The aim of the work was to design, manufacture, and characterize targeted multi-component dry powder aerosols of (non-destructive) mucolytic agent (mannitol), antimicrobial drug (tobramycin or azithromycin), and lung surfactant mimic phospholipids (DPPC:DPPG=4:1 in molar ratio). The targeted dry powder for inhalation formulation for deep lung delivery with a built-in rationale of specifically interfering several disease factors of chronic infection diseases in deep lungs such as cystic fibrosis, pneumonia, chronic bronchitis, and etc. The dry powder aerosols consisting of selected chemical agents in one single formulation was generated by using spray drying from organic solution. The physicochemical properties of multi-component dry powder inhaler (DPI) formulation were characterized by a number of techniques. In addition, the in vitro aerosol dispersion performance, storage stability test, and in vitro drug release of selected spray-dried (SD) multi-component systems were conducted. The physicochemical study revealed that multi-component aerosol particles possessed essential particle properties suitable for deep lung delivery. In general, the multi-component particles (typically 0.5 to 2 µm) indicated that the designed SD aerosol particles could potentially penetrate deep lung regions (such as respiratory bronchiolar and alveolar regions) by sedimentation and diffusion, respectively. The essential particle properties including narrow size distribution, spherical particle and smooth surface morphologies, and low water content (or water vapor sorption) could potentially minimize interparticulate interactions. The study of in vitro aerosol dispersion performance showed that majority of SD multi-component aerosols exhibited low values (less than 5µm) of MMAD, high values (approximately above 30% up to 60.4%) of FPF, and high values (approximately above 90%) of ED, respectively. The storage stability study showed that azithromycin–incorporated multi-component aerosol particles stored at 11 and 40% RH with no partial crystallization were still suitable for deep lung delivery. Compared to SD pure azithromycin particles, the azithromycin-incorporated multi-component particles exhibited an enhanced initial release. The targeted microparticulate and nanoparticulate multi-component dry powder aerosol formulations with essential particle properties for deep lung pulmonary delivery were successfully produced by using spray drying from organic solution. The promising experimental data suggest the multi-component formulations could be further investigated in in vivo studies for the purpose of commercialization.

Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against cocaine by ~8- and ~2000-fold, respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans.

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation of force and the role of adenosine and phosphate in motor motion were demonstrated. It was concluded that phi29 genomic DNA is pushed to traverse the motor channel section by section with the aid of ATPase gp16, similar to the hexameric AAA+ family in the translocation of dsDNA. A new model of "Push through a One-way Valve" for the mechanism of viral DNA packaging motor was coined to describe the coordinated interaction among the hexameric packaging ATPase gp16 and the revolution mechanism of the dodecameric channel which serves as a control device to regulate the directional movement of dsDNA.

<p>There are today strong incentives for an increased understanding of material properties and manufacturing processes to facilitate the development of new technologies in the pharmaceutical industry. The purpose of this thesis was to suggest methods requiring a low sample amount for characterization of technical properties of powders.</p><p>Compression analysis was used to evaluate the formulation relevance of some compression equations. Using the mechanics of single granules to estimate powder functionality was part of this work. It was concluded that the formability of granular solids and the plasticity of single granules could be determined with compression analysis by using the Kawakita model for single components and binary mixtures of ductile granules.</p><p>Further on, the fragmentation propensity of solid particles could be estimated from compression analysis by using the Shapiro equation, enabling indicators of both the fragmentation and the deformation propensity of particles to be derived in one single compression test.</p><p>The interpretations of the compression parameters were only valid if the influence of particle rearrangement was negligible for the overall compression profile. An index indicating the extent of particle rearrangement was developed and a classification system of powders into groups dependent on the incidence of particle rearrangement was suggested as tools to enable rational interpretations of compression parameters.</p><p>The application of compression analysis was demonstrated by investigating the relevance of the mechanics of granular solids for their tableting abilities. The plasticity of single gran-ules was suggested to influence both the rate of compactibility and the mode of deformation, and consequently the maximal tablet strength. The degree of granule bed deformation was shown to be a potential in line process indicator to describe the tableting forming ability.</p><p>This thesis contributes to a scheme, suitable in formulation work and process control, to describe manufacturability of powders for an enhanced tablet formulation technology.</p>

This dissertation aimed at developing an inhibitor strategy to improve the oral bioavailability (Foral) and systemic exposure (AUC∞) of buprenorphine (BUP) as well as reduce the variability associated with them. Twenty-seven generally recognized as safe (GRAS) compounds or dietary substances were evaluated for their potential to inhibit the oxidative and conjugative metabolism of BUP, using pooled human intestinal and liver microsomes. In both the organs, oxidation appeared to be the major metabolic pathway with a 6 fold (intestine) and 4 fold (liver) higher intrinsic clearance than glucuronidation. Buprenorphine was predicted to show low and variable Foral, AUC∞, and a large total clearance. The biorelevant solubilities of 5 preferred inhibitors were incorporated in the final model. An inhibitor dosing strategy was identified to increase Foral and reduce the variability in oral BUP AUC∞. These results demonstrate the feasibility of the approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. This inhibitor strategy has promising applicability to a variety of drugs suffering from low and variable oral bioavailability due to extensive presystemic oxidative and conjugative metabolism.

Alzheimer’s disease is a devastating neurodegenerative disorder and the leading cause of dementia. The disease manifests via several pathologies including neuroinflammation, oxidative stress, metal ion dyshomeostasis, and cell death. To address the multifaceted nature of this disorder, the design of several diverse compounds, targeting many pathological effects, was generated. First, a series of compounds based on curcumin and diosgenin were synthesized following the bivalent design strategy. Two compounds were discovered to have neuroprotective ability, anti-oxidative function, and anti-Aß oligomerization (AßO) properties. A second set of molecules was also designed, wherein a hybrid compound strategy was utilized. Three hybrids were to shown to protect MC65 cells from Aß-induced toxicity and to have significant anti-oxidative activity. Mechanistic studies propose that protection is through disruption of interactions between AßOs and partner proteins. Furthermore, one hybrid was also shown to be able to pass the BBB. Lastly, studies of glyburide, an anti-diabetic medication, have shown an off-target anti-inflammatory effect specific for the NLRP3 inflammasome, which has been implicated in AD development. Therefore, a series of glyburide analogs were synthesized and characterized. One analog was able to successfully inhibit the NLRP3 inflammasome and reduce IL-1ß expression without affecting blood glucose. In vivo studies demonstrated an ability to prevent or ameliorate adverse inflammation-related outcomes in murine inflammatory models. Altogether, these investigations have yielded three novel series of compounds, all capable of modifying Alzheimer’s disease pathology. These results warrant future investigations into the development, optimization, and characterization of these analogs as potential treatments for Alzheimer’s disease.

Phenylephrine (PE) is the most commonly used over-the-counter nasal decongestant. The problem associated with phenylephrine is that it undergoes extensive first pass metabolism in the intestinal gut wall leading to its poor and variable oral bioavailability. This research project aims at developing strategies in order to increase the oral bioavailability of PE by co-administration of GRAS compounds. A HILIC assay method was developed to detect the parent drug, phenylephrine (PE) and its sulfate metabolite (PES).The enzyme kinetic studies were done with phenolic dietary or GRAS compounds using LS180 human intestinal cell model, recombinant SULT enzymes and human intestinal cytosol (HIC). From the screening studies done, one inhibitor was selected in order to study the mechanism of inhibition. In conclusion the studies done in vitro provided a basis in order to predict in vivo intrinsic clearance through the sulfation pathway.

A manutenção da qualidade do solo como um recurso natural vital à humanidade tem impulsionado a pesquisa sobre a dinâmica ambiental de resíduos de antibióticos veterinários no ambiente. Os processos de sorção, degradação e lixiviação da sulfadiazina (SDZ) foram avaliados em quatro solos com atributos físico-químicos distintos, conforme o uso de técnicas radiométricas e os protocolos da OECD para pesticidas. Os coeficientes de sorção (Kd) da SDZ variaram de baixo a muito alto (2,6 a 80 L kg-1). O seu baixo potencial de dessorção (< 24,2% do sorvido ou < 12,1% do aplicado) ratifica a existência de mecanismos específicos envolvidos na sorção da SDZ, afora as interações eletrostáticas e a partição hidrofóbica apontadas pelos estudos prévios. Em concentrações residuais próximas às encontradas em campo (??84 ?g kg-1), o potencial de sorção da SDZ foi consideravelmente maior do que em altas concentrações. A dissipação da SDZ foi rápida em todos os solos (t1/2 < 2,7 dias) e a formação de resíduos ligados foi a principal via de dissipação, correspondendo a > 78% do aplicado após 7 dias. A taxa média de mineralização foi < 3% do aplicado. A presença do antibiótico não impactou a biomassa do solo, indicando que em doses crônicas os efeitos sejam imperceptíveis em curto prazo. Apesar do potencial de mobilidade, a SDZ apresentou baixo potencial de lixiviação nos solos (< 0,11% do aplicado). Já a redistribuição da SDZ foi maior no perfil dos solos recém-aplicados do que nos envelhecidos e a grande maioria permaneceu nos primeiros 5 cm (> 95 % do aplicado). Nos solos envelhecidos, a sua mobilidade foi praticamente nula (0,06% do aplicado). Os resultados da pesquisa contribuem para elucidar a dinâmica de contaminantes orgânicos em solos intemperizados e ácidos de regiões tropicais, o que poderá auxiliar modelos de avaliações de risco, ações de mitigação, entre outros. / The soil quality as a vital resource for humanity has driven research on the occurrence, environmental fate and behavior of antibiotic residues in the environment. Sorption, leaching and degradation tests were performed on four soils with distinct physic-chemical attributes with 14C-sulfadiazine, following guidelines developed by OECD (USA) for laboratory studies. As the development of research on the subject has its origins in temperate countries, the soil and climatic conditions occurring in the tropics resulted in variations in the dynamic of sulfadiazine, mainly due to conditions of weathered and acidize.The results showed higher Kd (from 3, 5 to 80 L kg-1) for the residual concentration detected in monitoring studies, rapid dissipation (DT 50 between 2 and 3 days) and reduced leaching potential, in contrast with rates reported in the literature. The formation of bound residues was the major route of dissipation in all soils, reaching values between 78 % and 89 % of the total in just 7 days, whereas the mineralization did not exceed 1.5% throughout the experiment. The analysis of the variation of microbial-biomass carbon as an negative effect of antibiotic, present no difference with the content in the control, as reported by several studies that argue that the effects of chronic doses are imperceptible in short term. Although the knowledge about processes transformation of organic chemicals in soils is very divergent, laboratory tests contributed to a first elucidation about environmental of antibiotic residues, contributing to the improved performance of models to compose risk assessments and mitigation actions in tropical soils, for example.

A manutenção da qualidade do solo como um recurso natural vital à humanidade tem impulsionado a pesquisa sobre a dinâmica ambiental de resíduos de antibióticos veterinários no ambiente. Os processos de sorção, degradação e lixiviação da sulfadiazina (SDZ) foram avaliados em quatro solos com atributos físico-químicos distintos, conforme o uso de técnicas radiométricas e os protocolos da OECD para pesticidas. Os coeficientes de sorção (Kd) da SDZ variaram de baixo a muito alto (2,6 a 80 L kg-1). O seu baixo potencial de dessorção (< 24,2% do sorvido ou < 12,1% do aplicado) ratifica a existência de mecanismos específicos envolvidos na sorção da SDZ, afora as interações eletrostáticas e a partição hidrofóbica apontadas pelos estudos prévios. Em concentrações residuais próximas às encontradas em campo (??84 ?g kg-1), o potencial de sorção da SDZ foi consideravelmente maior do que em altas concentrações. A dissipação da SDZ foi rápida em todos os solos (t1/2 < 2,7 dias) e a formação de resíduos ligados foi a principal via de dissipação, correspondendo a > 78% do aplicado após 7 dias. A taxa média de mineralização foi < 3% do aplicado. A presença do antibiótico não impactou a biomassa do solo, indicando que em doses crônicas os efeitos sejam imperceptíveis em curto prazo. Apesar do potencial de mobilidade, a SDZ apresentou baixo potencial de lixiviação nos solos (< 0,11% do aplicado). Já a redistribuição da SDZ foi maior no perfil dos solos recém-aplicados do que nos envelhecidos e a grande maioria permaneceu nos primeiros 5 cm (> 95 % do aplicado). Nos solos envelhecidos, a sua mobilidade foi praticamente nula (0,06% do aplicado). Os resultados da pesquisa contribuem para elucidar a dinâmica de contaminantes orgânicos em solos intemperizados e ácidos de regiões tropicais, o que poderá auxiliar modelos de avaliações de risco, ações de mitigação, entre outros. / The soil quality as a vital resource for humanity has driven research on the occurrence, environmental fate and behavior of antibiotic residues in the environment. Sorption, leaching and degradation tests were performed on four soils with distinct physic-chemical attributes with 14C-sulfadiazine, following guidelines developed by OECD (USA) for laboratory studies. As the development of research on the subject has its origins in temperate countries, the soil and climatic conditions occurring in the tropics resulted in variations in the dynamic of sulfadiazine, mainly due to conditions of weathered and acidize.The results showed higher Kd (from 3, 5 to 80 L kg-1) for the residual concentration detected in monitoring studies, rapid dissipation (DT 50 between 2 and 3 days) and reduced leaching potential, in contrast with rates reported in the literature. The formation of bound residues was the major route of dissipation in all soils, reaching values between 78 % and 89 % of the total in just 7 days, whereas the mineralization did not exceed 1.5% throughout the experiment. The analysis of the variation of microbial-biomass carbon as an negative effect of antibiotic, present no difference with the content in the control, as reported by several studies that argue that the effects of chronic doses are imperceptible in short term. Although the knowledge about processes transformation of organic chemicals in soils is very divergent, laboratory tests contributed to a first elucidation about environmental of antibiotic residues, contributing to the improved performance of models to compose risk assessments and mitigation actions in tropical soils, for example.

Chemotherapy is the most common cancer treatment. Traditionally, platinum-based drugs are used in chemotherapy. More recently, researchers have focused on ruthenium based compounds as a substitute for the platinum compounds. Ruthenium-based compounds appear to be less toxic to healthy cells than traditional platinum-based compounds. In this study, 7 ruthenium-based compounds were tested on HT-29 (colon) and MCF-7 (breast) human cancer cell lines with the specific aim of determining whether or not any of the ruthenium-based compounds exhibited cytotoxic properties. In addition, levels of vascular endothelial growth factor (VEGF) production were tested in supernate from the cancer cells treated with various ruthenium-based compounds to determine whether or not the ruthenium-based compounds had an effect their VEGF production. Our results indicate that none of the ruthenium based compounds tested had a cytotoxic effect on the cancer cell lines; however, some of the compounds did exhibit inhibition of cell growth. Results further indicate an initial decrease in VEGF production in the cell lines treated with the ruthenium compounds but that this effect was compound-cell line specific.