Anfotericina b pré-aquecida: um novo sistema terapêutico para infecções fúngicas

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(1)MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. ANFOTERICINA B PRÉ-AQUECIDA: UM NOVO SISTEMA TERAPÊUTICO PARA INFECÇÕES FÚNGICAS. SCHEYLA DANIELA VIEIRA DA SILVA SIQUEIRA. NATAL/RN 2013.

(2) SCHEYLA DANIELA VIEIRA DA SILVA SIQUEIRA. ANFOTERICINA B PRÉ-AQUECIDA: UM NOVO SISTEMA TERAPÊUTICO PARA INFECÇÕES FÚNGICAS SISTÊMICAS. Dissertação apresentada ao Programa de Pós-graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito para a obtenção de título de Mestre em Ciências da Saúde. Orientador: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito. NATAL/RN 2013.

(3) Ficha catalográfica. S619a Siqueira, Scheyla Daniela Vieira da Silva. Anfotericina B pré-aquecida: um novo sistema terapêutico para infecções fúngicas / Scheyla Daniela Vieira da Silva Siqueira. – Natal, 2013. 46f. Orientador: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito. Dissertação (Mestrado) – Programa de Pós-Graduação em Ciências da Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte. 1. Anfotericina B – Dissertação. 2. Liofilização – Dissertação. 3. Atividade/Toxicidade – Dissertação. I. Egito, Eryvaldo Sócrates Tabosa do. II. Título.. RN-UF/BS-CCS. CDU: 615.282(043.3). ii.

(4) MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. Coordenadora do Programa de pós-Graduação em Ciências da Saúde: Profª. Drª. Ivonete Batista de Araujo. iii.

(5) SCHEYLA DANIELA VIEIRA DA SILVA SIQUEIRA. ANFOTERICINA B PRÉ-AQUECIDA: UM NOVO SISTEMA TERAPÊUTICO PARA INFECÇÕES FÚNGICAS. Aprovada em 28/06/2013. Banca examinadora:. Presidente da Banca: Prof.Dr. Eryvaldo Sócrates Tabosa do Egito. Membros da Banca: Prof. Dr. Guilherme Maranhão Chaves Prof. Dr. Fernando Sérgio Escócio Drummond Viana de Faria. iv.

(6) AGRADECIMENTOS. A Deus. A minha família e em especial à minha mãe, pelo amor, carinho, dedicação e apoio às minhas decisões. À Universidade Federal do Rio Grande do Norte e CAPES, pelo apoio estrutural e financeiro. Ao meu orientador Sócrates Egito, pelo incentivo e entusiasmo contagiantes. À professora Ivonete Batista, pela atenção, momentos de descontração, exemplo de competência e amor à profissão. A Miguel Adelino pela amizade,ajuda e incentivo constantes. A Christian Assunção pelo esforço constante em me ajudar. Ao colega Gustavo pela ajuda na coleta de material. À professora Tereza Medeiros pelo apoio estrutural. Aos amigos de laboratório Andreza, Laura, Éverton, Júnior, Alexandrino, Thales, Raphael, Érica, Kátia, Luiza, Bartô, Juliana, Lucas, Walteçá, Belle, Nednaldo, André e Rosilene pelos momentos de descontração, ajuda e amizade.. v.

(7) RESUMO O aquecimento moderado (70ºC por 20 min) de soluções micelares de Anfotericina B (AmB) gera um rearranjo molecular que apresenta diminuição significativa na sua toxicidade sem comprometer sua atividade. O objetivo desse trabalho foi avaliar aspectos físico-químicos e farmacológicos das soluções micelares de Anfotericina B não-aquecida (AmB-M) e aquecida (AmBA) antes e após o congelamento e posterior liofilização. Para isso foram feitas avaliações antes e depois do congelamento e secagem. As amostras foram analisadas nas seguintes concentrações: 50 mg.L-1, 5 mg.L-1, 0,5 mg.L-1 e 0,05 mg.L-1. O teste de liberação de potássio e hemoglobina de eritrócitos foi realizado para avaliar a toxicidade aguda e crônica, respectivamente. A eficácia das soluções foi analisada por meio da liberação de potássio e do teste da concentração inibitória mínima em uma cepa de Candida albicans. Após o aquecimento, foi observada uma turvação característica do rearranjo estrutural molecular. da. AmB. aquecida.. Adicionalmente,. a. AmB-A. apresentou. deslocamento no comprimento máximo de absorção de 327 nm para 323 nm. Essas características se mantiveram após a liofilização, evidenciando estabilidade físico-química dessa nova forma estrutural micelar. Com relação à toxicidade, a AmB-M produziu significativamente maior valor de liberação de hemoglobina que a forma aquecida. Esses resultados foram observados independentes do tipo de amostra (antes e depois da liofilização). Porém, não houve diferença entre AmB-M e AmB-A quanto à atividade antifúngica. Portanto, foi demonstrado que a liofilização não mudou o comportamento da forma aquecida da AmB comprovando sua viabilidade para ser liofilizada e produzida em escala industrial.. Palavras-chaves: anfotericina B, liofilização, toxicidade, atividade.. vi.

(8) LISTA DE FIGURAS. Figura 1- Estrutura química da anfotericina B .................................................9 Figura 2- Esquema representativo da obtenção das amostras estudadas ....14. vii.

(9) SUMÁRIO. 1 Introdução .......................................................................................................9 2 Justificativa.....................................................................................................12 3 Objetivos ........................................................................................................13 3.1 Geral............................................................................................................13 3.2 Específicos..................................................................................................13 4 Métodos........................................................................................................14 4.1 Preparação das formulações de AmB........................................................14 4.2 Avaliação das propriedades físico-químicas............................................15 4.3 Avaliação da atividade e toxicidade de soluções micelares de AmB.........15 4.3.1 Preparação da suspensão de hemácias..................................................15 4.3.2 Padronização do inoculo de Candida albicans.........................................15 4.4 Ensaio de toxicidade....................................................................................16 4.5 Avaliação da atividade antifúngica..............................................................16 4.6 Análise estatística........................................................................................17 5 Artigo produzido .............................................................................................18 6 Comentários, críticas e sugestões .................................................................41 7 Referências ....................................................................................................42. viii.

(10) 9. 1 INTRODUÇÃO. Nas últimas décadas, tem-se tornado crescente o número de infecções fúngicas invasivas responsáveis por morbidade e mortalidade, principalmente em pacientes imunocomprometidos (1). A anfotericina B (AmB),. antifúngico poliênico isolado de cepas de. Streptomyces nodosus foi utilizada clinicamente em 1960 pela primeira vez. Esse fármaco ainda é ativo contra muitas espécies fúngi cas e protozoários do gênero Leishmania (2,3). A AmB reduz em 50 a 80% o risco de infecções fúngicas sistêmicas em pacientes imunocomprometidos e reduz a taxa de mortalidade de 23% para 45% (4). Ela também é frequentemente utilizada na terapia empírica em pacientes. com. granulocitopenia,. que. apresentam. febre. de. origem. desconhecida (5-7). Esse fármaco possui em sua estrutura química 37 átomos de carbono organizados em um anel macrocíclico fechado por lactonização, uma cadeia de duplas ligações conjugadas e na região oposta, uma cadeia com sete grupos hidroxila livres, o que lhe confere característica anfipática ( Figura 1) (8).. Figura 1. Estrutura da anfotericina B A anfotericina B, na sua formulação micelar, Fungizon® (AmB-M), consiste em um complexo de AmB com um sal biliar, o desoxicolato de sódio, cuja finalidade é solubilizá-la em água e estabilizar a suspensão na forma de micélios. O complexo é comercializado na forma de pó liofilizado contendo 50.

(11) 10. mg de AmB, 41 mg de desoxicolato de sódio e sais para constituir um tampão com pH fisiológico (12). Em meio aquoso, a AmB-M forma uma mistura de monômeros e agregados de moléculas, sendo a forma monomérica sua forma menos tóxica (9). A atividade antifúngica da AmB depende da sua ligação a uma porção esterol de membrana, o ergosterol presente na membrana dos fungos. Isso origina um aumento da permeabilidade da membrana e desencadeia a perda dos componentes citoplasmáticos (10). A alteração da permeabilidade celular permite o escape de pequenos íons, principalmente íons potássio, levando consequentemente à morte celular (11). Adicionalmente, lesões oxidativas tambêm ocorrem durante a ação da AmB e isso resulta em danos prejudiciais à sobrevida das células. Em menor intensidade, a AmB também se liga ao colesterol presente nas células humanas ocasionando efeitos adversos (9). A alta incidência de nefrotoxicidade (30-80%) dessa formulação levou ao desenvolvimento de três tipos de formulações lipídicas: complexo lipídico, dispersão coloidal e lipossomal (12). Apesar de essas formulações serem menos tóxicas, elas apresentam elevado custo, o que dificulta o seu acesso (12). Desenvolver novas formulações eficazes de AmB, com menor toxicidade e de baixo custo, continua a ser um grande desafio. Gaboriau et al.,(1997), descobriram que o aquecimento moderado do Fungizon®, permite um rearranjo molecular de maior tamanho, formado pela condensação das formas monoméricas e agregadas resultando em “superagregados”, que neste trabalho serão denominados AmB-A (Anfotericina B aquecida) que têm demonstrado maior estabilidade química, mantendo sua eficácia nas células fúngicas e menor toxicidade em células de mamíferos (13, 14,15). Considerando-se que a AmB-M é comercializada na forma liofilizada para que sua estabilidade seja aumentada, é de extrema importância avaliar a susceptibilidade dos superagregados de AmB-A sob o processo de liofilização,.

(12) 11. que é um método amplamente empregado para secagem e consequente melhora da estabilidade de produtos farmacêuticos. O ciclo de liofilização pode ser dividido em três estágios: congelamento (solidificação); secagem primária (sublimação do gelo) e secagem secundária (dessorção da água não congelada). Esses procedimentos podem danificar as estruturas dos produtos farmacêuticos (16). Nesse contexto é necessário investigar o comportamento dos superagregados de AmB sob esse processo. Tendo-se por base resultados de estudos prévios, em que foi observado que a estabilidade dessa nova estrutura organizacional micelar de AmB-A é maior (16), espera-se que esse sistema seja estável ao processo de liofilização..

(13) 12. 2 JUSTIFICATIVA Apesar das formulações menos tóxicas de AmB já desenvolvidas, ainda não existe uma alternativa ideal, economicamente viável para um sistema assistencial de saúde pública, tendo em vista o elevado custo das formulações menos tóxicas de AmB existentes (17). O aquecimento moderado de soluções micelares de AmB mostrou-se ser um sistema promissor para a solução desse problema, tendo em vista sua menor toxicidade e simplicidade no seu método de obtenção (14). Levando-se em consideração a importância do processo de liofilização para a comercialização da AmB, é de extrema importância a análise desse novo sistema frente a esse processo..

(14) 13. 3 OBJETIVOS. 3.1 GERAL. - Realizar um estudo comparativo das propriedades físico-químicas e biológicas entre a AmB-M e AmB-A frente ao processo de liofilização a fim de se determinar a viabilidade da AmB aquecida a esse processo.. 3.2 ESPECÍFICOS. - Comparar as propriedades biológicas quanto à atividade e toxicidade da AmB-A com a AmB-M após congelamento e secagem (liofilização). - Comparar as propriedades físico-químicas da AmB-A com a AmB-M após congelamento e secagem (liofilização)..

(15) 14. 4 MÉTODOS. 4.1 PREPARAÇÃO DAS FORMULAÇÕES DE AM B Uma solução de Fungizon® (Cristália, Itapira, Brazil) na concentração de 5x10-3M (5,000 mg.L-1) foi preparada adicionando-se 10 mL de água bidestilada ao pó liofilizado de AmB e agitando-se até completa dissolução. A partir dessa solução, foram preparadas quatro diluições: 5x10-5 M (50 mg.L-1), 5x10-6 M (5 mg.L-1), 5x10-7 M (0,5 mg.L-1) e 5x10-8 M (0,05 mg.L-1). A solução de AmB aquecida foi obtida aquecendo-se uma solução de Fungizon a 70ºC por 20 min. O congelamento das formulações foi realizado à -80ºC por 24h e a secagem foi realizada em um liofilizador por 24h a -65ºC sob pressão de 0,0018 mbar. Amostras aquecidas e não-aquecidas antes do congelamento, após o congelamento e após a secagem foram analisadas (ver esquema abaixo).. Figura 2. Esquema representativo da obtenção das amostras para estudo Esquema representativo do preparo das seis amostras estudadas. AmBM: anfotericina B micelar; AmB-A: anfotericina B aquecida; AmB-MC: anfotericina B micelar congelada; AmB-AC: anfotericina B aquecida congelada;.

(16) 15. AmB-ML: anfotericina B micelar liofilizada; AmB-AL: anfotericina B aquecida liofilizada.. 4.2 AVALIAÇÃO DAS PROPRIEDADES FÍSICO-QUÍMICAS. Por meio da espectrofotometria foram analisadas quatro concentrações diferentes de AmB com diferentes caminhos ópticos para obtenção de valores de absorbância menores que 0,8. Assim, para a concentração de 5x10-5 M (50 mg.L-1) foi escolhido o caminho óptico de 0,1 cm, 1 cm para 5x10-6 M (5 mg.L-1), 10 cm para as concentrações de 5x10-7 M (0,5 mg.L-1) e 5x10-8 M (0,05 mg.L-1). Os coeficientes de absortividade molar (ε) foram calculados utilizando-se a equação de Lambert-Beer. A faixa de comprimento de onda utilizado foi de 300–450 nm (18). 4.3 AVALIAÇÃO DA ATIVIDADE E TOXICIDADE DE SOLUÇÕES MICELARES DE AM B Para esta finalidade utilizaram-se dois modelos celulares: Candida albicans como modelo celular fúngico para avaliação da atividade e eritrócitos como modelo celular humano para avaliação da toxicidade. 4.3.1 PREPARAÇÃO DA SUSPENSÃO DE HEMÁCIAS Cinco mililitros de sangue de um doador saudável foram coletados em tudo contendo EDTA a 10%. Em seguida, o plasma e a camada de leucócitos foram retirados por meio de centrifugação. Então os eritrócitos foram lavados três vezes com solução salina a 0,9%. As hemácias foram resuspendidas em 4 mL de solução salina e contadas em uma câmara de Neubawer TM. A partir da contagem, uma concentração de hemácias de 5 x 10 7 células/mL foi obtida. As células foram utilizadas no dia em que foram coletadas (19). 4.3.2 PADRONIZAÇÃO DO INÓCULO DE Candida albicans Uma cepa de Candida albicans ATCC (22019) foi mantida em Agar Saborauddextrose-cloranfenicol em temperatura ambiente e passado para outro meio mensalmente. Antes dos experimentos, um inóculo da cultura foi transferido para um novo Agar com as mesmas características do descrito anteriormente e.

(17) 16. foi incubado a 37ºC por 24h. As células fúngicas foram então lavadas três vezes com solução fisiológica 0,9%, ressuspendidas, contadas no retículo central da câmara de NeubauerTM e foram resuspensas novamente para se obter a concentração desejada de 5 x 10 7 unidades formadoras de colônias por mililitros (ufc.ml-1)(20). 4.4 ENSAIO DE TOXICIDADE Para avaliar a toxicidade dos superagregados de AmB, 4mL da suspensão de hemácias foram incubados por uma hora a 37ºC com as respectivas preparações nas concentrações de 50, 5, 0,5 e 0,05 mg/L, representando 5.10 5. , 5.10-6, 5.10-7, 5.10-8M, respectivamente. Após a incubação, o meio com as. células e o fármaco foram centrifugados a 1500 rpm por 5 minutos, e lavados 3 vezes com NaCl a 0,9%. (p/v).. Em seguida, as hemácias foram lisadas com 4mL. de água destilada, e centrifugadas a 1500 rpm por 5 minutos, para remover resíduos das membranas. Em seguida, a hemoglobina presente no hemolisado foi avaliada por espectrofotometria, utilizando-se o comprimento de onda de 540 nm. O potássio foi medido por meio de um fotômetro de chama calibrado com um padrão de potássio de 5 mEq/L. Cada experimento foi realizado em triplicata e repetido três vezes (19). 4.5 AVALIAÇÃO DA ATIVIDADE ANTIFÚNGICA Para este fim, 2 mL de uma suspensão de células fúngicas contendo 5 x 107.mL-1 foram incubadas por 1h a 37ºC com AmB-A a AmB convencional nas concentrações de 50, 5, 0,5 e 0,05 mg.L-1. Posteriormente a suspenção foi centrifugada por 10 min a 2.200 g e lavadas três vezes em solução fisiológica. Em seguida foram adicionados 2 mL de água deionizada ao precipitado de células fúngicas. Uma alíquota desse precipitado foi lisado por meio de aquecimento por 5 minutos à 100ºC e então foram centrifugadas para remoção das membranas. Em seguida, o potássio livre foi medido. A liberação de K+ foi calculada de forma semelhante ao cálculo dos eritrócitos(19). A concentração inibitória mínima (IC 100) também foi determinada. O teste de susceptibilidade foi realizado utilizando uma adaptação da versão do Comitê Nacional para padronização de laboratórios clínicos (21)..

(18) 17. 4.6 ANÁLISE ESTATÍSTICA Todas as amostras foram realizadas em triplicatas e as variações analisadas por ANOVA e teste T de student pelo programa Prim 4 para Windows 4.02 (GraphPad Software, San Diego, CA)..

(19) 18. 5 ARTIGO PRODUZIDO 5.1 O artigo “Influence of the freeze-drying process on the physicochemical and biological properties of pre-heated amphotericin B micellar systems” será publicado no periódico AAPS PharmSciTech que possui fator de impacto 1,432 e Qualis B2 para área de Medicina II..

(20) 19. Influence of the freeze-drying process on the physicochemical and biological properties of pre-heated amphotericin B micellar systems. Scheyla D.V.S. Siqueira1, Miguel A. Silva-Filho1, Christian A. Silva2, Ivonete B. Araújo1,2 , Anselmo G. Oliveira3 and Eryvaldo Sócrates T. Egito1,2,* 1. Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do. Rio Grande do Norte (UFRN), Centro de Ciências da Saúde (CCS), Laboratório de Sistemas Dispersos (LASID), Natal, RN, Brazil. 2. Departamento de Farmácia, UFRN, CCS, Lasid, RN, Brazil.. 3. Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas (UNESP), Rodovia Araraquara, Jaú Km 01, 14801-902 Araraquara, SP, Brazil. E-mail: socratesegito@gmail.com. * Corresponding author: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito Laboratório de Sistemas Dispersos Rua Praia de Areia Branca, 8948 Natal – RN – Brazil Zip Code: 59094-450 Telephone: +55 84 9431 8816 Fax number: +55 84 3342 9817.

(21) 20. Abstract The moderate heat treatment of amphotericin B (AmB) in its micellar system (MAmB) results in superaggregates (H-AmB), which present a substantially lower toxicity and similar activity. The aim of this work was to evaluate the H-AmB behavior after a freeze-drying cycle. H-AmB and M-AmB micelles were evaluated before and after freeze-drying concerning their physicochemical and biological properties by spectrophotometry and activity/toxicity assay. The following concentrations of M-AmB and H-AmB were studied: 50 mg.L-1, 5 mg.L1. , 0.5 mg.L-1 and 0.05 mg.L-1. Then, potassium and hemoglobin leakage from. red blood cells was used to evaluate the acute and chronic toxicity, respectively. The efficacy of M-AmB and H-AmB was assessed by potassium leakage from Candida albicans and microdilution test. After heating, besides an evident turbidity, a slight blue-shift from 327 nm to 323 nm was also observed at the concentrations of 50 and 5 mg.L-1. Additionally, an increase in absorbance at 323 nm at the concentration of 0.5 mg.L-1 was detected. Concerning the toxicity, H-AmB caused significantly lower hemoglobin leakage than M-AmB. These results were observed for H-AmB both before and after freeze-drying. However, there was no difference between H-AmB and M-AmB as regards their activity. Accordingly, the freeze-drying cycle did not show any influence on the behavior of heated formulations highlighting the suitability of such a method to produce efficient and less toxic AmB carriers. KEY WORDS: Amphotericin B, heating, freeze-drying, activity, toxicity..

(22) 21. INTRODUCTION Amphotericin B (AmB) consists of hydrophilic polyhydroxyl and hydrophobic polyene domains (1-5). Its micellar system, Fungizone® (M-AmB), is used for the treatment of various systemic fungal infections (6-11). In fact, MAmB reduces the relative risk of invasive fungal infections from 50% to 80% and the overall mortality rate from 23% to 45% (12). It is also the most potent drug commercially available for treating leishmaniasis, affecting promastigotes and amastigotes forms, both in vivo and in vitro (13). It has been used as first choice in the treatment of patients with signs of gravity such as those aging lower than 6 years old or higher than 65 years old (14). Recently, its combination with miltefosine. has. demonstrated. to. be. more. cost-effective. than. most. monotherapies in the treatment of visceral leishmaniasis (15). Unfortunately, the M-AmB has its use limited by its toxic effects mainly nephrotoxicity which has been estimated to occur in up to 80% of patients (16). Lipid-associated formulations of AmB such as AmBisome®, Amphotec® and Abelcet® have been successfully developed. However, their high cost is a limiting factor for their use (17). Moderate heat treatment of M-AmB has been found to induce a molecular rearrangement that yields structures known as superaggregates (HAmB) and can be proved by changes in light scattering, circular dichroism, absorption spectra, cryo-transmission and electron microscopy (18-20). Among those changes, a larger size, a blue-shift and a significantly lower toxicity have been evidenced (18-20). Recently, Silva-Filho et al have also demonstrated that heated Fungizone® exhibits significantly lower in vitro toxicity against mammalian cells while keeping its antifungal activity (21). However, the aqueous medium that surrounds this micellar system hinders their long term stability in such a manner that its stability is decreased to 24h at room temperature and to one week under refrigeration at 2 to 8ºC (22). In order to solve such stability issue, water has to be removed. The most common strategy is the freeze-drying process, which is a suitable drying method for AmB because it is carried out under low temperatures, unlike other.

(23) 22. methods such as spray-drying, by which high temperatures are required leading to instability problems (23). Freeze-drying consists in the sublimation of water from a frozen sample and it comprises the sample freezing followed by primary and secondary drying (24). These steps may trigger both chemical reactions and mechanical stress causing destabilization of micelles and likely secondary aggregation or fusion, hindering its micellar structure after reconstitution (25). Thus, the use of a cryoprotector is generally required (24, 26). To date there is no report in the literature about the behavior of superaggregates under freeze-drying: whether this process could disturb the new organizational system and whether its lower toxicity is remained after reconstitution. Thus, the aim of this work was to evaluate the influence of freeze-drying on the physicochemical and biological properties of H-AmB micellar system by a spectroscopy study and in vitro activity/toxicity assays. MATERIALS AND METHODS Materials Micellar amphotericin B (M-AmB) was kindly provided as a gift from Cristália (Itapira, Brazil). Normal saline solution [NaCl at 0.9% (w/v)] was purchased. from. Braun. (São. Paulo,. Brazil). and. Sabouraud-Dextrose-. Chloramphenicol (SDC) agar was provided by MicroMed (São Paulo, Brazil).. Preparation of the samples The stock solution of M-AmB at the concentration of 5x10-3 M (5,000 mg.L-1) was prepared by adding 10 mL of water for injection into a vial containing 50 mg of AmB, approximately 41 mg of sodium deoxycholate, and phosphate buffer, pH 7.4. The mixture was subject to vortex shaking until dissolution. Subsequently, M-AmB was heated at 70ºC for 20 min in order to obtain the formulation H-AmB..

(24) 23. Both M-AmB and H-AmB were frozen at -80ºC for 24 h in order to yield F-AmB and FH-AmB, respectively (Scheme 1). Finally, the frozen samples (F-AmB and FH-AmB) were dried by a freeze-dryer (Christ Alpha 1-2 LD, Germany) for 24 h under -65ºC and 0.0018 mbar producing the samples D-AmB and DH-AmB, respectively (Scheme 1).. Physicochemical analysis of AmB The. spectroscopy. study. was. carried. out. using. a. UV-VIS. spectrophotometer (Biochrom Libra S32, United Kingdom). The analyzed concentrations were 5x10-5 M (50 mg.L-1), 5x10-6 M (5 mg.L-1), 5x10-7 M (0.5 mg.L-1) and 5x10-8 M (0.05 mg.L-1). Their molar extinction coefficients (ε) were calculated using the Beer–Lambert equation. All spectra were recorded at 25°C ± 0.1°C with a 300–450 nm range (3). Temperature and time exposure were controlled by a built-in thermometer (Incoterm, Brazil) and a chronometer (Model Labor, Germany).. Toxicity against mammalian cells Preparation of red blood cells (RBC) suspension In accordance with the Ethical Research Committee of the Universidade Federal do Rio Grande do Norte, protocol number 002/2009, one healthy female adult donor signed the informed consent and provided all normal RBC for the in vitro experiments in order to minimize sources of variability. Five milliliters of venous blood were collected in sterile EDTA [1 mg/mL, ethylenediamine-tetraacetate at 10% (w/v)] syringes and promptly centrifuged at a refrigerated centrifuge (ALC, Model PK121R, Italy) in tubes at 1,100 g for 10 minutes at 4°C. Plasma and the exposed buffy coat were removed and discarded. The RBC were washed three times under centrifugation (1,100 g for 5 minutes at 4°C) and suspended in 5 volumes of normal saline. They were then.

(25) 24. resuspended in 4 mL of saline, counted in a Neubauer™ chamber (Splabor, Brazil), and resuspended again until the desired concentration (5 × 107 cells . mL−1) was achieved. The cells were used on the day of collection (27). Evaluation of H-AmB toxicity Four milliliters of RBC (5 × 107 cells. mL−1) were incubated for 1 h at 37°C with the vehicle control and with 50, 5, 0.5, and 0.05 mg. L −1 of M-AmB and H-AmB on its three studied forms. The RBC were then centrifuged for 5 minutes at 1,100 g and washed three times with normal saline. The pellet was lysed with 4 mL of distilled water and then stirred and centrifuged (1,100 g for 10 minutes) for removing membranes. The potassium (K+) content of the supernatant was determined using a Flame Photometer 7000 (910M Analyser, Brazil) calibrated with K+ reference at 5 mEq. L−1. Hemoglobin was determined from its absorption at 540 nm recorded on a UV-VIS spectrophotometer (Biochrom Libra S32, United Kingdom). The total amounts of K+ and hemoglobin were measured for the control RBC tubes. Release of ions was calculated as the difference between control and treated cells and expressed as a percentage of the total hemoglobin or K+ content (28).. Activity against Candida albicans strain Preparation of Candida albicans suspension Inoculums of a strain of Candida albicans ATCC (22019) were transferred to a saboraud-dextrose-agar scope and incubated at 37°C for 24 h. The desired concentration of 5 × 107 cfu . mL−1 was obtained by counting using a Neubauer™ chamber (27). Evaluation of H-AmB efficacy Two milliliters of a fungal suspension containing 5 x10 7 cfu.mL-1 were incubated for 1 h at 37ºC with both M-AmB and H-AmB at the concentrations of 50, 5, 0.5, and 0.05 mg.L-1, separately. Cells were centrifuged for 10 minutes at 2,200 g and washed three times in normal saline, and 2 mL of purified water were added to the pellet of fungal cells. A fraction of this pellet was lysed by heating for 5 minutes at 100ºC and centrifuged to remove membranes, and free.

(26) 25. K+ was measured. The K+ leakage was calculated similarly to the calculation of the RBC (27). The microdilution method was also conducted in order to evaluate the efficacy of H-AmB formulations. It was carried out using an adapted version of the Clinical Laboratory Standards Institute (28). Statistical analysis The results were evaluated by means of one-way analysis of variance (ANOVA) and t test to analyze the variation response in the same group and in different groups, respectively, using Prism 4 for Windows 4.02 (GraphPad Software, San Diego, CA).. RESULTS Physicochemical behavior of H-AmB micelles was kept unchanged after freeze-drying After heating, a prominent change in the turbidity of the H-AmB was observed. This suggests a rearrangement in the structural organization of the micellar system that was evidenced by changes in physicochemical properties of H-AmB. The electronic absorption spectrum of the AmB showed to be concentration-dependent. At low concentrations (5x10-8 M), the monomeric form was predominant with absorption spectrum exhibiting maxima at 364, 385, and 408 nm, similar to that obtained with polar organic solvents such as methanol (Fig. 1). This pattern was similar for both M-AmB and H-AmB. However, as the concentrations increased, the spectra changed for both M-AmB and H-AmB. At 5x10-5 and 5x10-6 M, where there is a higher amount of aggregates, AmB presented a maximum peak centered at 327 nm (Fig.1A-C). However, for HAmB systems, a new band centered at 323 nm was observed (Fig. 1A). Interestingly, after freeze-drying these patterns were similar (Fig. 2B-C). M-AmB was also freeze-dried as a control. As expected, no changes were observed once the product is already marketed in a freeze-dried form (Fig. 1A-C). As.

(27) 26. previously observed by Silva-Filho et al, the most important difference between M-AmB and H-AmB was found at the concentration of 5x10 -7 M, at which the molar extinction coefficient is higher for all heated AmB samples (ε 60,800; ε. FH-AmB. = 59,800 and ε. DH-AmB=. H-AmB=. 58,800) at 323 nm (Fig. 2A-C). On the. other hand, for M-AmB, FM-AmB and DM-AmB, these values changed to 44,600, 41,600 and 40,600, respectively, with a maximum wavelength centered at 327 nm (Fig. 1A-C). The spectrum recorded at 5x10-8 M was analyzed only to show the AmB concentration dependence since there is no difference between the studied formulations. As a matter of fact, this behavior was expected because there is a predominance of monomeric form at this concentration, as previously described. Taking into account that an isosbestic point was obtained at around 340 nm, the region of aggregates can be observed on the left of the region of 340 nm and monomers on its right (Fig. 1 and 2). At higher concentrations (5x10 -5 and 5x10-6 M) the maximum of absorption was detected at 327 nm for M-AmB and 323 nm for heated one. As the AmB concentration decreases (5x10 -7 M and 5x10-8 M), the intensity of absorption changed to three peaks of absorption at 364, 385 and 408 nm showing that a decrease in the amount of aggregates occurs at the same time as an increase in the amount of monomeric forms (Fig. 1 and 2). This variation is related to the equilibrium between the monomeric and aggregated forms of AmB, which can be represented by the following equilibrium equation:. (1) Where: AmBag = AmB aggregates AmBm = AmB monomers k = constant of equilibrium between AmBag and AmBm.

(28) 27. According to the Fig. 2, it is observed that after freezing and drying, the observed pattern remained the same for H-AmB. Toxicity of H-AmB micellar systems against mammalian cells was significantly reduced The K+ and hemoglobin leakage following AmB administration has been extensively used as evidence of acute and chronic toxicity, respectively (21, 27). Our results are shown in Fig. 3 and 4. For K+ leakage, all samples of MAmB and H-AmB presented a similar profile. The freezing-drying did not modify the behavior of H-AmB samples: at the highest concentrations (50 mg.L-1 and 5 mg.L-1), the K+ leakage was found to be higher than 90% for the six analyzed AmB preparations (Fig. 3). However, a significant difference in hemoglobin leakage was found for MAmB and H-AmB. While M-AmB induced the release of 99.10% ± 0.02 and 81.78% ± 0.01 of hemoglobin for the concentrations of 50 and 5 mg.L-1, respectively, H-AmB formulations even at the highest concentration, presented lower than 20% of release (Fig. 4A). The same rate was found for the freezedried formulations (Fig. 4B and C). Activity of H-AmB and M-AmB against Candida albicans strains was similar The in vitro activity against Candida albicans was determined by means of minimum inhibitory concentration (MIC 90) and K+ release. The MIC was found to be 0.5 μg.mL-1 of AmB for our six preparations. Such data are in agreement with K+ release study in which all preparations showed the same profile with no statistical significance difference among them at all four concentrations (p>0.05) (Fig. 3A-C). In fact, at the highest concentration, a significant antifungal activity was observed reaching around 81.80 ± 0,06, 81.73 ± 0.64 and 86.42% ± 1.2 for H-AmB (Fig. 3A), FH-AmB (Fig. 3B) and DH-AmB (Fig. 3C) at 50 mg.L-1, respectively, with no statistical difference (p> 0.05). For unheated one, these values changed to 84.21 ± 3.17, 80.85 ± 1.86 and 84.91% ± 0.69 for M-AmB, FM-AmB and DM-AmB, respectively, at the same concentration (p>0.05)..

(29) 28. DISCUSSION The freeze-drying process consists in the removal of the solvent from a frozen product by vacuum sublimation (26). Organic molecules are subject to a variety of chemical reactions in aqueous solutions, many of which are unacceptable in terms of product stability. For instance, hydrolysis and oxidation are reactions which can reduce the biological activity (26). It has been proved that in the dry state such degradation reactions can be significantly delayed (26). Freeze-drying cycle can be divided into three steps: freezing, where the solidification occurs; primary drying in which the freeze sublimates; and a secondary drying, when desorption of unfrozen water happens (29). The first consequence of freezing is a rapid solute concentration. A secondary one consists in a higher chance of chemical reactions such as oxidation (30). The seven conjugated double bonds in the AmB molecule make it more susceptible to this process (31). Radical autoxidation of Fungizone® was demonstrated by Schreier and Lamy-Freund and the its kinetic is related to the aggregation state of the antibiotic (32). Gaboriau and co-workers have suggested that superaggregates are more stable to autoxidation. They pointed out a decrease of 20% in the total AmB content of an unheated AmB solution, as a consequence of the autoxidation processes while a heated AmB solution after incubation for 1h at 37°C under stirring did not undergo any relevant change in the drug concentration (18-19). In another study, while autoxidation process involved less than 10% of the heated solution, the unheated one presented a 25%-decrease, both after 2 h under incubation (18-19). In light of these findings, we can partially explain why the superaggregates presented the same performance after freeze-drying process with no need of a cryoprotective agent. After heating, an increase in the turbidity of the system was observed. The turbidity of a suspension is related to the reduction in intensity of the transmitted beam because of scattering. Consequently, as the particle radius becomes larger, the turbidity increases. Therefore, the change in the turbidity of the system after heating is explained by the larger size of H-AmB.

(30) 29. (approximately 300 nm) according to Van Etten et al (2000) as well as its reservoir behavior (21, 33-34). At 5x10-8 M, there was no difference between H-AmB and M-AmB. However, the absorption spectrum displays the direct influence of the drug concentration on the AmB form present in solution. Accordingly, the monomeric form predominates as the concentration decreases with a maximum of absorption centered at 408 nm. Gaboriau et al have demonstrated that after heating, the aggregates are condensed with the monomer yielding the superaggregates (18). As a matter of fact, our results corroborate with their findings because after heating, the three heated formulations (Fig. 2) showed a higher intensity at 323 nm, which is different from the characteristic wavelengths of aggregates for unheated AmB (Fig. 1). Baas et al have suggested that the heating accelerates the dissociation of AmB molecules from the deoxycholate molecules while AmB self associates (35). We believe that the blue-shift after heating is a consequence of a different pattern of organization of the AmB with the deoxycholate salt. In fact, because of the seven double conjugated bonds of AmB and the aggregates larger size after heating (34), the amount of double bonds that are exposed to the spectrum light might have changed as we could see by the blue-shift and different pattern of molar absorptivity, specially at the concentration of 5x10 -7 M. AmB acts at the membrane level by binding to sterols and is toxic to both fungal and mammalian cells (36). At low levels, AmB causes a reversible permeabilizing effect on erythrocytes, whereas at higher levels it causes lysis. These two mechanisms of action are not completely understood, but it is believed that a permeabilizing effect is caused by AmB ability to form transmembrane channels, while the lytic effect is related to the peroxidative action at membrane level (37) by free radical production from autoxidation process (18, 31-32, 38). The oxidation of unsaturated fatty acids leads to a change in the integrity of the membrane, which becomes more susceptible to the osmotic shock caused by the formation of channels (Gaboriau et al., 1997a). Taking into consideration that superaggregates are more stable to the.

(31) 30. autoxidation process, the consequent lower production of free radicals would result in a reduced incidence of peroxidative and lytic actions of AmB as previously described. Therefore, this fact is believed to partially explain the lower ability of superaggregates at inducing lysis on RBC membranes. In order to assess M-AmB and H-AmB activities, we chose to replace the colony-forming unity survival rate (21), which is a laborious and timeconsuming assay, for the microdilution method. It is a reference method for antimicrobial susceptibility used when a more accurate result is required for clinical management (39). The activity of both H-AmB and M-AmB at their three studied forms was similar with no statistical difference among them. These results are in agreement with the findings by Lambing et al who demonstrated that the drug aggregates are responsible for channel-inducing species against cholesterol, but not for ergosterol-containing membranes (40).. CONCLUSION The heating process is an excellent approach to reduce the toxicity of AmB while keeping its activity by a simple and low-cost procedure. For the first time, it was demonstrated that AmB micellar system is able to be freeze-dried, with no change in its physicochemical and activity/toxicity properties. Thus, the freeze-drying technique may be a suitable method to increase the long term stability of H-AmB systems.. Acknowledgements: The authors wish to thank CAPES for the financial support and Cristália for gentle gift of Fungizone® samples. The authors are also grateful to Glenn Hawes, from the University of Georgia, American Language Program, for editing this manuscript..

(32) 31. Legend to figures Scheme 1: Graphical representation of the studied samples highlighting their treatment methods before analysis. Abbreviations: M-AmB: AmB micellar system; H-AmB: heated AmB; FM-AmB: frozen M-AmB; FH-AmB: frozen HAmB; DM-AmB: dried M-AmB and DH-AmB: freeze-dried H-AmB. Figure 1. Absorptivity spectra of M-AmB before freeze-drying (A); after freezing (B) and after freeze-drying (C) at 5x10-5M, 5x10-6M, 5x10-7M and 5x10-8M. Figure 2. Absorptivity spectra of H-AmB before freeze-drying (A); after freezing (B) and after freeze-drying (C) at 5x10-5M, 5x10-6M, 5x10-7M and 5x10-8M. Figure 3. In vitro release of potassium from human red blood cells and Candida albicans induced by M-AmB and H-AmB. (A) before freeze-drying; (B) after freezing and (C) after freeze-drying. Abbreviations: See Scheme 1. Figure 4. In vitro release of hemoglobin from human red blood cell and Candida albicans induced by M-AmB and H-AmB. (A) before freeze-drying; (B) after freezing and (C) after freeze-drying. Abbreviations: See Scheme 1..

(33) 32. Figure 1 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450. 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450. 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450.

(34) 33. Figure 2 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450. 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450. 120000. ε (M-1.cm-1). 100000 80000 60000 40000 20000 0 300. 350 400 Wavelength (nm). 450.

(35) 34. Figure 3. 120. K+ leakage (%). 100 80 60 40 20 0 0.05. 0.5 5 50 Concentration of AmB (mg.L-1). Concentration of AmB (mg.L-1) 120. K+ leakage (%). 100 80 60 40 20 0 0.05. 0.5. 5. 50. 120. K+ leakage (%). 100 80 60 40 20 0 0.05. 0.5 5 50 Concentration of AmB (mg.L-1).

(36) 35. Figure 4 120 Hemoglobin leakage (%). 100 80 60 40 20 0 0.05. 0.5 5 Concentration of AmB (mg.L-1). 50. Hemoglobin leakage (%). 120 100 80 60 40 20 0 0.05. 0.5. 5. 50. Concentration of AmB (mg.L-1). 120. Hemoglobin leakage (%). 100 80 60 40 20 0 0.05. 0.5. 5. Concentration of AmB (mg.L-1). 50.

(37) 36. Scheme 1.

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(41) 40. 36.. Bolard J, Legrand P, Heitz F, Cybulska B. One-sided action of. amphotericin-B on cholesterol-containing membranes is determined by its selfassociation in the medium. Biochemistry. 1991;30(23):5707-15. 37.. Brajtburg J, Elberg S, Schwartz DR, Vertutcroquin A, Schlessinger D,. Kobayashi GS, et al. Involvement of oxidative damage in erythrocyte lysis induced by amphotericin-B. Antimicrob Agents Chemother. 1985;27(2):172-6. 38.. Leon C, Taylor R, Bartlett KH, Wasan KM. Effect of heat-treatment and. the role of phospholipases on Fungizone((R))-induced cytotoxicity within human kidney proximal tubular (HK-2) cells and Aspergillus fumigatus. Int J Pharm. 2005;298(1):211-8. 39.. European Comm Antimicrobial S. Determination of minimum inhibitory. concentrations (MICs) of antibacterial agents by agar dilution. Clinical Microbiology and Infection. 2000 Sep;6(9):509-15. 40.. Lambing HE, Wolf BD, Hartsel SC. Temperature effects on the. aggregation state and activity of amphotericin-B. Biochim Biophys Acta. 1993;1152(1):185-8. 41.. Cheron M, Petit C, Bolard J, Gaboriau F. Heat-induced reformulation of. amphotericin B-deoxycholate favours drug uptake by the macrophage-like cell line J774. J Antimicrob Chemother. 2003 Dec;52(6):904-10. 42.. Sivak O, Bartlett K, Wasan KM. Heat-treated Fungizone retains. amphotericin B antifungal activity without renal toxicity in rats infected with Aspergillus fumigatus. Pharm Res 2004;21(9):1564-6..

(42) 41. 6 COMENTÁRIOS, CRÍTICAS E SUGESTÕES. A etapa inicial do mestrado foi iniciada com o estudo das técnicas de cromatografia líquida de alta eficiência para validação do método analítico e posterior quantificação do fármaco de estudo para análise de sua estabilidade após um tempo pré-determinado. Essa etapa consumiu um bom tempo do cronograma. e. não. forneceu. uma. adequada. reprodutibilidade.. Esse. comprometimento na reprodutibilidade pode estar associado com o uso da coluna cromatográfica para análise de várias moléculas, com diferentes fases móveis. Com parte do cronograma comprometido e após uma avaliação do trabalho feito com o orientador foi traçada uma nova estratégia de trabalho que consistiu no estudo de efeito da liofilização do sistema de anfotericina B aquecido, já que não há nada descrito na literatura sobre isso. Os dados iniciais obtidos com a validação do sistema gerou um trabalho no Congresso Latino-Americano de Cromatografia e Técnicas Relacionadas, evento que ocorreu em Florianópolis em 2012, com convite para publicação numa revista de edição especial. No mesmo ano também foram divulgados resultados por meio de quatro trabalhos apresentados no XIV Congresso Científico UNP e outro no congresso internacional NanoFlórida 2012. No ano de 2011, foram publicados dois trabalhos no 1 Workshop Brasileiro de Tecnologia Farmacêutica e Inovação, em Aracaju. Um trabalho no XXII CIC Congresso de Iniciação Científica da UFRN e outro no IX Congresso Brasileiro de Farmácia, em Olinda. Os resultados obtidos até o presente momento permitem ter como perspectiva o estudo mais minucioso desse novo sistema que se demonstra promissor para uso clínico quando se considera o não comprometimento das suas propriedades físico-químicas e farmacotoxicológicas após o processo de liofilização, requisito essencial para sua estabilidade físico-química..

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