<p> The pediatric mental health burden in the United States (US) is substantial, with more than 4 million children meeting diagnostic criteria for a mental health disorder. As of 2014, this number represented 20% of US children and adolescents. In 2010, mental health disorders are estimated to cost children and their families $247 billion dollars annually and severely impact quality of life for children and their families. From 2007 to 2010, inpatient admissions for mental health disorders in pediatric patients increased 24% and mood disorder admissions in pediatric patients increased 80% from 1997&ndash;2010. An estimated $11.6 billion was spent on pediatric mental health hospitalizations from 2006 through 2011, with public sources such as Medicaid and Medicare responsible for approximately 50% of the payments, leaving 50% to private payers. This economic and clinical concern has led pediatric medical associations and health quality agencies to increase support and funding for pediatric mental health research and treatment. </p><p> Medication therapy is a common intervention in mental health treatment and atypical antipsychotics are increasing in utilization, often becoming first-line therapy. Despite available data describing the need to treat pediatric mental health conditions, the available evidence for clinical effectiveness and economic impact of atypical antipsychotics (AAPs) has many shortfalls. Most available research is derived from patients utilizing publicly-funded medical care, such as Medicaid or Medicare resources, with little data available about patients with privately-funded care. To help address this gap in the literature, we used a large, privately-insured, US population for our analysis. We examined if the increased trend in AAP utilization from previous research is also present in this pediatric population. Considering the payer perspective, we evaluated the cost of AAP medication therapy based on most recent utilization. </p><p> Available studies lack information about the direct costs of pediatric mental health treatment and efficacy of psychiatric medications in the pediatric population. Most efficacy studies are based on clinical trials necessary for pediatric indication approval from regulatory agencies such as the Food and Drug Administration (FDA). Many of the AAP medications do not have pediatric clinical trial evidence available and are frequently utilized without pediatric indications. The available data suggests that off-label prescribing is not an uncommon practice in the pediatric patient population. </p><p> Approximately half of atypical antipsychotics do not have pediatric indications but are increasingly used, particularly in treating behavior disorders, due to such factors as improved patient compliance and improved side effect profiles. Limited formal studies examining atypical antipsychotic use compared to other agents in the class have been conducted. Studies with direct comparisons have yet to be conducted in the pediatric population with mental health disorders. </p><p> The manuscripts that comprise this dissertation aim to provide new insights into available trend and utilization patterns of atypical antipsychotic medication use in children. This research characterized the prevalence of atypical antipsychotic use in pediatric patient with mental health conditions in a large, privately insured US population, evaluating the diagnoses associated with treatment and estimate the cost of AAP medication therapy in this population. This research determined if the trends observed in publicly-insured children persist in the privately-insured, pediatric patient. The analysis evaluated annual trends in prevalent use of atypical antipsychotic medication over 6-year period in this pediatric population and evaluated the appropriate use of AAPs for mental health diagnoses. Lastly, an evaluation determined if specific antipsychotic therapy delayed time to readmission among privately-insured children following a psychiatric hospital admission. The results of this dissertation will provide new insights regarding the trends and direct medication costs of atypical antipsychotic agents when utilized in pediatric patients with mental health disorders. </p><p> <u>Manuscript 1:</u> This analysis focused on characterizing the most recent (2015) AAP use in the pediatric population, using a large, US population of privately- insured children. The study evaluated if the prevalence data observed among publicly insured children persists. Characterization of the prescribing trends for atypical antipsychotics and the medication costs of the use in this population were examined. Patterns of use across demographics and associated mental health diagnoses were characterized by the class of medication. This study focused on the prevalent use of AAPs in pediatric patients, evaluated the mental health diagnoses associated with AAPs and the direct cost burden of medication therapy associated with this use of AAP in the pediatric population to the private payer. </p><p> <u>Manuscript 2:</u> This research evaluated the trends in the prescribing of atypical antipsychotic medications from 2010 through 2015 in this privately-insured pediatric population. The trends of AAP use in the pediatric population over six years were examined. The associated mental health diagnoses corresponding with AAP prescribing were described to examine the off-label diagnoses treatment prevalence in this population. (Abstract shortened by ProQuest.) </p><p>

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.

<p> Exemestane (EXE) is an endocrine therapy used to combat postmenopausal breast cancer. Several studies have reported substantial differences in clinical outcomes between EXE-treated patients, as well as inexplicable variability in serum concentrations of EXE and its major metabolite, 17&beta;-dihydroexemestane (17&beta;-DHE). For many pharmaceuticals, drug response is influenced by patient-specific genetic factors related to xenobiotic metabolism. Thus, it is possible that allelic variation in genes involved in EXE metabolism contributes to inter-individual differences in patient outcomes, possibly through differential EXE clearance or varied rates of metabolite formation. Historically, knowledge of phase I EXE metabolism has been extremely limited with significant ambiguity surrounding the identity of the specific hepatic enzymes involved. To address this gap in knowledge, <i>in vitro</i> studies were undertaken to better characterize hepatic phase I EXE metabolism and in particular, to assess the impact of genetic variation in drug-metabolizing enzymes on the production of EXE metabolites with inhibitory activity against aromatase. </p><p> The first part of this dissertation describes the identification of phase I EXE metabolites and details their capacity to suppress estrogen synthesis. Four metabolites, including 17&beta;-DHE, were detected in incubations of EXE with pooled human liver microsomes. 17&beta;-DHE and a novel metabolite, 17&alpha;-DHE, were formed in incubations of EXE with pooled human liver cytosol. The identities of phase I EXE metabolites were confirmed through comparison to reference compounds using UPLC/MS/MS. Anti-aromatase activity assays (AAA) revealed that 17&beta;-DHE is the only phase I EXE metabolite formed by human liver fractions that appreciably impedes estrogen formation. AAA also suggest that the inhibitory potency of EXE is unaffected by common nonsynonymous polymorphisms in aromatase. The latter half of this dissertation identifies hepatic enzymes that are likely to participate in phase I EXE metabolism. <i>In vitro </i> assays show that CBR1, AKR1Cs, and multiple hepatic CYP450s predominantly reduce EXE to 17&beta;-DHE with minor formation of additional inactive metabolites. Kinetic assays comparing 17&beta;-DHE formation by each wildtype enzyme to its common variant allozymes show that specific genotypes are associated with altered EXE metabolism <i>in vitro</i>. However, additional investigations are needed to determine the prognostic value of these associations for predicting <i> in vivo</i> EXE response.</p><p>

<p> Pancreatic cancer is the one of the leading causes of cancer-related deaths in the United States and is characterized with low survival rate and high drug resistance. Because of the redundant and highly mutated signaling pathways in pancreatic cancer, numerous combinational therapies have been sought. Currently the selection of drug combinations is largely empirical and methods of evaluating and optimizing drug combinations have not been standardized. An important reason for this is the lack of comprehensive characterization of drug mechanisms of action and causes for drug resistance. </p><p> The purposes of this dissertation are: first, to set up a paradigm for evaluating drug combinations mathematically and translating the evaluation methods from <i>in vitro</i> to <i>in vivo</i> preclinical systems; second, to serve as an example for characterizing the biological signaling pathways and drug pharmacology comprehensively with systems modeling approaches, supported with &ldquo;big data&rdquo; from advanced techniques such as proteomic analysis; and third, using such systems models, further selecting and optimizing drug combinations to reverse drug resistance and enhance efficacy. </p><p> The two drugs selected are gemcitabine, a major component in the therapies for pancreatic cancer treatment, and birinapant, an antagonist of inhibitor of apoptosis proteins (IAP). In Chapter 1, the efficacy of this drug combination was evaluated in PANC-1 cells. A basic pharmacodynamic (PD) model was developed to characterize the temporal changes in the numbers of attached and floating cells after treatments, and synergistic effects were observed for both proliferation inhibition and death induction. Measurements of cell cycle distributions and apoptosis were then obtained and a mechanism-based PD model was developed to reveal more details and capture the major features of the beneficial interactions. From the mechanism-based PD model, different exposure schedules were tested and an optimal one to achieve maximal efficacy was proposed. </p><p> Assumptions were made in developing the mechanism-based PD model in Chapter 1. In Chapter 2, a proteomic approach was utilized for a comprehensive, unbiased study of proteins perturbed by gemcitabine and birinapant to test previous hypotheses. The mechanisms of action for both drugs were characterized more intensively, and additional details were incorporated into the interaction knowledge described previously. Based on the proteomics data, reasons for gemcitabine resistance were discussed, and regulators of DNA damage responses involving DNA repair, anti-apoptosis, and pro-migration and invasion proteins were proposed as promising candidates for therapeutic targeting. </p><p> With the rich quantitative proteomics data, a network modeling approach was attempted in Chapter 3. Quantitative relationships were developed for selected signaling pathways of cell cycle regulation, DNA damage responses, DNA repair, apoptosis, NF-&kappa;B, and MAPK-p38, which were then linked to describe the cell cycle progression and apoptosis, and finally to changes in cell numbers. Based on the developed network model, simulations were made under different conditions and compared with observations, serving as a validation process. The impact of p53 mutation and p53 silencing on the efficacy of gemcitabine was tested with this model. Sobol Sensitivity Analysis was applied to select promising targets to be combined with gemcitabine. In addition, the efficacy of curcumin combined with gemcitabine was evaluated based on the model simulation. </p><p> With extensive evaluation and comprehensive characterization of the mechanisms of this drug combination in cell culture, efforts were continued to investigate the effects of the combination in a mice xenograft model. In Chapter 4, pharmacokinetic information for gemcitabine and birinapant was gathered from the literature and full physiologically-based pharmacokinetic models (PBPK) were developed to characterize drug distribution in the body and into the pancreatic tumor. The tumor concentrations then were used to drive inhibition in tumor growth and a semi-mechanistic PBPK/PD model was developed to evaluate the efficacy of the drug combination <i>in vivo.</i> Their joint effects were revealed as merely additive. The network model developed in Chapter 3 was introduced to bridge the PBPK and PD models, and reasons for the discrepancies <i> in vitro</i> and <i>in vivo</i> were explored. Model predictions showed that simultaneous dosing was preferable to sequential dosing <i> in vivo</i> with stronger suppression of the DNA repair signaling. </p><p> In summary, this dissertation proposed a paradigm for evaluating drug combinations quantitatively in preclinical systems of cell lines and xenograft models. Comprehensive characterization of drug mechanisms of action and biological systems through network modeling can facilitate the selection and optimization of candidates for anti-cancer combination therapy. The bridging of knowledge in different scales with mathematical models in different complexity helps to minimize the gap of translating from <i>in vitro</i> to <i> in vivo</i> or even from preclinical to clinical research.</p><p>

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.

<p> Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (Z_HER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of Z_HER2:342 has limited its pharmaceutical application. Human serum albumin (HSA), as the main protein in plasma, has been commonly used to extend the small peptides serum half-life. Its high solubility, stability and excellent ability to carry multiple ligands in blood stream make it a good candidate for drug delivery.</p><p> Two HSA and Z_HER2:342 fusion proteins, one with a single Z_HER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and the other with two tandem copies of Z_HER2:342 (rHSA-(ZHER2)₂), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)₂, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)₂ treatment has been observed. However, the inhibition effect on HER2-overexpressing cells is cell linedependent. Fusion protein rHSA-(ZHER2)₂ showed preferred accumulation in tumor tissues in xenograft model.</p><p> HSA fusion proteins are easy and economical to express, purify, and formulate. Two formulation strategies have been explored, one is to complex the fusion protein with FA modified chemo drugs, and the other is to make them into nanoparticles. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified fluorescein isothiocyanate (FITC) could be efficiently taken up by cells. FA-Taxol/albumin formulation showed its advantages over Taxol/albumin treatments on <i>in vitro</i> cell growth inhibition. Nanoparticles containing rHSA-ZHER2 produced by desolvation method displayed optimal size distribution, satisfactory stability and preferred binding/uptake on HER2-overexpressing cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.</p><p>

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.

Lipid-based formulations (LBFs) can be an effective formulation strategy to improve the bioavailability of lipophilic drugs. However, conventional single-compartment in vitro lipolysis assays often underestimate LBF performance in vivodue to the absence of absorption sink. Recently, a two-compartment setup separated by an absorptive membrane provided good in vivo in vitro relationship (IVIVR). The goal of this study was therefore to investigate the use of the artificial membrane “LiDo” in two-compartment assays with regards to digestive enzymes, donor volume and different pH-control methods. The in vivo relevance of the assay was investigated using a single LBF and carvedilol, varying drug-to-formulation ratio and physical state of carvedilol. These formulations were already examined in vivo in a previous study. Digestion was performed using three different enzymes: porcine pancreatin, porcine lipase and Novozyme 435. Due to the incompatibility of pH electrodes with LiDo membrane, high strength lipolysis buffer was tested as alternative to the normal lipolysis buffer. The latter cannot counteract pH decreases caused by lipid digestion and requires dynamic titration.  In vitro assays were conducted in full-size as well as small-size setup. The barrier integrity was evaluated using Lucifer Yellow as a membrane marker. It was found that use of porcine pancreatin best reflected in vivo drug plasma exposure. In contrast, no relationship was observed between in vitro data obtained from lipolysis assays with lipase and Novozymes 435 and in vivo data. Results from assays with lipase may not reliably compare to assays with Novozyme 435 where the latter showed tendency to aggregate in low donor volume, resulting in poor lipid digestion. The outcomes from assays performed in the small-scale setup did not produce good IVIVR either. This outcome could be caused by insufficient agitation in the donor compartment, leading to poor dispersion of LBFs. However, “LiDo'' membrane retained its integrity during lipolysis by all three enzymes. Hence, we can demonstrate further evidence on the utility of LiDo membrane for in vitro lipolysis-permeation.

No description available.