doses of DNA, it is possible to determine the state of polyplexed DNA in the bloodstream after various periods of circulation. The goal of this thesis was to overcome the first barrier of a systemically administered gene delivery system by developing a carrier molecule that reversibly binds to DNA and stabilizes it against metabolism in the bloodstream while avoiding undesirable biodistribution properties. The PEGylated polyacridine peptides presented herein were modified in response to each polyplex's in vivo performance based on pharmacokinetics, biodistribution, and gene expression by HS in mice after intravenous dosing. Modifications to the DNA-binding motif of the peptide were addressed initially along with various formulation strategies. Because PEG is installed to stealth polyplex surface properties, the effect of PEG attributes was also examined through optimization of PEG conjugation, size, and position. The results demonstrate the development of long circulating polyplexes that completely stabilize 1 µg of DNA in the bloodstream for five hours. This result provides a necessary prerequisite to allowing targeted accumulation of a polyplex at the site of action, which is the next step toward a fully-effective, systemically-administered non-viral gene delivery system.

The role of integrins in various aspects of tumor biology including tumor growth, angiogenesis, invasion and metastasis is well established. However, the integrin-specific involvement in these processes in non-small cell lung cancer (NSCLC) cells has not been thoroughly investigated. This study focuses on the role of integrins in cell motility and anoikis in NSCLC with an in vitro model system using the lung adenocarcinoma cell line A549 and its paclitaxel resistant derivative A549-T24. This research includes the following three parts: In order to demonstrate whether stimulation of the signaling mediated through specific integrins can differentially sensitize A549 and T24 cells to anchorage dependent apoptosis (anoikis), flow cytometry was used to detect the percentage of cells in certain apoptotic stages. T24 cells treated with an antibody to αvβ3 integrin showed more early apoptosis than A549 treated with the same anti-integrin antibody. To determine the involvement of integrins in mediating the motility of A549 and T24 cells, μ-slide chemotaxis chambers was used to detect chemotactic responses of migrating cells with or without treatment with anti-αvβ3 integrin. When treated with the antibody, both T24 and A549 cells attach less and move slower compared to untreated cells. In addition, the anti-integrin antibody caused a greater reduction of velocity of cell movement in T24 cells compared with A549 cells. To understand the survival signaling pathway activated by specific integrin extracellular matrix (ECM) interactions, a western blot analysis was performed to demonstrate that the PI3K pathway was involved in integrin-ECM interactions inactivating BAD for cell survival in T24 cells. Overall, specific integrin modulation in T24 cells resistant to paclitaxel may acquire some of their more-aggressive characteristics including rescuing from anoikis and accelerating cell movement.