Avaliação de complexos de inclusão e micropartículas poliméricas como alternativas de estabilização do composto antiprotozoário, antibacteriano e antifúngico 2-(2-nitrovinil) furano (G-0)
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Data
2019-02-19
Autores
Ruz Sanjuan, Vivian
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Universidade Estadual Paulista (Unesp)
Resumo
2-(2-nitrovinil)furano (G-0) apresenta atividade antiprotozoária, antibacteriana e antifúngica frente a elevado número de patógenos porém, a capacidade de sublimação, baixa velocidade de dissolução em meio aquoso, problemas de compatibilidade com excipientes e fotodegradação, tem limitado sua incorporação em formas farmacêuticas para uso humano e/ou veterinário. O propósito do presente trabalho foi avaliar duas alternativas para a estabilização do candidato a fármaco, através da formação de complexos de inclusão liofilizados (FD) com hidroxipropil-β-ciclodextrina e sulfobutiléter-β-ciclodextrina sódica e utilizando a microencapsulação com etilcelulose pelo método de emulsificação/evaporação do solvente. Uma vez formados e previamente caracterizados, os complexos foram submetidos ao ensaio de estabilidade acelerada a 40°C/75 %HR e ensaios de fotoestabilidade acelerada. A câmara de fotoestabilidade também foi utilizada para confirmar o perfil de degradação dos complexos. Os fotoprodutos foram isolados em coluna analítica e o maioritário foi caracterizado por espectroscopia de RMN. Foi avaliada também a volatilidade do G-0 na forma de complexos comparado com misturas físicas equivalentes através de TGA e CG-HS-FID, complementando assim um estudo desenvolvido anteriormente no modo isotérmico. As propriedades biofarmacêuticas da substância livre e na forma complexada foram estudadas em relação ao comportamento de dissolução em Fluido Vaginal Simulado e a permeação/retenção em mucosa vaginal bovina. Finalmente foi avaliada a influência dos complexos na atividade antifúngica in vitro frente a Candida spp. assim como na citotoxicidade frente às linhagens celulares de fibroblastos e queratinócitos pelo método do MTT. Por outro lado, as micropartículas poliméricas foram obtidas com a ajuda de um planejamento fatorial 24. A caracterização dos sistemas selecionados foi desenvolvida usando DSC, DRX, MEV, FTIR-ATR e RMN 1H. Posteriormente as amostras foram submetidas aos ensaios de estabilidade acelerada a 40°C/75 %HR, ensaio de fotoestabilidade acelerada, analise da volatilidade do G-0 usando TGA/DTA e ensaio de liberação em solução tampão acetato, pH 4,2. Como resultados principais da avaliação, determinou-se que os complexos de inclusão com HP-β-CD e SBE-β-CD apresentaram-se como a melhor alternativa de estabilização porque garantiram ótima estabilidade química de G-0 em condições aceleradas de temperatura e umidade durante os seis meses de estudo porém, ambos os complexos são fotossensíveis. Também diminuíram as perdas por volatilização da substância em comparação com misturas físicas e aumentaram a velocidade de dissolução em fluido vaginal. Não foi quantificado G-0 no compartimento receptor durante o ensaio de permeação em nenhuma das amostras estudadas porém, verificou-se retenção do fármaco na mucosa vaginal bovina, sem influência significativa da formação de complexos. O teste "in vitro" de atividade antifúngica mostrou que o complexo FD G-0/HP-β-CD manteve a atividade da substância encapsulada frente a C. albicans (padrão e isolado clínico) e melhorou a atividade frente à cepa isolado clínico azol resistente de C.krusei (CIM=12,5 μg.mL-1) comparado com o G-0 (CIM=25 μg.mL-1) e anfotericina B (CIM=20 μg.mL-1). O complexo FD G-0/SBE-β-CD melhorou a atividade em todos os casos, atingindo CIM=8 μg.mL-1 frente a C.albicans e CIM=10 μg.mL-1 frente a C.krusei. A viabilidade celular de fibroblastos e queratinócitos seguiu a ordem FD G-0/SBE-β-CD > FD G-0/HP-β-CD > G-0, mostrando maior susceptibilidade para os queratinócitos. As micropartículas poliméricas selecionadas apresentaram forma esférica, com diminuição da cristalinidade do G-0, principalmente no sistema com maior proporção do polímero, e ausência de interações químicas do G-0 e a etilcelulose. Ainda quando as micropartículas melhoraram a fotoestabilidade do G-0 em condições aceleradas comparado com o fármaco livre, só o sistema G-0/EC 1:1,5 m/m conseguiu diminuir as perdas atribuídas à volatilização porém, também foi o sistema que liberou menor quantidade de G-0 (72,7 % em 12 h). Contrariamente, o sistema que apresentou maior estabilidade química em condições aceleradas foi o G-0/EC 1:1 m/m (102,7 ± 2,4 %), não sendo assim para o sistema com maior proporção polimérica (74,1 ± 4,2 %) durante os três meses de estudo.
2-(2-nitrovinyl)furan (G-0) has antiprotozoal, antibacterial and antifungal activity against a large number of pathogens, although the sublimation, low dissolution rate in aqueous medium, low compatibility with excipients and photodegradation, have hindered the incorporation in pharmaceutical dosage forms, to use in human and/or veterinary medicine. The aim of this work was to evaluate two alternatives for the stabilization of the drug candidate through freeze-drying inclusion complex formation (FD) with hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin sodium salt and through microencapsulation with ethylcellulose by mean of emulsion/solvent evaporation method. Once the complexes were prepared and fully characterized previously, they were subjected to an accelerated stability study at 40°C/75 %RH, and a photostability assay under forced irradiation conditions. A photostability chamber was also used in order to confirm the degradation profile for the complexes. Photoproducts were isolated in analytical column and the main degradation product was characterized aided by NMR. Drug volatility was also evaluated when G-0 was complexed, and compared with equivalent physical mixtures through TGA and GC-HS-FID. Biopharmaceutical properties of free drug and in the complexes were studied in terms of dissolution rate in Simulated Vaginal Fluid and permeation/retention in bovine vaginal mucosa. Finally, the influence of both complexes on the "in vitro" antifungal activity against Candida spp. and the cytotoxicity on fibroblast and keratinocyte cell lines were evaluated using the MTT method. Polymeric microparticles were obtained with the aid of a 24 factorial experimental design. Characterization for the selected systems was performed using DSC, XRD, SEM, FTIR-ATR and 1H NMR. Further, they were subjected to an accelerated stability study at 40°C/75 %RH, a forced photostability assay, TGA/DTA experiments to assess the drug volatility and drug release assay in acetate buffer medium, pH 4,2. As a main result, inclusion complexes with HP-β-CD and SBE-β-CD were the best stabilization alternative as they ensure optimum chemical stability of G-0 even under accelerated conditions of humidity and temperature for six months, however both were photosensitive. The formation of inclusion complexes reduced the volatilization of the drug compared with physical mixtures and increased the dissolution rate in vaginal fluid. During the permeability assay G-0 was not quantified in the receptor compartment for any of the samples applied, nevertheless retention in bovine vaginal mucosa was verified for all of them, without significant influence of the complexes formation. "In vitro" antifungal activity assay showed that FD G-0/HP-β-CD complex maintained the activity for the encapsulated drug against C. albicans (standard and clinical isolated strains) and improved the activity against a clinical isolated azole resistant strain of C.krusei (MIC=12,5 μg.mL-1), compared with free drug (MIC=25 μg.mL-1) and amphotericin B (MIC=20 μg.mL-1). FD G-0/SBE-β-CD complex improved the activity in all cases, with MIC= 8 μg.mL-1 against C.albicans and MIC=10 μg.mL-1 against C.krusei. Cellular viability of fibroblasts and keratinocytes followed the ranking order FD G-0/SBE-β-CD > FD G-0/HP-β-CD > G-0, showing higher susceptibility for keratinocytes. Polymeric microparticles selected resulted spherical in shape, with decreased crystallinity for the encapsulated substance (mainly for the system with higher polymer content) and lack of G-0-ethylcellulose chemical interactions. Even when microparticles improved the drug photostability under forced conditions compared with free drug, only G-0/EC 1:1,5 w/w system was able to decrease the weight loss attributed to drug volatilization, however it showed the lower amount of G-0 released (72,7 % in 12 h). In contrast, the system with better chemical stability was G-0/EC 1:1, w/w (102,7 ± 2,4 %), not having the same behavior the system with higher polymer content (74,1 ± 4,2 %), during three months under accelerated conditions.
2-(2-nitrovinyl)furan (G-0) has antiprotozoal, antibacterial and antifungal activity against a large number of pathogens, although the sublimation, low dissolution rate in aqueous medium, low compatibility with excipients and photodegradation, have hindered the incorporation in pharmaceutical dosage forms, to use in human and/or veterinary medicine. The aim of this work was to evaluate two alternatives for the stabilization of the drug candidate through freeze-drying inclusion complex formation (FD) with hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin sodium salt and through microencapsulation with ethylcellulose by mean of emulsion/solvent evaporation method. Once the complexes were prepared and fully characterized previously, they were subjected to an accelerated stability study at 40°C/75 %RH, and a photostability assay under forced irradiation conditions. A photostability chamber was also used in order to confirm the degradation profile for the complexes. Photoproducts were isolated in analytical column and the main degradation product was characterized aided by NMR. Drug volatility was also evaluated when G-0 was complexed, and compared with equivalent physical mixtures through TGA and GC-HS-FID. Biopharmaceutical properties of free drug and in the complexes were studied in terms of dissolution rate in Simulated Vaginal Fluid and permeation/retention in bovine vaginal mucosa. Finally, the influence of both complexes on the "in vitro" antifungal activity against Candida spp. and the cytotoxicity on fibroblast and keratinocyte cell lines were evaluated using the MTT method. Polymeric microparticles were obtained with the aid of a 24 factorial experimental design. Characterization for the selected systems was performed using DSC, XRD, SEM, FTIR-ATR and 1H NMR. Further, they were subjected to an accelerated stability study at 40°C/75 %RH, a forced photostability assay, TGA/DTA experiments to assess the drug volatility and drug release assay in acetate buffer medium, pH 4,2. As a main result, inclusion complexes with HP-β-CD and SBE-β-CD were the best stabilization alternative as they ensure optimum chemical stability of G-0 even under accelerated conditions of humidity and temperature for six months, however both were photosensitive. The formation of inclusion complexes reduced the volatilization of the drug compared with physical mixtures and increased the dissolution rate in vaginal fluid. During the permeability assay G-0 was not quantified in the receptor compartment for any of the samples applied, nevertheless retention in bovine vaginal mucosa was verified for all of them, without significant influence of the complexes formation. "In vitro" antifungal activity assay showed that FD G-0/HP-β-CD complex maintained the activity for the encapsulated drug against C. albicans (standard and clinical isolated strains) and improved the activity against a clinical isolated azole resistant strain of C.krusei (MIC=12,5 μg.mL-1), compared with free drug (MIC=25 μg.mL-1) and amphotericin B (MIC=20 μg.mL-1). FD G-0/SBE-β-CD complex improved the activity in all cases, with MIC= 8 μg.mL-1 against C.albicans and MIC=10 μg.mL-1 against C.krusei. Cellular viability of fibroblasts and keratinocytes followed the ranking order FD G-0/SBE-β-CD > FD G-0/HP-β-CD > G-0, showing higher susceptibility for keratinocytes. Polymeric microparticles selected resulted spherical in shape, with decreased crystallinity for the encapsulated substance (mainly for the system with higher polymer content) and lack of G-0-ethylcellulose chemical interactions. Even when microparticles improved the drug photostability under forced conditions compared with free drug, only G-0/EC 1:1,5 w/w system was able to decrease the weight loss attributed to drug volatilization, however it showed the lower amount of G-0 released (72,7 % in 12 h). In contrast, the system with better chemical stability was G-0/EC 1:1, w/w (102,7 ± 2,4 %), not having the same behavior the system with higher polymer content (74,1 ± 4,2 %), during three months under accelerated conditions.
Descrição
Palavras-chave
2-(2-nitrovinil) furano, estabilização de fármacos, ciclodextrinas, complexos de inclusão, micropartículas, etilcelulose, 2-(2-nitrovinyl) furan, drug stabilization, cyclodextrins, inclusion complexes, microparticles, ethylcellulose