Dissertation (Ph.D.)--Auburn University, / Abstract. Vita. Includes bibliographic references (p.175-179).

Proefschrift--Groningen. / With a summary in English.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O monitoramento da sensibilidade antifÃngica em espÃcies de Candida de origem veterinÃria à uma prÃtica recente e os mecanismos envolvidos ainda nÃo foram completamente elucidados. Considerando que o arsenal de drogas antifÃngicas à limitado e que o fenÃmeno de resistÃncia tem se tornado mais freqÃente, a compreensÃo deste fenÃmeno e a busca por alternativas terapÃuticas se fazem necessÃrias. Dessa forma, o presente trabalho teve como objetivo monitorar a sensibilidade in vitro de Candida spp. oriundas de animais, com Ãnfase na resistÃncia aos azÃlicos mediada por bombas de efluxo e na avaliaÃÃo da atividade do imidazÃlico levamisol sobre o crescimento destas leveduras. Para tanto, em um primeiro momento, foram avaliados 126 isolados de Candida, sendo 19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis, dos quais 22 foram isolados de rapinantes, 32 de periquitos do sertÃo, 56 de papagaios, 7 de araras canindà e 3 de um ouriÃo-cacheiro. Todas as cepas foram submetidas ao teste de microdiluiÃÃo em caldo, ante a anfotericina B, itraconazol, fluconazol, segundo metodologia padronizada pelo Clinical Laboratory Standards Institute (documento M27-A3). As concentraÃÃes inibitÃrias mÃnimas (CIMs) variaram de 0,03125 a 2 Âg/mL, 0,125 a 250 Âg/mL e de 0,03125 a 125 Âg/mL para anfotericina B, fluconazol e itraconazol, respectivamente. Dos 126 isolados avaliados por microdiluiÃÃo, 33 (26,2%) foram resistentes aos azÃlicos, sendo 7 (5,6%) resistentes somente a fluconazol, 1 (0.8%) resistente somente ao itraconazol e 24 (19%) resistentes a ambas as drogas. Em um segundo momento, todas estas cepas resistentes aos derivados azÃlicos, mais 20 C. albicans e 3 C. tropicalis resgatadas da nossa coleÃÃo de leveduras resistentes de origem veterinÃria, foram submetidas ao teste de inibiÃÃo de bomba de efluxo com prometazina, perfazendo um total de 56 cepas resistentes a azÃlicos. Dessa forma, as CIMs de fluconazol e itraconazol sofreram reduÃÃes significativas de 2 a 250 e de 16 a 4000 vezes, respectivamente. Investigou-se, ainda, a atividade antifÃngica do levamisol contra 12 C. albicans, 12 C. krusei, 12 C. parapsilosis e 12 C. tropicalis, sendo obtidas CIMs e concentraÃÃes fungicidas mÃnimas que variaram de 0,58 a 2,34 mg/mL e de 2,34 a 9,37 mg/mL, respectivamente. Paralelamente, foi demonstrado que o levamisol inibe a formaÃÃo do biofilme de C. albicans e C. tropicalis, bem como interfere na manutenÃÃo do biofilme maduro. Os dados apontam que a resistÃncia aos derivados azÃlicos Ã, pelo menos em parte, mediada por bombas de efluxo, bem como demonstram o potencial antifÃngico do imidazÃlico levamisol e sua capacidade de inibir o biofilme de cepas de Candida spp. oriundas de animais. / Monitoring of the antifungal susceptibility of Candida species from veterinary sources is a recent practice and the mechanisms involved in antifungal resistance have not been completely elucidated. Considering that the antifungal arsenal is limited and that antifungal resistance has become more frequent, the comprehension of this phenomenon and the pursuit for therapeutic alternatives are necessary. Thus, the present work aimed at monitoring the in vitro susceptibility of Candida spp. isolated from animals, with emphasis on efflux pump-mediated azole resistance and on the evaluation of the effect of the imidazole levamisole on the growth of these yeasts. For such, in a first approach, 126 Candida isolates (19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis), out of which 22 were recovered from raptors, 32 from cactus parakeets, 56 from Amazon parrots, 7 from blue-and-gold macaws and 3 from a Brazilian porcupine. All isolates were submitted to broth microdilution test against amphotericin B, itraconazole and fluconazole, according to the methodology recommended by the Clinical Laboratory Standards Institute (document M27-A3). The MICs ranged from 0.03125 to 2 Âg/mL, 0.125 to 250 Âg/mL and 0.03125 to 125 Âg/mL for amphotericin B, fluconazole and itraconazole, respectively. Out of 126 evaluated isolates, 33 (26.2%) were resistant to azoles, with 7 (5.6%) isolates resistant to fluconazole, 1 (0.8%) isolate resistant to itraconazole and 24 (19%) resistant to both drugs. In a second approach, all these azole resistant isolates, plus 20 C. albicans and 3 C. tropicalis that were recovered from our collection of resistant yeasts from veterinary sources, were submitted to the efflux pump inhibition assay with promethazine, with a total of 56 azole resistant isolates. Thus, MICs for fluconazole and itraconazole significantly reduced from 2 to 250 times for fluconazole and from 16 to 4000 times for itraconazole. The antifungal activity of levamisole against 12 C. albicans, 12 C. krusei, 12 C. parapsilosis and 12 C. tropicalis, was also evaluated, and MICs and minimum fungicidal concentrations varying from 0.58 to 2.34 mg/mL and from 2.34 to 9.37 mg/mL were obtained, respectively. Parallelly, it was demonstrated that levamisole significantly inhibits biofilm formation and interferes with the maintenance of mature biofilms. These data show that azole resistance is partially mediated by efflux-pumps and demonstrate the antifungal potential of the imidazole levamisole and its capacity of inhibiting the biofilm of strains of Candida spp. from animals.

<p>Opioid addiction has become a global epidemic and a national health crisis in recent years. In 2016, approximately 64,000 Americans under 50 years old were killed because of opioid overdoses. The aim of developing an abuse deterrent opioid is to render any form of manipulation that encourages abuse challenging and therefore, non-profitable. With this goal, the Food and Drug Administration (FDA) is extensively supporting research into the development of abuse deterrent technologies and prioritizing their production as a public health necessity. Abuse deterrent approaches include but are not limited to the following: (1) using a physical barrier (e.g., Polyethylene oxide PEO) that basically limit the release of the drugs in the blood or the digestive tract and prevent mechanical alteration of the drugs by crushing, grating, grinding, chewing etc, (2) using chemical barriers that employ gelling agents that prevent the aqueous or organic extraction of the drugs, and (3) combining the drug with an antagonist that blocks the post-abuse euphoria.</p> <p>PEO is a popular polymer used as a matrix in these complex opioid products. The polymer is responsible for the abuse deterrent properties as well as extended release behavior of opioid drugs. PEO hinders the extraction of Opioid drugs from Abuse Deterrent Formulations (ADF), makes it challenging to be injected, and resists mechanical stress and pulverization when crushed. PEO can be subjected to thermal processing such as thermal curing, compression molding, melt extrusion, and injection molding owing to its thermoplasticity.</p> <p>Assessment of the impact of using various manufacturing processes to develop ADFs and the effect of using various grades of this polymer is essential to improve upon the next generation of ADFs. There are three main categories of premarket studies: Category 1 – laboratory based (in-vitro manipulations and drug extractions), category 2 – pharmacokinetic and category 3 –clinical. These studies are required by the FDA to demonstrate that a given formulation exhibit abuse deterrent properties before a drug product is released to the market. In vitro laboratory based manipulation and extraction studies which are used to assess AD properties of these products are challenging, but essential for product development and generic abuse deterrent product approvals. It is important to realize that there is a great correlation between the laboratory based in vitro manipulation and extraction studies and the expectations of potential abuse and misuse of opioid drugs. The ability of these studies to mimic the manipulation techniques applied by abusers to defeat the abuse deterrent properties of a given formulation optimizes predictions on post-market abuse and misuse potential of ADFs. These studies should also correlate well with <i>in-vivo</i> studies since there is a direct correlation with the concentration (mg/mL in water) and the “high” obtained by an abuser. </p> <p>This research aimed to conduct laboratory based in vitro manipulation and extraction studies to investigate failure modes of PEO-based prescription opioids and Promethazine Hydrochloride PMZ HCl tablets. It highlighted the formulation components and the manufacturing parameters that might affect the dose dumping of Active Pharmaceutical Ingredients (APIs). Furthermore, this research identified model compounds that can be used as surrogates for Oxycodone and the best experimental setup that can be used to conduct smoking simulation experiments. Moreover, it provided an overview of the societal impacts of the opioid crisis in the state of Indiana.</p> <p>Investigations of the failure modes of the PEO-based prescription opioids and PMZ HCL tablets showed that physical manipulation techniques via chopping or grinding are much more effective in the destruction of the PEO matrix than thermal manipulation via the application of heat thus promoting the fast release. The factor with the most significant effect on the failure modes of PMZ HCL tablets was the application of physical manipulation, while the one with the lowest impact was the polymer grade. Moreover, producing PEO-based matrix tablets via Direct Compression DC significantly affected dose dumping behavior of the API from the drug products. The production of the PEO-based matrix tablets via DC was found to be favored over the usage of the melt extrusion method and molding techniques. It was clear that DC kept the integrity of the polymer, allowed for slow and controlled release fashion of the API, and rendered the extraction process relatively hard compared to the Hot Melt Extrusion HME and Molding techniques.</p> <p>Furthermore, the release profile of the investigated PMZ HCL products consisted of various phases of polymer swelling and API release. Thermal manipulations via the application of heat were found to accelerate the dose dumping behavior (90% release) of the APIs from the compressed, extruded, and molded PEO-based matrix formulations similarly. On the other hand, heating was much more effective in the extraction of APIs than chopping or grinding thus promoting the ability to<b> </b>draw a solution containing the API into a syringe for injection relatively easy and facilitate higher % API recovery.</p> <p>Among the formulation components that might have an impact on the AD properties of the PEO-based drug products are; the choice of the antioxidant, the use of complexing agents, chelating agents, and plasticizers. On the other hand, manufacturing process variables that might have a critical impact on AD properties of the PEO-based drug products include but are not limited to; processing temperature compared to the melting point of the polymer and time of exposure</p> <p>PMZ HCl was used as a model drug for Oxycodone in dissolution and extractability studies, while Caffeine and L-Nicotine were used as model drugs in smoking simulation experiments. The combination of the propane torch and Kugelrohr apparatus mimic the real-world scenario for smoking Opioids; however, this experimental setup caused thermal degradation rather than vaporization of some model drugs.</p> <p>According to the National Center for Health Statistics; a statistically significant increase in drug overdose death rates was reported in 2016 in the state of Indiana among other states. The number of deaths related to opioid pain relievers increased by 3732 folds in 2017 compared to the number of deaths in 2014. Moreover, Males were more affected by the opioid crisis than females. On the other hand, the age group 25-44 years, and white people were the most affected by the opioid crisis in Indiana. </p>

The adsorption isotherm of two weak bases, Promethazine hydrochloride and Propranolol hydrochloride, were determined with isocratic reversed-phase liquid chromatography, with a 60 w% methanol in 20 mM sodium acetate buffer pH 4 as the mobile phase, and calculated by the elution by characteristic points method. The data obtained from the method were then fitted into the Langmuir isotherm and the electrostatically modified Langmuir. Propranolol fitted reasonably good into the models while Promethazine was not as good. When Promethazine and Propranolol were together in the same sample, there was indication of competition of the adsorption sites. For comparing retention and peak shape between a C18 column and a mixed mode column, Waters XBridge C18 and Thermo Scientific Acclaim WCX-1, were tested in gradient elution with 11.32 mM sodium acetate buffer and 10–70 % methanol. The mixed-mode column gave significantly better peak shapes, while the retention time were longer compared to the C18 column. / Adsorptions-isotermerna för två svaga baser, Prometazin hydroklorid och Propranolol hydroklorid, bestämdes med isokratisk omvänd-fas vätskekromatografi, med w% 60 metanol i en 20 mM natriumacetatbuffert pH 4 som mobil fas, och beräknad med metoden elution by characteristic points.  Från metoden erhållna data passades till Langmuir isotherm och den elektrostatiskt modifierade Langmuir. Propanolen passade ganska bra till de olika isotermerna, medan Prometazin var något sämre passad. När Prometazin och Propranolol var tillsammans i samma prov, fanns det indikationer på konkurrens om adsorptionsställen. För jämförelse av topparnas form och retentionstid mellan en C18-kolonn och en mixed-mode-kolonn, användes Waters XBridge C18 och Thermo Scientific Acclaim WCX-1, som testades i gradient eluering med 11, 32 mM natriumacetatbuffert och 10–70 % metanol. Mixed-mode-kolonnen gav åtskilligt bättre toppar, medan retentionstiden var längre jämfört med C18-kolonnen.

The aim of this thesis has been design, construction and application of a flow-through nephelometric detection cell based on photodiode as a sensor and a laser source. The developed device has been tested and its function has been proven on the determination of sulphate by flow injection analysis with nephelometric detection after precipitation by barium chloride. The results achieved were comparable with those obtained by spectrometric detection. Furthermore, the device has been successfully applied to the determination of promethazine based on formation of its insoluble ion associate with bromophenol blue. Powered by TCPDF (www.tcpdf.org)