Background The mechanism by which allergies trigger asthma occurs through the interaction of antigen, IgE and the FcεR1 receptor on mast cells resulting in the release of mediators that exert their effects on various surrounding tissues causing bronchoconstriction, plasma exudation and mucus hypersecretion. The response is usually maximal within 30 minutes and resolves spontaneously within two hours. At least half of the individuals who exhibit this so called early response also manifest a late response which is a subsequent episode of bronchoconstriction that is usually maximal around six hours following exposure and involves airway inflammation. Montelukast has proven efficacious in the management of asthma and desloratadine is effective in the treatment of allergic rhinitis and chronic idiopathic urticaria. Since the early response involves the actions of multiple mediators, including histamine and the leukotrienes, the question of whether concurrent mediator blockade would be superior to either agent alone was raised. Additionally, the recent evidence supporting anti-inflammatory activity for these agents suggested potential efficacy against the late airway response. Methods Two double-blind, randomized, placebo-controlled, 4-way crossover allergen inhalation challenge investigations were conducted in twenty (10 per investigation) mild atopic asthmatics. The early response investigation involved the administration of either 5 mg desloratadine, 10 mg montelukast, the combination , or placebo (Vitamin B1) at 26 hours and 2 hours prior to allergen inhalation. The late response investigation involved single dose administration of each agent, alone or in combination, 2 hours prior to allergen inhalation. Measurements of changes in airway responsiveness and inflammation were also conducted. Results The early response was significantly inhibited by montelukast and the combination. Desloratadine did not differ from placebo. The late response was significantly decreased by desloratadine and montelukast and completely blocked with the combination. Desloratadine decreased sputum eosinophils at 7 hours, montelukast at 24 hours, and the combination at both time points. Airway responsiveness to methacholine trended lower with montelukast and the combination. Montelukast was the only treatment to significantly decrease exhaled nitric oxide levels. Conclusion The combination of desloratadine and montelukast provides inhibition that is superior to both monotherapies on the early and the late airway responses to inhaled allergen in people with mild atopic asthma.

Background The mechanism by which allergies trigger asthma occurs through the interaction of antigen, IgE and the FcεR1 receptor on mast cells resulting in the release of mediators that exert their effects on various surrounding tissues causing bronchoconstriction, plasma exudation and mucus hypersecretion. The response is usually maximal within 30 minutes and resolves spontaneously within two hours. At least half of the individuals who exhibit this so called early response also manifest a late response which is a subsequent episode of bronchoconstriction that is usually maximal around six hours following exposure and involves airway inflammation. Montelukast has proven efficacious in the management of asthma and desloratadine is effective in the treatment of allergic rhinitis and chronic idiopathic urticaria. Since the early response involves the actions of multiple mediators, including histamine and the leukotrienes, the question of whether concurrent mediator blockade would be superior to either agent alone was raised. Additionally, the recent evidence supporting anti-inflammatory activity for these agents suggested potential efficacy against the late airway response. Methods Two double-blind, randomized, placebo-controlled, 4-way crossover allergen inhalation challenge investigations were conducted in twenty (10 per investigation) mild atopic asthmatics. The early response investigation involved the administration of either 5 mg desloratadine, 10 mg montelukast, the combination , or placebo (Vitamin B1) at 26 hours and 2 hours prior to allergen inhalation. The late response investigation involved single dose administration of each agent, alone or in combination, 2 hours prior to allergen inhalation. Measurements of changes in airway responsiveness and inflammation were also conducted. Results The early response was significantly inhibited by montelukast and the combination. Desloratadine did not differ from placebo. The late response was significantly decreased by desloratadine and montelukast and completely blocked with the combination. Desloratadine decreased sputum eosinophils at 7 hours, montelukast at 24 hours, and the combination at both time points. Airway responsiveness to methacholine trended lower with montelukast and the combination. Montelukast was the only treatment to significantly decrease exhaled nitric oxide levels. Conclusion The combination of desloratadine and montelukast provides inhibition that is superior to both monotherapies on the early and the late airway responses to inhaled allergen in people with mild atopic asthma.

Unidad de práctica para optar al título de Químico Farmacéutico / Autor no autoriza el acceso a texto completo de su documento / Las normas GMP para productos medicinales, de acuerdo a la OMS y a la legislación en la materia de la Unión Europea se encargan de asegurar que los productos farmacéuticos sean consistentemente producidos y controlados en base a los criterios de calidad apropiados para su uso y como lo requieren sus especificaciones de diseño. Estas normas ayudan a evitar la contaminación cruzada, la confusión y la variabilidad en los procesos, además de contribuir a la trazabilidad en la fabricación. Una parte importante de las normas GMP se centra en las validaciones. Cada etapa considerada crítica dentro del proceso de manufactura de un producto debe estar validada. Para asegurar, con un alto grado de confianza, que el producto terminado cumpla con todas las especificaciones de calidad previstas en su diseño de manera reproducible. Para la validación de un proceso de fabricación se requiere, previamente, contar con: validaciones de metodologías analíticas, validaciones de limpieza de equipos, validaciones de sistemas de apoyo crítico, calificación de equipos de fabricación, entre otras. A su vez, la validación de un proceso de fabricación es un requisito para desarrollar los estudios de bioequivalencia de un medicamento. La presente unidad de práctica optativa reúne todo el trabajo estadístico, documental y empírico que se llevó a cabo, acorde a las exigencias vigentes de la entidad regulatoria nacional, para validar el proceso de fabricación del producto Montelukast Comprimidos Recubiertos 10 mg. Esta validación, de tipo concurrente, se realizó considerando tres lotes productivos fabricados de forma consecutiva en el Área de Producción de Sólidos de Laboratorios Saval

Estudiamos 24 niños de entre 6 y 14 años que comenzaron a tomar Montelukast para el control del broncoespasmo inducido por ejercicio tras ser diagnosticados con la prueba de ejercicio físico en tapiz rodante, 12 de ellos por la mañana y 12 por la noche. Después de catorce días se invirtió la hora de la toma tras realizar una nueva prueba de ejercicio en tapiz. Tras otros catorce días, 28 en total, se realiza la prueba de ejercicio final. Encontramos una significativa disminución en la caída del FEV1 del 32% para la mañana y la noche sin diferencias entre las horas de toma al estudiar la máxima caída del FEV1 ni el Área bajo la curva para este parámetro. Montelukast tiene la misma efectividad cuando se toma por la mañana o la noche. Supone un índice de protección para el FEV1 del 32%, tras tomarlo entre 14 y 28 días. / Montelukast was recommended to be taken in the evening with no evidence for that recommendation. We studied 24 children between 6 and 14 years of age to test whether the timing of the administration modified the effectiveness of Montelukast to control exercise induced bronchospasm (EIB). Children diagnosed of EIB after performing a challenge test using standardized exercise on a treadmill, received treatment for a total of 28 days in two periods of 14 days in a clinical trial with a cross over design. Dosage administration were randomly assigned to the morning or night for half the children in each study phase. Montelukast was equally effective to prevent exercise-induced bronchospasm irrespective of the timing of its administration.

<p>The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.</p><p>Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.</p><p>S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. </p><p>Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.</p><p>Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.</p>

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

O montelucaste (MTC) sódico é um agente antagonista do receptor de cisteinilleucotrienos, utilizado para o tratamento da asma e comercializado no Brasil sob a forma de comprimidos revestidos, comprimidos mastigáveis e grânulos orais com o nome de Singulair®. Embora seja um medicamento amplamente comercializado, há poucas referências relativas aos métodos de análise da forma farmacêutica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade do montelucaste sódico nos comprimidos revestidos, bem como a realização de estudos preliminares de estabilidade térmica e de fotoestabilidade. A análise qualitativa do MTC sódico foi realizada por cromatografia em camada delgada (CCD), espectrofotometria na região do ultravioleta (UV), cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC). Para análise quantitativa, foram validados os métodos por espectrofotometria na região do UV, CLAE e EC. A validação foi efetuada de acordo com as guias de validação disponíveis na literatura. No estudo preliminar de estabilidade térmica utilizaram-se amostras de comprimidos revestidos submetidos à temperatura de 60° C por 35 dias. A fotoestabilidade foi realizada em câmara de luz ultravioleta de 352 nm, por um período de 72 horas. Estas amostras foram analisadas por CLAE para determinação de MTC sódico e seus produtos de degradação. Os métodos qualitativos foram efetivos para identificação do MTC sódico. Os métodos quantitativos propostos foram validados de acordo com os parâmetros analíticos preconizados, podendo ser perfeitamente intercambiáveis. O estudo preliminar de estabilidade térmica não apresentou degradação da amostra nas condições de análise empregadas. O estudo de fotoestabilidade apresentou degradação intensa nas condições testadas. / The sodium montelukast is a selective leukotriene receptor antagonist used for the treatment of asthma, available in Brazil as a coated tablets, cheweble tablets and oral granules named Singulair®. Even though, it is widely commercialized, there are few references related to the quality control methods of it pharmaceutical dosage form. The aim of this work was the development and validation of analytical methodology for the quality control of sodium montelukast in coated tablets, as well the preliminary thermical and photostability studies. The qualitative analysis was performed by thin layer chromatography, ultraviolet spectrophotometry, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). For quantitative analysis, the methods of ultraviolet spectrophotometry, HPLC and CE were validated. The validation of the methods was performed according to the parameters of validation guidances available in the literature. The preliminary thermical studies were evaluated by exposing samples of coated tablets to temperatures of 60 °C for 35 days. The photostability was verified in an ultraviolet light chamber of 352 nm, during 72 hours. The sodium montelukast and their degradation products were determined in the samples by HPLC. The methods employed for the qualitative analysis demonstrated to be usefull for the sodium montelukast identification. The quantitative methods were validated in accordance to the analytic parameters related in the validation guides, allowing perfect interchanges. The thermical stability study had not presented sample degradation in the applied analysis conditions. The photostability study demonstrated an accentuated degradation in the described test conditions.

O montelucaste (MTC) sódico é um agente antagonista do receptor de cisteinilleucotrienos, utilizado para o tratamento da asma e comercializado no Brasil sob a forma de comprimidos revestidos, comprimidos mastigáveis e grânulos orais com o nome de Singulair®. Embora seja um medicamento amplamente comercializado, há poucas referências relativas aos métodos de análise da forma farmacêutica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade do montelucaste sódico nos comprimidos revestidos, bem como a realização de estudos preliminares de estabilidade térmica e de fotoestabilidade. A análise qualitativa do MTC sódico foi realizada por cromatografia em camada delgada (CCD), espectrofotometria na região do ultravioleta (UV), cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC). Para análise quantitativa, foram validados os métodos por espectrofotometria na região do UV, CLAE e EC. A validação foi efetuada de acordo com as guias de validação disponíveis na literatura. No estudo preliminar de estabilidade térmica utilizaram-se amostras de comprimidos revestidos submetidos à temperatura de 60° C por 35 dias. A fotoestabilidade foi realizada em câmara de luz ultravioleta de 352 nm, por um período de 72 horas. Estas amostras foram analisadas por CLAE para determinação de MTC sódico e seus produtos de degradação. Os métodos qualitativos foram efetivos para identificação do MTC sódico. Os métodos quantitativos propostos foram validados de acordo com os parâmetros analíticos preconizados, podendo ser perfeitamente intercambiáveis. O estudo preliminar de estabilidade térmica não apresentou degradação da amostra nas condições de análise empregadas. O estudo de fotoestabilidade apresentou degradação intensa nas condições testadas. / The sodium montelukast is a selective leukotriene receptor antagonist used for the treatment of asthma, available in Brazil as a coated tablets, cheweble tablets and oral granules named Singulair®. Even though, it is widely commercialized, there are few references related to the quality control methods of it pharmaceutical dosage form. The aim of this work was the development and validation of analytical methodology for the quality control of sodium montelukast in coated tablets, as well the preliminary thermical and photostability studies. The qualitative analysis was performed by thin layer chromatography, ultraviolet spectrophotometry, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). For quantitative analysis, the methods of ultraviolet spectrophotometry, HPLC and CE were validated. The validation of the methods was performed according to the parameters of validation guidances available in the literature. The preliminary thermical studies were evaluated by exposing samples of coated tablets to temperatures of 60 °C for 35 days. The photostability was verified in an ultraviolet light chamber of 352 nm, during 72 hours. The sodium montelukast and their degradation products were determined in the samples by HPLC. The methods employed for the qualitative analysis demonstrated to be usefull for the sodium montelukast identification. The quantitative methods were validated in accordance to the analytic parameters related in the validation guides, allowing perfect interchanges. The thermical stability study had not presented sample degradation in the applied analysis conditions. The photostability study demonstrated an accentuated degradation in the described test conditions.

O montelucaste (MTC) sódico é um agente antagonista do receptor de cisteinilleucotrienos, utilizado para o tratamento da asma e comercializado no Brasil sob a forma de comprimidos revestidos, comprimidos mastigáveis e grânulos orais com o nome de Singulair®. Embora seja um medicamento amplamente comercializado, há poucas referências relativas aos métodos de análise da forma farmacêutica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade do montelucaste sódico nos comprimidos revestidos, bem como a realização de estudos preliminares de estabilidade térmica e de fotoestabilidade. A análise qualitativa do MTC sódico foi realizada por cromatografia em camada delgada (CCD), espectrofotometria na região do ultravioleta (UV), cromatografia líquida de alta eficiência (CLAE) e eletroforese capilar (EC). Para análise quantitativa, foram validados os métodos por espectrofotometria na região do UV, CLAE e EC. A validação foi efetuada de acordo com as guias de validação disponíveis na literatura. No estudo preliminar de estabilidade térmica utilizaram-se amostras de comprimidos revestidos submetidos à temperatura de 60° C por 35 dias. A fotoestabilidade foi realizada em câmara de luz ultravioleta de 352 nm, por um período de 72 horas. Estas amostras foram analisadas por CLAE para determinação de MTC sódico e seus produtos de degradação. Os métodos qualitativos foram efetivos para identificação do MTC sódico. Os métodos quantitativos propostos foram validados de acordo com os parâmetros analíticos preconizados, podendo ser perfeitamente intercambiáveis. O estudo preliminar de estabilidade térmica não apresentou degradação da amostra nas condições de análise empregadas. O estudo de fotoestabilidade apresentou degradação intensa nas condições testadas. / The sodium montelukast is a selective leukotriene receptor antagonist used for the treatment of asthma, available in Brazil as a coated tablets, cheweble tablets and oral granules named Singulair®. Even though, it is widely commercialized, there are few references related to the quality control methods of it pharmaceutical dosage form. The aim of this work was the development and validation of analytical methodology for the quality control of sodium montelukast in coated tablets, as well the preliminary thermical and photostability studies. The qualitative analysis was performed by thin layer chromatography, ultraviolet spectrophotometry, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). For quantitative analysis, the methods of ultraviolet spectrophotometry, HPLC and CE were validated. The validation of the methods was performed according to the parameters of validation guidances available in the literature. The preliminary thermical studies were evaluated by exposing samples of coated tablets to temperatures of 60 °C for 35 days. The photostability was verified in an ultraviolet light chamber of 352 nm, during 72 hours. The sodium montelukast and their degradation products were determined in the samples by HPLC. The methods employed for the qualitative analysis demonstrated to be usefull for the sodium montelukast identification. The quantitative methods were validated in accordance to the analytic parameters related in the validation guides, allowing perfect interchanges. The thermical stability study had not presented sample degradation in the applied analysis conditions. The photostability study demonstrated an accentuated degradation in the described test conditions.

Montelukast (ML) is primarily an antagonist of type 1 cysteinyl leukotriene receptors (CysLT1R), an activity which underpins its therapeutic efficacy in bronchial asthma. However, ML has also been reported to be useful in the treatment of acute and chronic inflammatory disorders of both infective and non-infective origin in which CysLTs are unlikely to be the predominant mediators of harmful inflammatory responses. These include conditions such as chronic obstructive pulmonary disease and cystic fibrosis in which the neutrophil is believed to be the primary offender, suggesting that ML may possess neutrophil-targeted, CysLT1R-independent mechanisms of anti-inflammatory activity. Accordingly, the laboratory research presented in this thesis was designed with the primary objectives of characterizing possible CysLT1R-dependent and – independent neutrophil-targeted anti-inflammatory activities of ML in vitro, and consisted of 3 phases. These were investigation of: i) the effects of the CysLTs, LTC4 and LTD4 (in the absence and presence of ML) on mobilization of intracellular Ca2+ stores, generation of reactive oxygen species (ROS) and release of primary and secondary granule proteinases; ii) the effects of ML on a series of pro-inflammatory activities of neutrophils following activation of the cells with the chemoattractants FMLP and platelet-activating factor (PAF); and iii) the interactive, anti-inflammatory effects on neutrophils of ML in combination with the long-acting beta-2 agonist, formoterol. In addition to the aforementioned activities, measurement of the production and expression of CR3, as well as generation of inositol triphosphate (IP3), cyclic AMP, and activities of the enzymes cAMP- and cGMP-phosphodiesterases (PDEs) in isolated neutrophil cytosol and membrane fractions, were also included. The following assays were used: i) chemiluminescence procedures for the detection of ROS; ii) a colourimetric procedure for the detection of elastase; iii) ELISA procedures for the detection of the matrix metalloproteinases (MMPs) 8- and -9, LTB4, and cyclic AMP; iv) fura-2-based spectrofluorimetry and a radiometric procedure for monitoring cytosolic Ca2+ fluxes; v) flow cytometry for CR3; and vi) radioassays for IP3 and activity of cAMP- and cGMP-PDEs. Exposure of neutrophils to LTD4, but not LTC4, activated a very modest and transient increase in cytosolic Ca2+, but failed to initiate the generation of ROS or release of elastase or MMP-8. However, brief pre-treatment with either LTC4 or LTD4 sensitized the cells for increased production of ROS and release of granule proteinases following activation with FMLP, which was partially attenuated by inclusion of ML. In the second part of the study, pre-treatment of neutrophils with ML, at therapeutically relevant concentrations, resulted in dose-related inhibition of the FMLP- or PAF-activated generation of ROS and LTB4, as well as the release of elastase, with the former being unaffected by an inhibitor of 5-lipoxygenase (MK886), compatible with a CysLT1R-independent mechanism of anti-inflammatory activity. From a mechanistic perspective, these interactions of ML with neutrophils were associated with accelerated clearance of Ca2+ from the cytosol of the cells which could not be attributed to inhibition of production of IP3, but was, however, associated with increased levels of cAMP, apparently as a consequence of non- specific inhibition of cyclic nucleotide phosphodiesterases. In the third part of the study, combining ML with formoterol caused (in most cases) additive inhibitory effects on the generation of ROS and LTB4, release of granule proteinases, as well as expression of CR3, which again were associated with elevations in cAMP and interference with Ca2+ mobilization. In conclusion, ML appears to attenuate neutrophil activation by CysLT1R-dependent and –independent mechanisms. In the case of the former by interfering with the modest sensitizing (priming) interactions of LTC4 and LTD4 with neutrophils, and in the latter by inhibition of PDEs, leading a to sustained elevation in cAMP, resulting in rapid clearance of Ca2+ from the cytosol and decreased uptake of the cation from the extracellular milieu. / Thesis (PhD)--University of Pretoria, 2011. / Immunology / Unrestricted