名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水谷武史氏の博士論文として提出された

In asthma therapy, the use of theophylline to prevent bronchial spasm and glucocorticoids to decrease inflammation is widely indicated. Apart from the acute asthma attack oral theophylline is treated for chronic therapy in order to minimize inflammation and to enhance the efficiency of corticosteroids and recover steroids’ anti-inflammatory actions in COPD treatment. The preferred application route for respiratory disease treatment is by inhalation, such as dry powder inhalers (DPI) being the delivery systems of first choice. As shown recently, there is an advantageous effect if the drugs are given simultaneously which is caused by a synergistic effect at the same target cell in the lung epithelia. Therefore, it seems rational to combine both substances in one particle. This type of particle has the advantage over a combination product containing both drugs in a physical mixture which occurs rather randomly deposition leading to API segregation and non-dose-uniformity. Dry powder inhalers (DPIs) is a type of therapeutic pharmaceutical formulations usually present in the solid form. Due to the nature of the solid-state, an understanding of chemical and physical properties must be established for acquiring optimum performance of the active pharmaceutical ingredients (APIs). In recent year, generation of DPIs is a destructive procedure to meet the micron size. Such processes are inefficient and difficult to control. Moreover, according to current researches on combination APIs formulation, this type of DPIs performed a greater variability in does delivery of each active, leading to poor bioavailability and limit clinical efficient. This result suggest that combination formulations require advanced quality and functionality of particles with suitable physicochemical properties. Hence, in order to production of binary and combination DPIs products, the aim of this study was to develop the spray drying and ultrasonic process for engineering of combination drug particles that will be delivered more efficiently and independently of dose variations to the lung. Microparticles were produced by spray drying or/and ultrasonic technique. The processing parameters and addition of excipients (polymers) were optimized using a full factorial design such that microparticles were produced in a narrow size range suitable for inhalation. Employing excipients resulted in high saturation environment leading to minimized sphere particles when compared to conventional solvent. Solid state characterization of microparticles using powder x-ray diffraction and differential scanning calorimetry indicated that the particles contained crystalline but no cocrystal. The combination particles comparable to or better than micronized drug when formulated as a powder blended with lactose. It was concluded that the use of HPMC enhanced crystallinity suitable for inhalation; and combination particles improved uniform distribution on the stage of NGI.

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-25Bitstream added on 2014-06-13T20:25:00Z : No. of bitstreams: 1 silva_ml_dr_botfm.pdf: 1815885 bytes, checksum: 46fea01e7f48aa87bb45665d17541f32 (MD5) / Objetivos: Investigar a eficácia e segurança da budesonida comparada à prednisona na crise aguda de sibilância. Métodos: Estudo prospectivo, duplo-cego, randomizado comparou a budesonida nebulizada (2mg diários com redução gradual para 0,5mg, n=30, grupo A) com a prednisona (n= 30, grupo B) e placebo (n=15, grupo C) durante sete dias, envolvendo crianças de 40 dias a 36 meses em Santo André, SP, Brasil. Os critérios de inclusão foram: crianças nutridas, sem uso de corticosteróide, hospitalizadas por crise aguda grave de sibilância. Escore clínico foi mensurado nos períodos: 20, 40, 60 e 90 minutos, 2,4,6,12,24 horas e, após diariamente por uma semana. A função adrenal foi avaliada por dosagem de cortisol salivar nos dias de acompanhamento 3,5,7,10 e 15. Saliva foi coletada pela manhã e tarde. Cortisol foi dosado por radioimunoensaio com anticorpo anticortisol- 3-oxima albumina bovina. Resultados: Budesonida proporcionou melhora clínica mais rápida do que prednisona ou placebo nas primeiras 24h do tratamento (A 56 e B 282, p= 0,0000, Mann Whitney) (C 434 minutos). O percentil de interrupção por piora clínica foi: A: 3,3, B: 43, C: 40. Todos os grupos exibiram concentração de cortisol salivar mais alta de manhã do que tarde. Grupo A mostrou diminuição da dosagem vespertina no 10º dia de acompanhamento (p< 0,0001). Conclusão: Budesonida nebulizada proporcionou efeitos benéficos mais precocemente (1h) em exacerbações graves de broncoespasmo, sem alterar o ritmo circadaino. / Background: Inhaled steroids are an effective and recommended prophylactic treatment for moderate to severe asthma, but in acute exacerbations are not advocated as conventional therapy. Objectives: To investigate the efficacy of budesonide as compared to prednisone in severe acute wheezing.Methods: In prospective, double-blind, randomized study nebulized budesonide (2mg with gradual reduce to 0,5mg, n= 30, group A) was compared with oral prednisone (n= 30, group B) and placebo (n= 15, group C) during seven days, enrolling children aged 40 days to 36 months in Santo André, SP, Brazil. Inclusion criteria were: well-nourished, free from corticosteroid use and hospitalized for severe acute wheezing. Index score clinical was measured in the periods: 20, 40, 60 and 90 minutes; 2,4,6,12,24 h and then daily for a week. The adrenal function was availably for cortisol salivary dosage in the 3,5,7,10 and 15 days of treatment. Saliva was collected in the morning and afternoon. Cortisol was radioimmunoassayed with cortisol 3- oxime-bovine albumin antiserum. Results: Budesonide displayed a faster clinical improvement compared with prednisone or placebo into 24h of treatment (A 56 and B 282 minutes, p < 0,0000). The percentile of interruption by worse clinical was: A 3.3; B 43; C 40. All groups exhibited higher cortisol concentration during the morning than afternoon (p < 0.0001). Group A showed lower afternoon value in the 10th day (p < 0,001). Conclusion: nebulized budesonide brings early beneficial effects (1h) in severe acute exacerbations of wheezing without adrenal suppression, without changing the circadian rhythm.

In asthma therapy, the use of theophylline to prevent bronchial spasm and glucocorticoids to decrease inflammation is widely indicated. Apart from the acute asthma attack oral theophylline is treated for chronic therapy in order to minimize inflammation and to enhance the efficiency of corticosteroids and recover steroids’ anti-inflammatory actions in COPD treatment. The preferred application route for respiratory disease treatment is by inhalation, such as dry powder inhalers (DPI) being the delivery systems of first choice. As shown recently, there is an advantageous effect if the drugs are given simultaneously which is caused by a synergistic effect at the same target cell in the lung epithelia. Therefore, it seems rational to combine both substances in one particle. This type of particle has the advantage over a combination product containing both drugs in a physical mixture which occurs rather randomly deposition leading to API segregation and non-dose-uniformity. Dry powder inhalers (DPIs) is a type of therapeutic pharmaceutical formulations usually present in the solid form. Due to the nature of the solid-state, an understanding of chemical and physical properties must be established for acquiring optimum performance of the active pharmaceutical ingredients (APIs). In recent year, generation of DPIs is a destructive procedure to meet the micron size. Such processes are inefficient and difficult to control. Moreover, according to current researches on combination APIs formulation, this type of DPIs performed a greater variability in does delivery of each active, leading to poor bioavailability and limit clinical efficient. This result suggest that combination formulations require advanced quality and functionality of particles with suitable physicochemical properties. Hence, in order to production of binary and combination DPIs products, the aim of this study was to develop the spray drying and ultrasonic process for engineering of combination drug particles that will be delivered more efficiently and independently of dose variations to the lung. Microparticles were produced by spray drying or/and ultrasonic technique. The processing parameters and addition of excipients (polymers) were optimized using a full factorial design such that microparticles were produced in a narrow size range suitable for inhalation. Employing excipients resulted in high saturation environment leading to minimized sphere particles when compared to conventional solvent. Solid state characterization of microparticles using powder x-ray diffraction and differential scanning calorimetry indicated that the particles contained crystalline but no cocrystal. The combination particles comparable to or better than micronized drug when formulated as a powder blended with lactose. It was concluded that the use of HPMC enhanced crystallinity suitable for inhalation; and combination particles improved uniform distribution on the stage of NGI.

No / Budesonide is a mixture of 22R and 22S epimers. The epimeric content of budesonide was reported in both British and European pharmacopoeias to be within the range of 60-49/40-51 for R and S epimers, respectively. In this work, contribution of the two epimers to the overall infrared spectrum of budesonide has been investigated by quantum chemical calculations.

No / Experimental solubilities of budesonide, hydrocortisone, and prednisolone in ethanol + water mixtures at 298.2 K are reported. The solubility of drugs was increased with the addition of ethanol and reached the maximum values of the volume fractions of 90 %, 80 %, and 80 % of ethanol. The Jouyban-Acree model was used to fit the experimental data, and the solubilities were reproduced using previously trained versions of the Jouyban-Acree model and the solubility data in monosolvents in which the overall mean relative deviations (OMRDs) of the models were 5.1 %, 6.4 %, 37.7 %, and 35.9 %, respectively, for the fitted model, the trained version for ethanol + water mixtures, and generally trained versions for various organic solvents + water mixtures. Solubilities were also predicted by a previously established log-linear model of Yalkowsky with the OMRD of 53.8 %.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 5 uppsatser.

Yes / Two different types of dry powder inhalers (Easyhaler® and RS01®) were used in this work to evaluate the ex vivo and in vitro performance of a budesonide inhaled formulation with recrystallised mannitol, commercial DPI-grade mannitol, or lactose. The aerodynamic performance of the budesonide formulation with recrystallised mannitol was superior when RS01® was used (FPF = 45.8%) compared to Easyhaler® (FPF = 14%). However, the aerodynamic profile was very poor in both devices when commercial mannitol was used. Interestingly, the aerosol performance of the marketed budesonide formulation significantly improved when RS01® was used compared to Easyhaler® (the original device for the formulation). Due to the significant increases in the surface energy of the commercial mannitol formulation, the aerodynamic performance of the formulation was very poor. This work demonstrates the impact of inhaler devices on the performance of inhaled formulations and considers the particle surface energy during formulation development.

Over recent years the delivery of nanosized drug particles has shown potential in improving bioavailability. Drug nanosizing is achieved by 'top-down' and by 'bottom-up' approaches. Owing to limitations associated with the top-down techniques, such as high energy input, electrostatic effects, broad particle size distributions and contamination issues, great interest has been directed to alternative bottom up technologies. In this study, the hypothesis that microreactors can be used as a simple and cost-effective technique to generate organic nanosized products is tested using three steroids (hydrocortisone, prednisolone and budesonide). Arrested antisolvent nanoprecipitation using ethanol (solvent) and water (antisolvent) was conducted within the microreactors. To enable experimental design for the microreactor studies, solubility profiles in different ethanol-water combinations at 25 °C were explored. All three drugs' solubility increased with increasing ethanol concentration showing maxima at 80-90 % v/v ethanol-water mixtures. Because of the complex multivariate microfluidic process, artificial neural network modelling was then employed to identify the dominant relationships between the variables affecting nanoprecipitation (as inputs) and the drug particle size (as output). The antisolvent flow rate was found to have the major role in directing drug particle size. Based on these successful findings, the potential of preparing pharmaceutical nanosuspensions using microfluidic reactors was researched. A hydrocortisone (HC) nanosuspension (NS) was prepared by introducing the generated drug particles into an aqueous solution of stabilizers stirred at high speed with a propeller mixer. A tangential flow filtration system was then used to concentrate the prepared NS. Results showed that a stable narrow sized HC NS of amorphous spherical particles 500 ± 64 nm diameter and zeta potential -18 ± 2.84 mV could be produced. The ocular bioavailability of a microfluidic precipitated HC NS (300 nm) was assessed and compared to a similar sized, milled HC NS and HC solution as a control. The precipitated and the milled NS achieved comparable AUC0-9h of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, significantly (P < 0.01) higher than HC solution (15.86 ± 2.7). These results illustrate the opportunity to design sustained release ophthalmic formulations. Going nano via microfluidic precipitation was also exploited to tailor budesonide (BD) NS for pulmonary administration. The in vitro aerosolization by nebulization of a BD NS was studied in comparison with a commercial BD microsuspension. Overall, the fine particle fraction generated from BD NS (56.88 ± 3.37) was significantly (P < 0.05) higher than the marketed BD (38.04 ± 7.81). The mean mass aerodynamic diameter of BD NS aerosol (3.9 ± 0.48 μm) was significantly smaller (P < 0.05) than the microsuspension (6.2 ± 1.09 μm) indicating improved performance for BD NS. In conclusion, findings of this study support the hypothesis of using microfluidic nanoprecipitation as a promising and economical technique of drug nanosizing.

A asma é definida como uma doença inflamatória crônica de caráter multifatorial, caracterizada pela obstrução reversível das vias aéreas, denso infiltrado inflamatório e hiper-reatividade brônquica a estímulos externos. Clinicamente, a doença é marcada por sintomas episódicos de dispneia, sibilo, tosse seca e sensação de aperto no peito. A terapia convencional da asma compreende o uso de anti-inflamatórios e broncodilatadores. A budesonida é um glicocorticoide esteroide e é dos fármacos mais utilizados na terapêutica da asma. No entanto, a budesonida apresenta baixa biodisponibilidade oral e o uso prolongado pode levar a efeitos adversos graves como afinamento da pele e supressão adrenocortical. No desenvolvimento de novas formulações, a avaliação da toxicidade é de extrema importância. Por conseguinte, o uso de cultura celular é de grande valia no desenvolvimento de protocolos para avaliação da toxicidade de novas formulações. Adicionalmente, a nanotecnologia é uma ferramenta importante para resolver problemas de biodisponibilidade e para contornar efeitos adversos da terapêutica convencional. Desta forma, o objetivo desta tese foi desenvolver um novo sistema nanoestruturado na forma de pó seco para inalação (Dry powders inhalers – DPI), obtido por aspersão contendo budesonida encapsulada, visando o tratamento da asma aguda e crônica. Essa proposta foi baseada na obtenção de um sistema pulverulento nanoestruturado com tamanho reduzido e controlado, visando a entrega pulmonar da budesonida. Na etapa de pré-formulação foi realizado um estudo fatorial avaliando diferentes métodos de preparação das nanocápsulas e os adjuvantes de secagem utilizados. As análises de tamanho de partícula, da formulação selecionada (nanocápsulas contendo budesonida e secas por aspersão com leucina) mostraram um tamanho reduzido e adequado para a administração pulmonar (2,7 μm). A morfologia demonstrou que estas partículas possuem um tamanho reduzido, forma esférica e superfície irregular, características importantes para a administração pulmonar. Quando analisada a distribuição pulmonar in vitro, em Impactador de Andersen, a formulação apresentou uma fração de partículas finas (Fine Particle Fraction – FPF) de 28%. Analisando os resultados dos experimentos em modelos de asma aguda e crônica induzidos por ovalbumina, os resultados da mecânica respiratória e função pulmonar mostraram uma diminuição na resistência e na elastância pulmonar, quando a budesonida nanoencapsulada foi utilizada, quando comparada com uma formulação comercial de budesonida, nas duas doses utilizadas (0,5 e 1,0 mg/Kg). Esse tratamento com nanocápsulas também mostrou eficiência na redução da inflamação, pela redução do número de leucócitos totais no fluido de lavagem bronco alveolar (Broncho Alveolar Lavage Fluid – BALF) e, principalmente, redução significativa no número dos eosinófilos no infiltrado pulmonar. Corroborando esses resultados, a quantificação da eotaxina – 1 e das citocinas pró-inflamatórias foram reduzidas, quando comparadas ao tratamento comercial. A análise histopatológica mostrou que quando o tratamento com as nanocápsulas foi utilizado, a produção de muco foi reduzida, bem como a produção de fibrose sub-epitelial, sugerindo um possível efeito sobre o remodelamento tecidual. Os resultados de toxicidade utilizando linhagem celular epitelial pulmonar (H441) mostrou uma redução na toxicidade da budesonida, quando encapsulada nas nanopartículas, tanto na forma de suspensão como na forma pulverulenta. Essa redução da toxicidade foi de 75% e de 50%, na dose de 100 μg/mL, para a suspensão e para o DPI, respectivamente. O conjunto dos resultados obtidos mostrou a potencial aplicabilidade da budesonida nanoencapsulada para o tratamento da asma, utilizando esse novo sistema DPI. / Asthma is characterized as a chronic inflammatory disease developed by multifactorial aspects such as genetic predisposition and exposure to environmental factors such as pollution, smoke and microorganisms. The conventional asthma therapy comprises the use of bronchodilators and anti-inflammatory. Budesonide is a glucocorticoid and is the most frequently used therapy in the treatment of asthma. However, this drug has low oral bioavailability and long term use may lead to adverse effects such as skin thinning and adrenal suppression. The evaluation of the toxicity of new formulation has critical role in the pharmaceutical development. The use of cell culture experiments can help this aspect. Additionally, nanotechnology is an important tool to solve problems regarding bioavailability and to circumvent adverse effects of conventional therapy. The aim of this work was to develop a nanostructured system as dry powder inhaler (DPI) containing budesonide loaded, obtained by spray-drying, targeting the treatment of acute and chronic asthma. This proposal was based on obtaining a nanostructured powder system with reduced and controlled size, aiming an alternative to treatment of asthma. A factorial study comparing different methods to produce the nanocapsules as well as the type of drying adjuvants was performed. The particle size of the selected formulation was 2.7 μm, an adequate reduced size suitable for pulmonary administration. The morphology of these particles showed a small size, spherical shape and irregular surface. All these characteristics are important for pulmonary administration. When analyzed the in vitro pulmonary distribution of the DPI, using an Andersen Cascade Impactor, showed a fine particle fraction (FPF) of 28%. Analyzing the results of the biological experiments, the mechanical respiratory and pulmonary function showed a decrease in lung elastance and resistance when budesonide was used nanoencapsulated compared with a commercial formulation of budesonide in two doses (0.5 and 1.0 mg / kg). Both treatments also showed nanocapsules efficiency in reduction of inflammation by reducing the total of leukocytes in the bronchial alveolar lavage fluid (BALF) and especially significant reduction in eosinophil infiltration in the lung tissue. Corroborating with these results, the quantification of eotaxin - 1 and proinflammatory cytokines was reduced when compared to commercial budesonide treatment. Histopathological analysis showed that when treatment with the nanocapsules was used, mucus production was reduced and reversed the phenomena of airway remodeling. The cytotoxicity assay by Alamar blue using the bronchial epithelium cell line (H441) showed a reduction on the toxicity of budesonide when the nanocapsules were used even in suspension or in the DPI. The cytotoxicity reduction were 75 and 50%, at 100 μg/mL, for the suspension and the DPI, respectively. All these results show that budesonide-loaded nanocapsules in dry powder inhaler is a promising approach for the treatment of asthma.