Análise químico-farmacêutica e estudos microbiológico e de estabilidade de preparações injetáveis de daptomicina
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Data
2016-09-29
Autores
Belavenuto, Eliane Gandolpho Tótoli [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A daptomicina é o primeiro membro aprovado de uma nova classe de antimicrobianos, a dos lipopeptídeos cíclicos, e apresenta ação seletiva contra bactérias Gram-positivas, incluindo cepas resistentes à meticilina e à vancomicina. É distribuída comercialmente como Cubicin®, na forma farmacêutica de pó liofilizado para solução injetável. Por ser um medicamento de recente inserção no mercado mundial, existem poucos estudos na literatura em relação ao desenvolvimento de metodologias analíticas e estudo de estabilidade aplicados a este lipopeptídeo cíclico. Desta forma, pesquisas nesta área são de fundamental importância e altamente relevantes para otimizar sua análise na indústria e garantir a qualidade do produto já comercializado. Este trabalho desenvolveu e validou métodos analíticos para análise qualitativa e quantitativa de daptomicina em pó liofilizado, bem como estudos de estabilidade e microbiológico. Em relação à análise qualitativa, a daptomicina foi analisada quanto às suas características físicas e ponto de fusão. Também foram desenvolvidos métodos por cromatografia em camada delgada (CCD) indicativo de estabilidade, espectrofotometria na região do ultravioleta (UV), cromatografia líquida de alta eficiência (CLAE), eletroforese capilar (EC) e espectrofotometria na região do infravermelho (IR) médio, possibilitando a identificação da amostra. Para quantificação do fármaco, foram validados os métodos de espectrofotometria na região do UV, ensaio microbiológico pelo método turbidimétrico, CLAE, EC e espectrofotometria na região do IR médio. O método espectrofotométrico na região do UV foi desenvolvido em 221 nm, com faixa de linearidade de 6,0 a 21,0 µg/mL, utilizando água como solvente. O ensaio microbiológico pelo método turbidimétrico utilizou o micro-organismo Staphylococcus aureus ATCC 6538 IAL 2082, com faixa linear de 8,0-18,0 µg/mL. O método por CLAE utilizou comprimento de onda de detecção de 221 nm, fase móvel composta por água e etanol (45:55, v/v), com pH ajustado para 4,5 com ácido acético glacial, obtendo-se um tempo de retenção médio de 5,7 minutos e capacidade de separação de produtos de degradação. A faixa linear avaliada foi de 20 a 70 μg/mL. O método de EC foi validado em 200 nm, com eletrólito de corrida composto por solução tampão fosfato de potássio (15 mM) pH 7,0, obtendo-se um tempo de migração médio de 1,7 minutos e capacidade de separar produtos de degradação. A faixa linear foi de 50,0 a 175,0 µg/mL. A espectrofotometria na região do IR médio foi desenvolvida na faixa espectral de 1700 a 1600 cm-1. A faixa linear foi de 0,2 a 0,6 mg/150 mg. Os métodos desenvolvidos destacam-se por não utilizarem solventes orgânicos em suas análises, enquadrando-se nos parâmetros da Química Verde. Estudos de estabilidade da daptomicina também foram realizados. O produto farmacêutico apresentou boa estabilidade nas condições aceleradas (25 oC ± 2 oC/ UR 60% ± 5%) e de longa duração estudadas (5 ºC ± 3 ºC). A daptomicina na forma pura mostrou-se instável em condições alcalinas, ácidas e oxidativas. No estudo microbiológico, a daptomicina demonstrou ser muito ativa contra cepas de S. aureus resistentes à meticilina, S. pneumoniae e enterococos resistentes à vancomicina. No entanto, 15% das cepas de S. aureus sensíveis à meticilina mostraram-se resistentes. Quanto aos estudos de sinergia, combinações entre daptomicina + LP9666 e daptomicina + NCL são promissoras e merecem um estudo mais aprofundado. Os métodos desenvolvidos neste trabalho contribuem para avaliação da qualidade, segurança e eficácia terapêutica de daptomicina em pó liofilizado, além de ampliar os conhecimentos científicos relacionados a este novo antimicrobiano.
Daptomycin is the first approved member from a new class of antimicrobial agents, the cyclic lipopeptides, and presents selective action against Gram-positive bacteria, including methicillin and vancomycin resistant strains. This antimicrobial agent is commercially distributed in lyophilized powder as CubicinTM. Since daptomycin is a new drug in the market, there are few studies in the literature regarding the development of analytical methodologies and stability studies applied to this cyclic lipopeptide. In this way, researches in this area are essential and highly relevant to optimize the analysis of this drug in the industry and ensure the quality of the marketed product. This work developed and validated analytical methods for qualitative and quantitative analysis of daptomycin in pharmaceutical formulation and its stability and microbiological studies. Regarding the qualitative analysis, the physical characteristics and melting point of daptomycin were studied. The following qualitative methods were also performed: thin layer chromatography, spectrophotometry in ultraviolet (UV) region, high performance liquid chromatography (HPLC), capillary eletrophoresis (CE) and spectrophotometry in medium infrared (IR) region, which allowed the daptomycin identification. For the quantitative analysis, the following methods were developed and validated: spectrophotometry in the UV region, microbiological method by turbidimetric assay, HPLC, CE and spectrophotometry in medium infrared (IR) region. The UV spectrophotometric method was developed in a wavelength of 221 nm, with concentration linear range from 6.0 to 21.0 μg/mL, using the water as solvent. The microbiological method employed Staphylococcus aureus ATCC 6538 IAL 2082 as the microorganism-test, and presented a concentration linear range from 8.0 to 18.0 μg/mL. The HPLC method used detection wavelength of 221 nm and mobile phase consisted of water and ethanol (45:55, v/v) with the pH adjusted to 4.5 with glacial acetic acid, obtaining a retention time of 5.7 minutes. This method also showed its stability-indicating ability. The evaluated linear range was from 20.0 to 70.0 μg/mL. The CE method was performed at 200 nm with an eletrolite consisting of potassium phosphate buffer solution (15 mM), pH 7.0, yielding an average migration time of 1.7 minutes and ability to separate degradation products. The IR spectrophotometry was carried out in the spectral range from 1700 to 1600 cm-1. The linear range was from 0.2 to 0.6 mg/150 mg. The developed methods stand out for not using organic solvents in their analysis, fitting the parameters of Green Chemistry. Stability studies of daptomycin in lyophilized powder and in the pure form were conducted. The pharmaceutical product showed to be stable under the accelerated (25 oC ± 2 oC/ UR 60% ± 5%) and long term (5 ºC ± 3 ºC) studied conditions. On the other hand, daptomycin in pure form proved to be unstable in alkaline, acidic and oxidative conditions. Microbiological studies of daptomycin were also performed and daptomycin showed to be very active against strains of methicillin-resistant S. aureus, S. pneumoniae, and vancomycin-resistant enterococci. However, 15% of tested S. aureus strains (sensitive to methicillin) showed to be resistant. Regarding the synergy studies, combinations of daptomycin + LP9666 and daptomycin + NCL 195 are promising and deserve further studies in this regard. The studies conducted in this work will contribute to assess the quality, safety and therapeutic efficacy of daptomycin in lyophilized powder, in addition to expanding the scientific knowledge related to this antimicrobial agent.
Daptomycin is the first approved member from a new class of antimicrobial agents, the cyclic lipopeptides, and presents selective action against Gram-positive bacteria, including methicillin and vancomycin resistant strains. This antimicrobial agent is commercially distributed in lyophilized powder as CubicinTM. Since daptomycin is a new drug in the market, there are few studies in the literature regarding the development of analytical methodologies and stability studies applied to this cyclic lipopeptide. In this way, researches in this area are essential and highly relevant to optimize the analysis of this drug in the industry and ensure the quality of the marketed product. This work developed and validated analytical methods for qualitative and quantitative analysis of daptomycin in pharmaceutical formulation and its stability and microbiological studies. Regarding the qualitative analysis, the physical characteristics and melting point of daptomycin were studied. The following qualitative methods were also performed: thin layer chromatography, spectrophotometry in ultraviolet (UV) region, high performance liquid chromatography (HPLC), capillary eletrophoresis (CE) and spectrophotometry in medium infrared (IR) region, which allowed the daptomycin identification. For the quantitative analysis, the following methods were developed and validated: spectrophotometry in the UV region, microbiological method by turbidimetric assay, HPLC, CE and spectrophotometry in medium infrared (IR) region. The UV spectrophotometric method was developed in a wavelength of 221 nm, with concentration linear range from 6.0 to 21.0 μg/mL, using the water as solvent. The microbiological method employed Staphylococcus aureus ATCC 6538 IAL 2082 as the microorganism-test, and presented a concentration linear range from 8.0 to 18.0 μg/mL. The HPLC method used detection wavelength of 221 nm and mobile phase consisted of water and ethanol (45:55, v/v) with the pH adjusted to 4.5 with glacial acetic acid, obtaining a retention time of 5.7 minutes. This method also showed its stability-indicating ability. The evaluated linear range was from 20.0 to 70.0 μg/mL. The CE method was performed at 200 nm with an eletrolite consisting of potassium phosphate buffer solution (15 mM), pH 7.0, yielding an average migration time of 1.7 minutes and ability to separate degradation products. The IR spectrophotometry was carried out in the spectral range from 1700 to 1600 cm-1. The linear range was from 0.2 to 0.6 mg/150 mg. The developed methods stand out for not using organic solvents in their analysis, fitting the parameters of Green Chemistry. Stability studies of daptomycin in lyophilized powder and in the pure form were conducted. The pharmaceutical product showed to be stable under the accelerated (25 oC ± 2 oC/ UR 60% ± 5%) and long term (5 ºC ± 3 ºC) studied conditions. On the other hand, daptomycin in pure form proved to be unstable in alkaline, acidic and oxidative conditions. Microbiological studies of daptomycin were also performed and daptomycin showed to be very active against strains of methicillin-resistant S. aureus, S. pneumoniae, and vancomycin-resistant enterococci. However, 15% of tested S. aureus strains (sensitive to methicillin) showed to be resistant. Regarding the synergy studies, combinations of daptomycin + LP9666 and daptomycin + NCL 195 are promising and deserve further studies in this regard. The studies conducted in this work will contribute to assess the quality, safety and therapeutic efficacy of daptomycin in lyophilized powder, in addition to expanding the scientific knowledge related to this antimicrobial agent.
Descrição
Palavras-chave
CLAE, Controle de qualidade, Daptomicina, Eletroforese capilar, Espectrofotometria, Estabilidade, Estudo microbiológico, Método turbidimétrico, Métodos analíticos, Sinergia, Analytical methods, Capillary electrophoresis, Daptomycin, HPLC, Microbiological study, Quality control, Spectrophotometry, Stability, Synergy, Turbidimetric assay