Thesis (M.S.)--University of Toledo, 2010. / Typescript. "Submitted to the Graduate Faculty as partial fulfillment of the requirements of the Master of Science degree in Pharmaceutical Sciences with Industrial Pharmacy Option." "A thesis entitled"--at head of title. Title from title page of PDF document. Bibliography: p. 87-102.

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Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-147).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 144-154).

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-15T11:48:36Z No. of bitstreams: 2 Dissertação - Romayne Paniago França - 2016.pdf: 1501835 bytes, checksum: 413c74416f9a069ed5fcaa317d37d106 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-15T11:49:02Z (GMT) No. of bitstreams: 2 Dissertação - Romayne Paniago França - 2016.pdf: 1501835 bytes, checksum: 413c74416f9a069ed5fcaa317d37d106 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-15T11:49:02Z (GMT). No. of bitstreams: 2 Dissertação - Romayne Paniago França - 2016.pdf: 1501835 bytes, checksum: 413c74416f9a069ed5fcaa317d37d106 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-07 / Polymorphism is the capability of an element or compound in crystallizing itself into more than one distinct crystal species, it affects not only the speed in which a substance acts in the organism, but also its chemical stability along time. There is a great problem that has been of considerable importance in the pharmaceutical industry, it is the development of a new drug. The realization of this research is justified by the fact that, according to the literature, the crystalline form or presence of polymorphism alters the solubility and the physical-chemical properties of drugs, being able to cause deviations of quality by the bioavailability of drugs, influencing the performance of the medicines and the bioequivalence. The objective of the research is to synthesize and characterize crystalline forms of the drugs Glicosamine and Tolbutamide, therefore having the polymorphism and the crystalline structure of these drugs analyzed. The experiments were performed in the Laboratory of Development of New Drugs of UFG/Regional Jataí, where the essays of crystallization for the obtainment of cocrystals of the drugs Glicosamine and Tolbutamide were performed. I was chosen the Tolbutamide, drug used as oral hypoglycemic agent and the Glycosamine used in the treatment of arthrosis, articular rheumatism and regeneration of damaged cartilage. The choice of these had as basis the availability of these substances in the Laboratory of Development of New Drugs of UFG/Regional Jataí, and the fact of not having been, yet, totally explored by the literature. Essays of crystallization for obtainment of cocrystals in different solvents were performed and the structures were characterized by monocrystal X-ray diffraction (MXRD) and Infra-red. The raw material Glicosamine and Tolbutamide were acquired at Pharma/China Zhejiang Golden- Shell and Sigma-Aldrich Co., USA, respectively. The behavior of solubility of Glicosamine and Tolbutamide were observed experimentally in the solvents: deionized water, methanol, ethanol, isopropyl alcohol, butyl alcohol, acetone, dimethyl formamide, ethyl acetate, ethyl ether, acetonitrile, chloroform, dichloromethane, tetrahydrofuran, hexane and n-heptane. It has been observed that Glicosamine is slightly soluble in the solvents deionized water, isopropyl alcohol, butyl alcohol, dimethyl formamide, and chloroform at room temperature, after the heating of the solutions, Glicosamine was only soluble in deionized water. However, it is insoluble in the solvents: methanol, ethanol, acetone, ethyl acetate, ethyl ether, acetonitrile, dichloromethane, tetrahydrofuran, hexane and n-heptane. In the results of the essays of crystallization for obtainment of cocrystals of Glicosamine, from the solution of Glicosamine and deionized water with salicylic acid and other solvents there was the formation of 2-aminobenzoic acid. It was observed that the Tolbutamide is soluble, at room temperature, in methanol, ethanol, isopropyl alcohol, acetone, dimethyl formamide, ethyl acetate, acetonitrile, chloroform, dichloromethane and tetrahydrofuran. It is slightly soluble in the solvents: deionized water, butyl alcohol, ethyl ether and insoluble in the solvents hexane and n-heptane and became soluble in butyl alcohol after heating. In the results of crystallization for obtainment of cocrystals of Tolbutamide, from the solution of Tolbutamide and methanol with salicylic acid in the solvents methanol, ethanol, isopropyl alcohol, acetonitrile and dichloromethane, there was the formation of 2aminobenzoic acid, however, there was no crystallization in the solvents ethyl acetate, chloroform and tetrahydrofuran. In the results of the essays of crystallization for obtainment of cocrystals of Tolbutamide, from the solution of Tolbutamide and ethanol with salicylic acid in the solvents ethanol, ethyl acetate, acetonitrile, dichloromethane and tetrahydrofuran there was the formation of 2aminobenzoic acid, however, there was no crystallization in the solvents methanol, isopropyl alcohol and chloroform. In the essays of crystallization it was found the polymorph I and the polymorph III of Tolbutamide. In the results of the essays of crystallization for obtainment of cocrystals of the solution of Tolbutamide, from the solution of Tolbutamide and ethanol with maleic acid in the solvents methanol and ethyl acetate there was the crystallization of Tolbutamide in the monoclinic system and in the spatial group /n group, while in the solvents butyl alcohol, acetone and tetrahydrofuran, there was the crystallization of Tolbutamide in the orthorhombic system and spatial group in the . The essays performed with tartaric acid and oxalic acid have great evidence of the formation of cocrystals of Tolbutamide with oxalic acid and Glicosamine with tartaric acid, identified by the measures of Infrared Spectroscopy. / O polimorfismo é a capacidade de um elemento ou composto em cristalizar-se em mais do que uma espécie distinta de cristal, este afeta não apenas a velocidade com que uma substância age no organismo, mas também sua estabilidade química ao longo do tempo. Existe um grande problema que tem sido de importância considerável na indústria farmacêutica, é o desenvolvimento de um novo fármaco. A realização dessa pesquisa se justifica pelo fato de que, de acordo com a literatura, a forma cristalina ou presença de polimorfismo alteram a solubilidade e as propriedades físico-químicas de fármacos, podendo ser capazes de causar desvios de qualidade através da biodisponibilidade dos fármacos, influenciando o desempenho dos medicamentos e a bioequivalência. O objetivo da pesquisa é sintetizar e caracterizar formas cristalinas dos fármacos Glicosamina e Tolbutamida, desta forma analisou-se o polimorfismo e a estrutura cristalina destes fármacos. Os experimentos foram realizados no laboratório de desenvolvimento de novos fármacos da UFG/Regional Jataí, onde se realizou os ensaios de cristalização para a obtenção de cocristais dos fármacos Glicosamina e Tolbutamida. Foram escolhidos a Tolbutamida, fármaco utilizado como hipoglicemiante oral e a Glicosamina usada no tratamento de artrose, reumatismo articular, e regeneração de cartilagem danificada. A escolha destes teve como base a disponibilidade destas substâncias no Laboratório de Desenvolvimento de Novos Fármacos da UFG/Regional Jataí, e o fato de não terem sido, ainda, totalmente explorados na literatura. Foram realizados ensaios de cristalização para obtenção de cocristais em diferentes solventes e caracterizadas as estruturas por difração de raios X por monocristal (DRXM) e infravermelho (IV). As matérias primas Glicosamina e Tolbutamida, foram adquiridas na Pharma/China Zhejiang Golden- Shell e Sigma-Aldrich Co., USA, respectivamente. Os comportamentos de solubilidade da Glicosamina e Tolbutamida foram observados experimentalmente para os solventes: água deionizada, metanol, etanol, isopropanol, butanol, acetona, dimetil-formamida, acetato de etila, éter etílico, acetonitrila, clorofórmio, diclorometano, THF, hexano e heptano. Observou-se que a Glicosamina é pouco solúvel nos solventes água deionizada, isopropanol, butanol, dimetil-formamida e clorofórmio à temperatura ambiente, após aquecimento das soluções a Glicosamina, apresentou ser solúvel apenas em água deionizada. Entretanto, é insolúvel nos solventes: metanol, etanol, acetona, acetato de etila, éter etílico, acetonitrila, diclorometano, THF, hexano, heptano. Nos resultados de ensaios de cristalização para obtenção de cocristais de Glicosamina, a partir da solução de Glicosamina e água deionizada com ácido salicílico e outros solventes obteve a formação do ácido 2aminobenzoico. Observou- se que a Tolbutamida se solubiliza, à temperatura ambiente, em metanol, etanol, isopropanol, acetona, dimetil-formamida, acetato de etila, acetonitrila, clorofórmio, diclorometano e THF. É pouco solúvel nos solventes: água deionizada, butanol e éter etílico e insolúvel nos solventes hexano e heptano e passou a ser solúvel em butanol após aquecimento. Nos resultados de ensaios de cristalização para obtenção de cocristais de Tolbutamida, a partir da solução de Tolbutamida e metanol com o ácido salicílico nos solventes metanol, etanol, isopropanol, acetonitrila e diclorometano, obteve-se a formação do ácido 2-aminobenzoico, porém não houve cristalização nos solventes acetato de etila, clorofórmio e THF. Nos resultados de ensaios de cristalização para obtenção de cocristais de Tolbutamida, a partir da solução de Tolbutamida e etanol com ácido salicílico nos solventes, etanol, acetato de etila, acetonitrila, diclorometano e THF, obteve a formação do ácido 2-aminobenzoico, porém não houve cristalização nos solventes metanol, isopropanol e clorofórmio. Nos ensaios de cristalização foram encontrados o polimorfo I e o polimorfo III da Tolbutamida. Nos resultados de ensaios de cristalização para obter cocristais da solução de Tolbutamida, a partir da solução de Tolbutamida e etanol com ácido maleico nos solventes metanol e acetato de etila ocorreu a cristalização da Tolbutamida no sistema monoclínico e no grupo espacial P21/n, enquanto nos solventes butanol, acetona e THF, ocorreu a cristalização da Tolbutamida no sistema ortorrômbico e no grupo espacial Pna21. Nos ensaios realizados com ácido tartárico e ácido oxálico, temos fortes evidências da formação de cocristais de Tolbutamida com ácido oxálico e Glicosamina com ácido tartárico, constatadas pelas medidas de Espectroscopia de Infravermelho.

No / Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

Increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells triggers insulin exocytosis. Among the Ca2+ channels present in the plasma membrane, the transient receptor potential (TRP) channels receptors are currently of great interest. The mechanisms by which the extracellular adenosine diphosphate ribose (ADPr) increases the [Ca2+]i is unknown. Our aims were to study the roles of the TRP channels in the tolbutamide induced [Ca2+]i increase and to identify the surface receptor that is activated by ADPr. We used S5 cells, a highly differentiated rat insulinoma cell line, as a model for β-cells. Single cell ratiometric microfluorometry was used to measure the [Ca2+]i changes in the Fura-2 loaded cells. Tolbutamide increased [Ca2+]i in the form of oscillations. After tolbutamide increased [Ca2+]i,capsazepine, a potent blocker of the transient receptor potential vanilloid subtype 1 (TRPV1) channel was added to the β-cells, which reduced the tolbutamide-induced [Ca2+]i increase. capsazepine, N-(p-Amylcinnamoyl) anthranilic acid (ACA),  TRPM2 channel blocker, and triphenyl phosphine oxide (TPPO), TRPM5 channel blocker were tested for their effect on potassium chloride (KCl) induced [Ca2+]i response. These blockers did not inhibit the KCl induced [Ca2+]i increase.   Adenosine diphosphate ribose (ADPr) increased [Ca2+]i in the form of initial transient peak followed by an elevated plateau. Application of ADPr shortly after a prior application and washout of Adenosine diphosphate (ADP) elicited only small [Ca2+]i increase  indicating desensitization of the receptor involved. 2´deoxy-N6-methyladenosine 3´5´bis-phosphate (MRS2179), and chloro N6-methyl-(N)-methanocarba 2´deoxyadenosine 3´5´ bis-phosphate (MRS2279), two selective inhibitors of P2Y1 receptor, abolished the ADPr-induced [Ca2+]i increase. Tolbutamide closes ATP sensitive potassium (KATP) channels. Our results demonstrate that besides the closure of the KATP channels, inward cation currents carried by Ca2+through the TRPV1 channel are necessary for depolarization to the threshold for the activation of the voltage gated calcium channels (VGCC) to increase the [Ca2+]i. Our results also show that ADPr increases [Ca2+]i by activating the P2Y1 receptor.

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca2+]i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from ob/ob-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (KATP channels), depolarization, and Ca2+ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca2+]i. When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca2+]i oscillations are present. By permanently opening or closing the KATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca2+]i, acting in synergy.