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MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
ELUCIDAÇÃO MOLECULAR E MICELAR DOS SUPERAGREGADOS DE
ANFOTERICINA B
Christian Assunção da Silva
Orientador: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito
NATAL/RN
2015
I
CHRISTIAN ASSUNÇÃO DA SILVA
ELUCIDAÇÃO MOLECULAR E MICELAR DOS SUPERAGREGADOS DE
ANFOTERICINA B
Orientador: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito
NATAL/RN
2015
Dissertação apresentada ao
Programa de Pós-Graduação em
Ciência da Saúde da Universidade
Federal do Rio Grande do Norte
como requisito para a obtenção do
título de Mestre em Ciências da
Saúde.
II
CATALOGAÇÃO NA FONTE
S586e Silva, Christian Assunção da.
Elucidação molecular e micelar dos superagregados de anfotericina B / Christian Assunção da Silva. – Natal, 2015.
50f.: il. 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 (AMB) - Dissertação. 2. Superagregados -
Dissertação. 3. Toxicidade - Dissertação. 4. Análises térmicas - Dissertação. I. Egito, Eryvaldo Sócrates Tabosa do. II. Título.
RN-UF/BS-CCS CDU: 615.282 (043.3)
III
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenador do Programa de Pós-Graduação em Ciências da Saúde: Prof.
Dr. Eryvaldo Sócrates Tabosa do Egito
IV
CHRISTIAN ASSUNÇÃO DA SILVA
ELUCIDAÇÃO MOLECULAR E MICELAR DOS SUPERAGREGADOS DE
ANFOTERICINA B
Aprovado em 20/07/2015
Branca examinadora:
Presidente da Branca: Prof. Dr. Eryvaldo Sócrates Tabosa do Egito
Membros da Branca: Profa. Dr. Franceline Reynaud
Prof. Dr. Ádley Antonini N. Lima
V
VI
AGRADECIMENTOS
São muitos os agradecimentos por essa vitória, sei que nem sempre
todos recebem mérito justo. Também sei da importância de vocês, então
dedico a vocês este momento.
Primeiramente agradeço aos meus pais, Edmilson e Maria das Graças
(in memoriam), que me deram o exemplo de dedicação e perseverança. A
minha madrasta Francisca que também sempre me apoiou nas minha
decisões. Agradeço pela compreensão, carinho e amor; por terem a minha
educação como prioridade; por me darem forças nos momentos difíceis, me
ajudarem nessa incrível jornada, fossem quais fossem os obstáculos.
Agradeço por tudo o que fizeram, mesmo quando eu reclamava. Mais, mas o
importante da vida é ter pessoas assim como vocês, sei que o é de mais
precioso você me deram, que foi a minha educação. Vocês são infinitamente
importante para minha vida.
As minhas irmãs, Eudenilza, Eliane e Débora, pela confiança na minha
capacidade, pelo acolhimento toda vez que eu voltava pra casa e por serem
pessoas em que eu sempre posso confiar e a minha sobrinha Leticia porque
sem ela não teria graça.
À toda minha família, especialmente as minha tia Ezineide, ao meu tio
Jailton por me darem forças e incentivo. Minha Avó Antônia por assumirem o
papel de cuidar de mim e das minhas irmãs logo quando minha mãe faleceu;
e aos meus primos Hézia, Hézica, Eduardo, Sirlene, Ciro, Sidney, Juliana,
Juliene, Diego, Jaderson, Stephany, Elaine, Ewerton, Davi, Linda, Larissa,
Layane, Luana e Guilherme, os quais eu considero irmãos.
VII
Aos colegas de sala que sempre me apoiaram nos estudos e me
ajudaram a crescer profissionalmente. E aqueles que não foram
simplesmente colegas, e sim amigos que estarão sempre guardados no
fundo do meu coração (Graziella, Rhadamés, Camila Carla, Philipe de
Castro, Mariana Campos, Julia Maria, Katiane Pereira, Alessandra Daniella,
José Renato e Paulo Henrique).
À todos os professores e funcionários que contribuíram para o meu
aprendizado.
Ao meu orientador Prof. Sócrates e a Gyselle por ter me mostrado o
árduo caminho da pesquisa, é por ter reconhecido meu trabalho.
À Miguel Adelino, Scheyla Daniella que não tenho nem palavras para
agradecer, mas agradeço imensamente por terem me ajudado a crescer
cientificamente e pessoalmente; pelos conselhos, paciência e puxões de
orelha. Vocês são mais que orientadores são eternos amigos. À Vanielle que
e nossa mais nova IC da equipe, desejo sucesso.
Aos meus colegas do LaSiD por me ajudarem nas manipulações,
resumos, artigos e pelos conhecimentos que me passaram; por me
escutarem ou simplesmente por “no intervalo discutirem ciência”. Entre estes
destaco Alexandrino, Caroline Aoqui, Rosilene, Alélia, Teresa, Italo, Everton,
Andreza, Christian, Gutember, Débora, Ibraim, Karen Cybelle, Marjorie, Sara,
Renata, Kátia, André e em especial a Bartô e meu Amigo/Irmão Lucas que
que foram imperecíveis para o meu sucesso, aos meus companheiros de
mestrado Sara Lima, Hennes Gentil e Quitéria porque sem eles não seria a
mesma coisa. As Pós Docs: Franceline, Francine e Julieta por formar uma
tríade perfeita e por sempre me apoiar.
VIII
Agradeço a Prof. Dr. Renata do instituto de química por ter me ajudado
nas análises de RMN.
Aos amigos Ronise, Francesco Romano, Mara Rúbia e Elias Ribeiro
agradecem por tudo, pelos conselhos, sempre me ajudando mesmo nos
momentos de alegrias e tristezas sempre ao meu lado “Amigos é um
tesouro”.
IX
“A descoberta consiste em ver o que todo
mundo viu e pensar o que ninguém pensou”
(Jonathan Swift).
X
RESUMO
A Anfotericina B (AmB) é amplamente utilizada no tratamento de
infecções fúngicas sistêmicas. No entanto, a sua utilização é limitada devido
a sua elevada toxicidade aguda e crônica. Os superagregados de AmB (H-
AmB) obtido pelo processo de aquecimento controlado, apresentam um
potencial reduzido de toxicidade quando comparados à Anfotericina B micelar
não aquecida (M-AmB). Diante do exposto, o objetivo deste estudo foi avaliar
as características físico-químicas do processo de formação dos
superagregados após a liofilização do H-AmB, por meio de técnicas tais
como a calorimetria de exploratória diferencial (DSC), termogravimetria (TG)
e análise térmica diferencial (DTA), ressonância magnética nuclear (RMN),
espalhamento dinâmico de luz (DLS) e difração de raios X (DRX). Os dados
de DLS indicaram um tamanho de aproximadamente 260 nm deste novo
sistema, sendo este tamanho compatível e viável para administração
intravenosa. A análise de DRX demonstrou a formação de um novo estado
cristalino do sistema micelar, podendo este estar relacionado a presença de
grande proporção de desoxicolato de sódio (NaDC) na formulação. O
aquecimento do NaDC resulta na formação de uma estrutura helicoidal
intermolecular, promovendo desta forma o aumento da agregação micelar e
consequentemente aumento de seu tamanho. Os estudos das análises
térmicas demonstraram que o processo de aquecimento não influência no
comportamento das amostras. Os dados de RMN da H-AmB demonstraram a
presença de ácido desoxicólico além do NaDC. O ácido desoxicólico é
formado após o processo de aquecimento e sugere-se que o equilíbrio entre
ambas as moléculas seja responsável pela redução da toxicidade da AmB.
Os resultados aqui apresentados sugerem que o processo de aquecimento
controlado altera a organização estrutural das micelas, resultando na
diminuição da toxicidade, na melhoria da estabilidade térmica e na
manutenção da atividade.
Palavras-Chaves: Superagregados; Aquecimento; toxicidade; AmB; Análises
térmicas; RMN; DLS; DRX.
XI
SUMÁRIO AGRADECIMENTOS ................................................................................................................. VI
RESUMO ............................................................................................................................................ X
1 INTRODUÇÃO ............................................................................................................... 11
2 JUSTIFICATIVA ............................................................................................................ 13
3 OBJETIVOS..................................................................................................................... 13
3.1 Objetivo geral ................................................................................................................ 13
3.2 Objetivos específicos ............................................................................................... 14
4 MÉTODOS ........................................................................................................................ 14
4.1 Preparação das formulações de AmB ........................................................... 14
4.2 Caracterização físico-química............................................................................. 15
4.2.1 Análise de tamanho dos superagregados ................................................... 15
4.2.2 Análises térmicas ....................................................................................................... 15
4.2.3 Difração de raio X ....................................................................................................... 16
4.3 Organização da estrutura química ................................................................... 16
5 ARTIGO PRODUZIDO ............................................................................................... 18
6 COMENTÁRIOS, CRÍTICAS E SUGESTÕES ............................................... 19
7 REFERÊNCIAS .............................................................................................................. 21
11
1 INTRODUÇÃO
A anfotericina B (AmB) é um antibiótico poliênico amplamente utilizado
no tratamento de infecções fúngicas sistêmicas e no tratamento de
Leishmania em seres humanos (1-3). Sua estrutura química é composta por
uma parte hidrofílica e outra hidrofóbica (4). O Fungizon®, uma preparação
comercial da AmB, é um sistema micelar (M-AmB) composto por AmB (50
mg), dedesoxicolato de sódio (NaDC) (41 mg), fosfato de sódio dibásico e
fosfato de sódio monobásico, estes últimos constituindo um sistema tampão
apresentando pH fisiológico (5). O Fungizon® apresenta uma acentuada
eficácia contra várias infecções fúngicas sistêmicas reduzindo de 80 a 50%
do risco de infecções fúngicas em pacientes imunocomprometidos, e com
isso favorecendo a redução da taxa de mortalidade de 45 para 23% (6, 7).
Apesar desta eficácia, a utilização da AmB é limitada devido a sua elevada
toxicidade tanto aguda como crônica, sendo estas caracterizadas por febre,
náuseas e principalmente nefrotoxicidade (7).
A associação da AmB à lípidos, tais como lipossomas unilamelares
(AmBisome®), dispersão coloidal (Amphocil®, ou Amphotec®), e complexo de
lípidos (Abelcet®), leva à uma redução na toxicidade da AmB, e tem sido o
objetivo de vários diferentes estudos nos últimos anos (8, 9). Apesar de estes
sistemas apresentarem uma toxicidade reduzida, a sua utilização acaba
sendo limitada devido ao custo elevado (8).
Na busca de alternativas eficazes e de baixo custo para diminuir a
toxicidade da AmB, alguns autores verificaram que o tratamento térmico
moderado apresenta-se alternativa viável. Existe na literatura relatos que o
tratamento térmico moderado do Fungizone® conduz à um rearranjo
molecular e micelar, conhecido como superagregados (H-AmB). Este
rearranjo apresenta, além da manutenção da atividade antifúngica, uma
elevada estabilidade química e ainda exibe uma menor toxicidade em células
de mamíferos (7, 10-12). De encontro com estes relatos, recentemente,
Siqueira e colaboradores (2014) demonstraram que o processo de liofilização
12
não altera as propriedades físico-químicas e biológicas do sistema micelar
aquecido (10).
A estrutura química da AmB (Figura 1) foi elucidada por métodos
químicos (13, 14), e indica a presença de 37 átomos de carbono organizado
em um anel macrocíclico fechado por lactonização, uma cadeia de duplas
ligações conjugadas e na região oposta, uma cadeia com sete grupos
hidroxilas livres, o que lhe confere característica anfifílica. A ligação dupla no
carbono 22 da AmB é responsável pela sua alta seletividade para as
membranas celulares fúngicas (15, 16).
Figura 1. Molecula de Anfotericina B (Adapatada)
A AmB é susceptível à diferentes processos de degradação, como
oxidação, fotólise e hidrólise (17), sendo tais eventos catalisados pela
presença de raios ultravioleta (luz), oxigênio (ar) e temperaturas elevadas
(18). A exposição da AmB a estes fatores favorecem o processo de
degradação levando a uma diminuição da sua atividade farmacológica. Sabe-
se que as propriedades e estabilidade térmica da AmB é de grande
importância para garantir assim a sua utilidade clínica.
Diante do exposto, este trabalho teve como objetivo a avaliação das
características físico-químicas do sistema micelar de AmB (M-AmB) após o
processo de aquecimento controlado, elucidação molecular e micelar após o
tratamento térmico e liofilização, e ainda elucidação da interação entre a AmB
e NaDC durante o processo de formação dos superagregados (H-AmB).
13
2 JUSTIFICATIVA
Apesar da existência de formulações de AmB com toxicidade reduzida,
o custo elevado das mesmas acabada limitando sua utilização(19). Diante
desta limitação, faz-se necessário buscar alternativas economicamente
viáveis para o Sistema de Assistência à Saúde Pública.
O aquecimento controlado 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 métodos de obtenção (7, 10-12).
O tratamento térmico controlado é uma importante etapa para
obtenção da formulação com baixa toxicidade. Porém, esta diminuição da
toxicidade pode ser decorrente de inúmeros aspectos tais como
conformações moleculares ou até mesmo produtos de degradação. Sabendo
que a AmB é passível de degradação térmica , tona-se necessário a
investigação do possível rearranjo molecular e micelar após o processo de
aquecimento.
3 OBJETIVOS
3.1 Objetivo geral
Avaliar as características físico-químicas do sistema micelar de AmB
após o processo de aquecimento controlado e elucidar molecularmente o
sistema micelar de AmB após o tratamento térmico e liofilização.
14
3.2 Objetivos específicos
Avaliar por meio de análises térmicas o comportamento térmico
do sistema micelar da AmB, aquecido e não aquecido;
Avaliar o estado cristalino do sistema micelar da AmB;
Determinar o tamanho dos superagregados formados pelo
aquecimento e correlacionar com uma possível administração
intravenosa;
Elucidar a estrutura molecular dos superagregados, através de
técnicas de RMN, com o intuito de identificar possíveis
alterações após o aquecimento do sistema.
4 MÉTODOS
4.1 Preparação das formulações de AmB
Um solução do sistema micelar de AmB (Cristália, Itapira, Brasil)
(5x10-3M) foi preparada adicionando água bidestilada (10 ml) ao pó liofilizado
de AmB e agitando até completa dissolução. O sistema micelar de AmB que
contém AmB (50 mg), desoxicolato de sódio (≈ 41 mg), fosfato dibásico de
sódio (10 mg) e fosfato de sódio monobásico (0,9 mg) (5), foi preparado sob
aquecimento controlado da solução a 70°C por 20 minutos em um banho
termostático sem agitação (7, 11). Foram realizados estudo com a AmB pura
para comparação (INDOFINE Chemical, Inc. New Jersey, EUA).
O sistema micelar antes da liofilização e reconstituição foi analisado
para comparação. As amostras de M-AmB e H-AMB foram congeladas a -
80°C (Brunswick INNOVA Ultra-Low congelador U525) durante 24 horas.
Finalmente, as amostras secas de H-AmB e M-AmB foram obtidos por
secagem em um liofilizador (Christ Alpha 1-2 LD, Alemanha), sob pressão
reduzida 0, 0018 mbar a -65˚C durante 24 h.
15
4.2 Caracterização físico-química
4.2.1 Análise de tamanho dos superagregados
A solução de M-AmB 5x10-3M foi diluída para 5x10-5M e então
aquecida a 70°C por 30 min. O aquecimento foi realizado em um
equipamento de espalhamento de luz dinâmico (DLS) (ZetasizerNanoZS,
Malvern Instruments Ltd., Worcestershire, RU). Neste equipamento foi
avaliada a distribuição do tamanho a cada 5 minutos durante o tempo de
aquecimento.
4.2.2 Análises térmicas
O estudo dos perfis térmicos dos sistemas foi realizado por
calorimetria exploratória diferencial - DSC (DSC 60-H celular, Shimadzu
Corporation, Quioto Japão), calibrado com índio e zinco. As análises de DSC
foram realizados para a AmB pura, M-AmB, H-AmB, NaDC e o sistema
micelar antes do processo de liofilização. Os parâmetros utilizados para a
análise foram: faixa de temperatura de 25 a 400˚C e razão de aquecimento
de 10°C/min-1(20). As amostras foram acondicionadas em cadinhos de
alumínio hermeticamente selados, com massas de aproximadamente 2mg.
As análises de Termogravimetria (TG) e análise térmica diferencial
(DTA) foram realizadas em um TGA-60 termobalança (Shimadzu
Corporation, Quioto, Japão), em uma atmosfera de N2 inerte, na faixa de 25
até 600°C tendo uma razão de aquecimento de 10°C/min-1. Foi realizado
método dinâmico por TG variando a razão de aquecimento de, 5˚C/min-1;
10˚C/min-1; 20˚C/min-1 e 30˚C/min-1 com massa de 4mg.
16
4.2.3 Difração de raio X
As análises de Difração de Raio X (DRX) foram realizadas em um
Difratômetro de Raios-X (Miniflex II, Rigaku Corporation, Japão), utilizando
Ni-filtrada, radiação Cu Kα com uma tensão de 30kV e uma corrente de 15
mA. A taxa de digitalização empregada foi 5 min-1, no angulo de 5 a 80˚ a 2θ
de intervalo (18, 20). Foram obtidos os padrões de DRX da AmB pura, H-
AmB, H-AmB, e o sistema micelar antes do processo de liofilização e do seu
principal constituinte o desoxicolato de sódio.
4.3 Organização da estrutura química
Os espectros de Ressonância Magnética Nuclear de Hidrogênio
(RMN 1H) e Carbono-13 (RMN 13C) uni e bidimensionais da H-AmB e M-AmB
foram obtidos em espectrômetro Bruker, modelo Avance DRX-500,
pertencente ao Centro Nordestino de Aplicação e Uso da Ressonância
Magnética Nuclear da Universidade Federal do Ceará (CENAUREMN-UFC).
Foram aplicadas freqüências de 500,13 MHz (1H) e 125,75 MHz
(13C), sob um campo magnético de 11,7 T, com sonda dual de 5 mm com
detecção direta. As amostras foram dissolvidas em alíquotas de 0,6 mL de
clorofórmio (CDCl3) deuterado. Os deslocamentos químicos () foram
expressos em partes por milhão (ppm) e referenciados, no caso dos
espectros de Hidrogênio, pelos picos dos hidrogênios pertencentes às
moléculas residuais não-deuteradas de clorofórmio ( 7,27). Nos espectros
de 13C, os deslocamentos químicos foram referenciados pelos picos centrais
dos 13C do clorofórmio ( 77,23).
As multiplicidades dos sinais de hidrogênio nos espectros de RMN
1H foram indicadas segundo a convenção: s (simpleto), d (dupleto), dd (duplo
dupleto), t (tripleto), q (quarteto), m (multipleto).
17
Nos experimentos unidimensionais de 1H e de 13C foram
estabelecidos os seguintes valores para os parâmetros de aquisição,
respectivamente: larguras espectrais de 24 e 260 ppm, intervalo para
relaxação de 1 s e largura de pulso de 90o de 9,60 s (0 dB) e 10,90 s (-3
dB) para 1H e 13C, respectivamente. Para todos os experimentos
unidimensionais foram utilizados 65356 pontos para a aquisição e 32768 para
o processamento, enquanto para os experimentos bidimensionais foram
utilizados 2048 x 256 pontos para a matriz de dados de aquisição e 2048 x
1024 pontos para o processamento. Predição linear para o processamento
2D, utilizando 80 coeficientes, foi usada quando necessária. O número de
transientes variou de 8, para 1H, a 16384, para 13C, para os experimentos
unidimensionais, e 2n (n ≥ 2) para bidimensionais, dependendo do
experimento e quantidade de amostra disponível. Os microprogramas
utilizados para a aquisição dos dados foram: 1H (zg), 13C-CPD (zgpg), 13C-
DEPT 135 (dept135).
A técnica DEPT (Distortionless Enhancement by Polarization
Transfer), com ângulos de nutação de 135, CH e CH3 com amplitudes em
oposição aos CH2, e 90, somente CH, foi utilizada na determinação do
padrão de hidrogenação dos carbonos em RMN 13C, descrito segundo a
convenção: C (carbono não-hidrogenado), CH (carbono metínico), CH2
(carbono metilênico ou metilidênico) e CH3 (carbono metílico). Os carbonos
não-hidrogenados foram caracterizados pela subtração do espectro DEPT
135 do espectro BB.
18
5 ARTIGO PRODUZIDO
Periódico: Molecules
ISSN: 1420-3049
Indexada em: Current Contents - Physical, Chemical & Earth Sciences
(Thomson Reuters), DOAJ - Directory of Open Access Journals, EMBASE
(Elsevier), Energy & Power Source (EBSCO), FSTA - Food Science and
Technology Abstracts (IFIS), Journal Citation Reports (Thomson Reuters),
MEDLINE (NLM), Polymer Library (Smithers Rapra), PubMed (NLM), Reaxys
- former Crossfire Beilstein (Elsevier), SCIE - Science Citation Index
Expanded (Thomson Reuters), SciSearch (Thomson Reuters), Scopus
(Elsevier), Web of Science (Thomson Reuters).
Fator de impacto: 2,7
Web Qualis: B1 para Medicina II
19
6 COMENTÁRIOS, CRÍTICAS E SUGESTÕES
A etapa inicial do metrado consistiu em estudos para avaliar a
formação dos superagregados de AmB em solução por técnicas
termoanalíticas. Nesta primeira etapa não foram obtidos resultados
satisfatórios, pois as análises térmicas de soluções são extremamente
complexas. Partindo desta problemática, optou-se em liofilizar as amostras
para então, com a obtenção dos sistemas na forma de pó, realizar as
análises térmicas para determinar o perfil térmico e possíveis alterações do
sistema após o aquecimento controlado.
Com o intuito de verificar possíveis alterações moleculares após o
aquecimento, as amostras foram submetidas a análise por espectroscopia no
infravermelho com transformada de Fourier (FTIR). Os resultados desta
análise não indicaram alterações após o aquecimento do sistema.
Considerando que dados da literatura indicam uma diminuição da toxicidade
da AmB após o aquecimento, e que este fator está relacionado a uma
alteração molecular do sistema o qual leva a redução da toxicidade da AmB,
decidiu-se utilizar técnicas mais robustas para a avaliação molecular do
sistema aquecido.
Diante do exposto, a etapa seguinte foi analisar os sistemas através
de Ressonância Magnética Nuclear (RMN). Esta análise possibilitou uma
visão detalhada da possível interação molecular entre a AmB e o NaDC.
Os dados obtidos no decorrer do mestrado, principalmente no que
concernem os dados de análises térmicas, DRX, e FTIR, foram apresentados
em diferentes eventos científicos de âmbito nacional e internacional, sendo:
dois resumos no XXI International Conference on Bioencapsulation, Berlin,
Alemanha em 2013; dois resumos no II Congress of The Brazilian Association
of Pharmaceutical Science - Pharmaceuticals of The Future: Tecnological and
Translational Estrategies. Búzios, Rio de Janeiro em 2014; e ainda dois
20
resumos no Congresso Científico da UnP XV, Natal, Rio Grande do Norte em
2014.
Os resultados obtidos até o momento nos permitem visualizar as
várias alterações moleculares após o aquecimento da AmB, isso possibilita a
realização de modelagens e/ou modificações moleculares por softwares
especializados para elucidar como se processa o mecanismo de redução da
toxicidade da AmB após seu aquecimento.
21
7 REFERÊNCIAS
1. Di Giorgio C, Faraut-Gambarelli F, Imbert A, Minodier P, Gasquet M, Dumon H. Flow cytometric assessment of amphotericin B susceptibility in Leishmania infantum isolates from patients with visceral leishmaniasis. The Journal of antimicrobial chemotherapy. 1999;44(1):71-6. 2. Caldeira LR, Fernandes FR, Costa DF, Frezard F, Afonso LC, Ferreira LA. Nanoemulsions loaded with amphotericin B: a new approach for the treatment of leishmaniasis. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2015 Apr 5;70:125-31. PubMed PMID: 25660615. Epub 2015/02/11. eng. 3. Sundar S, Chakravarty J. Liposomal Amphotericin B and Leishmaniasis: Dose and Response. Journal of Global Infectious Diseases. 2010 May-Aug;2(2):159-66. PubMed PMID: PMC2889656. 4. Selvam S, Mishra AK. Disaggregation of amphotericin B by sodium deoxycholate micellar aggregates. Journal of photochemistry and photobiology B, Biology. 2008 Nov 13;93(2):66-70. PubMed PMID: 18725181. Epub 2008/08/30. eng. 5. Sanchez-Brunete JA, Dea MA, Rama S, Bolas F, Alunda JM, Torrado-Santiago S, et al. Amphotericin B molecular organization as an essential factor to improve activity/toxicity ratio in the treatment of visceral leishmaniasis. Journal of drug targeting. 2004;12(7):453-60. PubMed PMID: 15621670. Epub 2004/12/29. eng. 6. Uehara RP, Sá VHLd, Koshimura ÉT, Prudente FVB, Tucunduva LTCdM, Gonçalves MS, et al. Continuous infusion of amphotericin B: preliminary experience at Faculdade de Medicina da Fundação ABC. Sao Paulo Medical Journal. 2005;123:219-22. 7. Silva-Filho MAd, Siqueira SDVdS, Freire LB, Ara˙jo IBd, Holanda e Silva KtGd, Medeiros AdC, et al. How can micelle systems be rebuilt by a heating process? International journal of nanomedicine. 2012;7:141-50. 8. Gangadhar KN, Adhikari K, Srichana T. Synthesis and evaluation of sodium deoxycholate sulfate as a lipid drug carrier to enhance the solubility, stability and safety of an amphotericin B inhalation formulation. Int J Pharm. 2014 Aug 25;471(1-2):430-8. PubMed PMID: 24907597. Epub 2014/06/08. eng. 9. Zhao M, Hu J, Zhang L, Zhang L, Sun Y, Ma N, et al. Study of amphotericin B magnetic liposomes for brain targeting. Int J Pharm. 2014 11/20/;475(1–2):9-16. 10. Siqueira SVS, Silva-Filho M, Silva C, Araújo I, Silva A, Fernandes-Pedrosa M, et al. Influence of the Freeze-Drying Process on the Physicochemical and Biological Properties of Pre-heated Amphotericin B Micellar Systems. AAPS PharmSciTech. 2014 2014/06/01;15(3):612-9. English. 11. Gaboriau F, Cheron M, Petit C, Bolard J. Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index. Antimicrobial Agents and Chemotherapy. 1997;41(11):2345-51. PubMed PMID: WOS:A1997YE74500005.
22
12. Gaboriau F, Cheron M, Leroy L, Bolard J. Physico-chemical properties of the heat-induced 'superaggregates' of amphotericin B. Biophysical Chemistry. 1997;66(1):1-12. PubMed PMID: WOS:A1997XE33600001. 13. Asher IM, Schwartzman G, Usasrg. Amphotericin B. Analytical Profiles of Drug Substances. 1977;6:1-42. 14. Ganis P, Avitabile G, Mechlinski W, Schaffner CP. Polyene macrolide antibiotic amphotericin B. Crystal structure of the N-iodoacetyl derivative. Journal of the American Chemical Society. 1971 1971/09/01;93(18):4560-4. 15. Vyas SP, Gupta S. Optimizing efficacy of amphotericin B through nanomodification. Int J Nanomedicine. 2006;1(4):417-32. PubMed PMID: 17722276. Pubmed Central PMCID: PMC2676632. Epub 2007/08/28. eng. 16. Cybulska B, Herve M, Borowski E, Gary-Bobo CM. Effect of the polar head structure of polyene macrolide antifungal antibiotics on the mode of permeabilization of ergosterol- and cholesterol-containing lipidic vesicles studied by 31P-NMR. Molecular pharmacology. 1986 Mar;29(3):293-8. PubMed PMID: 3951434. Epub 1986/03/01. eng. 17. Dicken CM, Rossi TM, Rajagopalan N, Ravin LJ, Sternson LA. A study of the chemical stability of amphotericin B in N , N -dimethylacetamide. Int J Pharm. 1988;46(3):223-9. 18. Aubkowski J, Bbazejowski J, Czerwinski A, Borowski E. Thermal behaviour and stability of amphotericin B. Thermochimica Acta. 1989;155:29-37. 19. Mistro S, Maciel Ide M, de Menezes RG, Maia ZP, Schooley RT, Badaro R. Does lipid emulsion reduce amphotericin B nephrotoxicity? A systematic review and meta-analysis. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2012 Jun;54(12):1774-7. PubMed PMID: 22491505. Epub 2012/04/12. eng. 20. Kim Y-T, Shin B-K, Garripelli VK, Kim J-K, Davaa E, Jo S, et al. A thermosensitive vaginal gel formulation with HP ϒCD for the pH-dependent release and solubilization of amphotericin B. European Journal of Pharmaceutical Sciences. 2010;41(2):399-406. 21. Gangadhar KN, Adhikari K, Srichana T. Synthesis and evaluation of sodium deoxycholate sulfate as a lipid drug carrier to enhance the solubility, stability and safety of an amphotericin B inhalation formulation. Int J Pharm. 2014 8/25/;471(1–2):430-8. 22. Pippa N, Mariaki M, Pispas S, Demetzos C. Preparation, development and in vitro release evaluation of amphotericin B–loaded amphiphilic block copolymer vectors. Int J Pharm. 2014 10/1/;473(1–2):80-6.
23
ANEXO
24
11
Artigo Produzido Molecules2015, 20,1-xmanuscripts; doi:10.3390/molecules200x0000x
molecules ISSN 1420-3049
www.mdpi.com/journal/molecules Article
MOLECULAR MICELLAR ELUCIDATION OF
THE “SUPERAGGREGATES” OF
AMPHOTERICIN B
Christian Silva1,4,†, Miguel Silva-Filho1,4,†, Bartolomeu Souza1,4,†, Lucas Machado2,4, Renata Araújo3 and Sócrates Egito1,2,4,†*
1 Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande do Norte (UFRN), Centro de Ciências da Saúde (CCS); E-Mails: [email protected] (C.S.) [email protected] (M.S.); [email protected] (B.S.); [email protected] (S.E.)
2 Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte (UFRN), Departamento de Farmácia. [email protected] (L.M.)
3 Instituto de Química, Universidade Federal do Rio Grande do Norte, (UFRN), Natal, RN, [email protected]
4 Laboratório de Sitemas Dispersos (LASID), Universidade Federal do Rio Grande do Norte (UFRN), Departamento de Farmácia, Natal, RN, Brazil.
† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: [email protected] Tel.: +55 84 994318816 (ext. 123); Fax: +55 84 3342 9817.
Academic Editor:
Received: / Accepted: /Published:
Abstract: Amphotericin B (AmB) is used in systemic fungal infection
treatment. However the use has been limited due its high toxicity. The
OPEN ACCESS
12
superaggregates of AmB (H-AmB) obtained by the controlled heating process
have shown to be less toxic than unheated amphotericin B micellar (M-AmB).
The aim of this study was to evaluate the characteristics of the superaggregation
after of freeze-drying H-AmB by techniques such as Differential Scanning
Calorimetry (DSC), Thermogravimetry (TG) and Differential Thermal Analysis
(DTG), Nuclear Magnetic Resonance (NMR), Dynamic Light Scattering (DLS) and
X-ray Diffraction (XRD). The DLS data have showed that the size of the
superaggregates at nearly 260mn of this new system is feasible to intravenous
administration. The XRD showed a new crystalline structure of the micellar
system, which can be explained by the behavior of the Sodium Deoxycholate
(NaDC) presents in a high proportion in the formulation. By heating the NaDC it
has the capacity to form a helical structure together with another NaDC
molecules. This promotes an increase of the micellar aggregation and its
respective increase in size. The thermal analyses studies showed that the heating
process does not affect the samples behavior. The NMR data of the H-AmB
showed the presence of deoxycholic acid besides of sodium deoxycholate. The
deoxycholic acid is produced after the heating process and perhaps the balance
between the both molecules be responsible by the decreasing of the toxicity.
These results suggest that the controlled heating process alters the structural
organization of the micellars, resulting in a decreasing of the toxicity, in a better
thermal stability and in the maintenance of the activity.
Keywords: Heating; Superaggregates; Thermal analysis; AmB; XRD;
NMR; DLS; Less toxicity
13
1. Introduction
Amphotericin B (AmB) is an polyene macrolide antifungal agent widely
used in the treatment of systemic fungal infections in humans(1). It consists of
hydrophilic polyhydroxyl and hydrophobic heptaene domains (4). The micellar
system, Fungizone® (M-AmB), is composed by AmB, sodium deoxycholate
(NaDC), dibasic sodium phosphate and monobasic sodium phosphate.
Fungizone® is a commercial preparation of AmB and it is an effective drug
available against several systemic fungal infections. Despite this, the use has
been limited by its acute and chronic toxicity, characterized by chills, fever,
nausea and nephrotoxicity (7).
Lipid-associated AmB formulations have demonstrated a reducing
toxicity such as small unilamellar liposomes (AmBisome®), colloidal dispersion
(Amphocil®, or Amphotec®), and lipid complex (Abelcet®). They have been
successfully developed with AmB. However their uses are limited because its
high cost (21).
The decrease the AmB toxicity has been the aim of several different researches
in the last years (9, 21, 22). One of the most effective and low cost alternative to
decrease the toxicity is the moderate heat treatment. It has been found that
moderate heat treatment of Fungizone® leads to a molecular micellar
rearrangement known as superaggregates (H-AmB). The new structure has
demonstrated a high chemical stability, activity maintenance in fungal cells and it
exhibits significantly lower toxicity in vitro, against mammalian cells (7, 11, 12).
Recently, Siqueira et al have also demonstrated that freeze-dying process does
not change the physicochemical and biological properties of the heated
Fungizone®(10).
14
Figure 1. Amphotericin B Molecule (Adapted)
AmB structure (Figure 1) has been elucidated by chemical methods (10).
Heptane domains is susceptible to many degradation processes like oxidizing
agents, light exposure, hydrolysis (11), ultraviolet rays, presence of oxygen (air)
and high temperatures (12), all these factors act on the degradation process
which lead to an activity decreasing. It is known that of the thermal properties
and stability of AmB is of great importance to ensure the clinical utility of the
compound. The double bond on carbon 22 of AmB is responsible for its high
selectivity for fungal cell membranes (13, 14).
The aim of this work is to obtain the H-AmB by the controlled heating
process and to evaluate the physicochemical behavior and the molecular
elucidation after the heat treatment and freeze-drying process, as well as to
elucidate how is the interaction between AmB and NaDC during the formation of
superaggregation process.
15
2. Results and Discussion
2.1.Superaggregates size
The size analysis of the M-AmB unheated (5x10-5 M) showed
superaggregates formation through the time (0 to 30 min) by heated 70˚C with a
temperature fixed for 30 minutes for Dynamic Light Scattering (Figure 2).
Heating process promoted a remarkable increase in size. At the beginning (0
min), the size was 87.3 nm, after 15 min the size increased to 164 nm. Finally, the
superaggregates size was about 289.6 nm (30 minutes).
Figure 2. Particle size distribution of M-AmB (5x10-5 M) superaggregates
formation with heated at 70oC during 30 min, ( ) size and ( ) polydispersity.
The heating process effect does not induce molecular dispersion, but it
seems to increase the size of aggregates until that the flocculation occurs.
Temperature and time are, especially, important parameters for superaggregates
formation (8). The temperature influence in the M-AmB particle size was
observed by the technique “trend of heating” where the sample was heated in a
DLS and its size was checked on the same equipment, as shown in the graph
(Figure 2). The results show that between 20 to 30 min the size of
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0
50
100
150
200
250
300
350
0 5 10 15 20 25 30
Poly
disp
ersity
Siz
e (n
m)
Time (minutes)
16
superaggregates did not significantly change, indicating that heating the samples
at 70 ˚C for 20 min was ideal for the formation of the superaggregates (around
260 nm). These results are similar as reported in literature (7, 8, 15). The
superaggregates size, about 260 nm, is an ideal and secure size to intravenous
administration because it is smaller than the blood cells size (16). The
polydispersity decrease is explained by the stabilization of superaggregates
formation, since the beginning of heating there are the presence of individual
NaDC molecules and superaggregates while after 20 minutes of heating there are
the stabilization of condensation of monomeric and aggregates forms of AmB
with NaDC (3, 8, 9).
2.2.Thermal Behavior
DSC Curves of AmB are characterized by two endothermic peaks (Figure
3), demonstrated in several studies (17, 18). DSC curve for pure AmB showed an
endothermic peak, at first event, in the range of 25 - 107˚C (∆H= -65.4 J.g-1), due
to sample dehydration. The sample dehydration was confirmed by a weight loss
of 7.7 % verified in the range of 25.8 – 108 ˚C in the TG/DTA (Figure 4). The
second endothermic peak, characteristic of pure AmB, was at range of 132.3 -
182.3˚C (∆H= -30.6 J.g-1). It showed a melting point of 173 ˚C. The third
endothermic event was at 188.6 - 204.7 ˚C (∆H= -36.4 J.g-1), which may be
attributed to drug decomposition just after its melting (12, 17, 19). TG/DTA
curves (Figure 4) for this molecule, demonstrated that the event of weight loss
only occurred over 195.3 ˚C. The results of DSC and TG/DTA for AmB obtained in
this work are in agreement with the reported data in the literature. Some studies
17
say that melting and decomposition are together, but in (figure 4), it is plain to
see that they are not together (10, 12, 18).
Figure 3. DSC curves of AmB pure, sample mass 2mg at heating rate of
10˚C/min-1.
18
Figure 4. TG/DTA curves of AmB pure in nitrogen atmosphere with a heating
ramp of 10˚C/min-1. TG (––) DTA (---).
DSC curves of H-AmB showed two specific endothermic peaks. The first
event was at range of 42.8 - 97˚C (∆H= -86.8 J.g-1), due to sample dehydration,
which was confirmed by weight loss of 9.6 % verified at 37.5 - 95˚C in the
TG/DTA (Table 1). The second event was at range of 152.4 - 181˚C (∆H= -30.2 J.g-
1), it showed the melting point of 167.2˚C, which was confirmed by weight loss of
8.3 % verified by the mid point of 169 ˚C in the TG/DTA. DSC curves for M-AmB
demonstrated two specific endothermic peaks. The first one was at range of 43.2
- 99˚C (∆H= -89.2 J.g-1), which is related to dehydration, confirmed with a weight
loss of 7.4 %, at 45.6 – 98 ˚C in the TG/DTA. The second event occurred at range
150.4 - 180˚C (∆H= -29.1 J.g-1), which the melting point was at 167˚C, confirmed
by weight loss of 8.4 % verified by the mid point of 163 ˚C in the TG/DTA. The
19
Fungizone sample showed in DSC curves two specific endothermic peaks. The
first event shows in the range of 42-103˚C (∆H=-90.2Jg-1), due to sample
dehydration, which was confirmed by weight loss of 10.8% verified in the 48-
102.3˚C in the TG/DTA. The second event shows in the range of 162.3-180.8˚C
(∆H=-33.1Jg-1), shows in the melting point of 168.5˚C, which was confirmed by
weight loss of 5.2% verified by the mid point of 170.6˚C in the TG/DTA. H-AmB,
M-AmB, and Fungizone demonstrated that the thermal characteristics are
similar.
The DSC curve for pure NaDC shows a first event endothermic peak in the
range of 112-131˚C (∆H=-0.68Jg-1), due to water crystallization into the crystal of
the sample(20), which was confirmed by a weight loss of 15.9% verified in the
range of 46-80˚C in the TG/DTA, in the second event shows exothermic peak in
the range of 173-178˚C (∆H=+0.01Jg-1), confirmed by DTA the similar the DSC
data, this exothermal process, probably due to reorganization of the solid phase,
is characteristic of some disordered crystallization or amorphous sample
phase(20). The third endothermic event shows in the range of 198-205˚C (∆H=-
0.10Jg-1), shows the melting point of 202˚C. The events of NaDC in the TG/DTA
showed only two steps of weight loss. The first one in the range of 46-80˚C had
weight loss at 15% and the second one in the range of 428-477˚C had weight loss
65%,.This last step is probably the decomposition event. These results of DSC
and TG/DTA for NaDC are in agreement with the data reported in the literature
(20).
The maintenance of the physicochemical properties of the AmB micellar
system after the thermal treatment is necessary for assuring the biological
activity of the drug. The DSC and TG/DTA analyses showed that thermal
20
properties are very similar to both, before and after the thermal treatment
(Table 1). The Fungizone before freeze-drying, demonstrated that the stress
conditions during the freeze-drying process did not affect the thermal properties
neither of the structural molecules of the drug (Figure 4). The thermal data
obtained by DSC analysis of pure AmB, H-AmB, M-AmB and Fungizone before
freeze-drying (Table 1) showed similar profiles.. The events of AmB is
characterized by two events, one at 173°C which can be attributed to the melting
point of the drug and the other at 198°C which is attributed to the total
decomposition (Figure 3). According to The Analytical Profiles of Drug
Substances (17) and in analyses with temperatures over 200°C is not found
evidence of the presence of the drug, possibly due to the decomposition. Studies
show that the decomposition of the amphotericin B is not clarified, and some
authors also said that possibly the peaks of melting and decomposition coincide
(17, 21). The data obtained in our analysis showed that these peaks are found at
different temperatures. The data of DSC of AmB showed clearly that the second
event is characterized by the total decomposition of the drug is confirmed by
TG/DTA data showing (Figure 4).
21
Figure 5. DSC curves of NaDC, sample mass 2mg at heating rate of 10˚C min-1.
Figure 6. TG/DTA curves of NaDC in nitrogen atmosphere with a heating ramp of
10˚C/min-1. TG (––) DTA (---).
22
The melting point of H-AmB, M-AmB, was about 168˚C, and it is very
similarly of pure AmB (17, 18), indicating that the formulation did not change
the thermal profile of the drug. The thermal events of weight loss were
confirmed by TG/DTA data (Table 1) the DSC and TG/DTA data, showing that the
heating process at 70˚C for 20 minutes do not change thermal profile of AmB
micellar system. It seems also that the presence of NaDC had no influence in the
thermal profile (Figure 5 and 6). NaDC is a kind of cholic salts which can forms a
micelle and complex with drugs (22), so that the NaDC it is used to form the AmB
micellar system. As the superaggregation formation was improved with heating
process, we can suggest that the complex formation of AmB and NaDC can be
improved by heating of the system (20). Different heating rates (5, 10, 20 e 30
oC.min-1) in TG curves of H-AmB and M-AmB (Figure 7 and 8), demonstrate not
to change the profile of weight loss, indicating that heating process don’t change
thermal stability of the drug. DTA data of H-AmB and M-AmB (Figure 9) both are
showing similarity, however the last events ( 400 ˚C) can indicate the presence
of a degradation product.
23
Figure 7. TG curves of H-AmB in nitrogen atmosphere with varying the heating ramp (–––) 30 ˚ C.min-1, (-----) 20 ˚C.min-1, (–––) 10 ˚C.min-1 and (-----) 5 ˚ C.min-1.
Figure 8. TG curves of M-AmB in nitrogen atmosphere with varying the heating ramp (–––) 30 ˚ C.min-1, (-----) 20 ˚C.min-1, (–––) 10 ˚C.min-1 and (-----) 5 ˚ C.min-1.
24
Figure 9. DTA curves of H-AmB (––), M-AmB ---).
2.3.Crystallinity of Superaggregates
X-ray diffraction of AmB, H-AmB, M-AmB, NaDC and Fungizone (before
freeze-drying) (Figure 11) was performed to determine the crystallinity of
superaggrates structure. All the samples showed a high degree of crystallinity
demonstrated by the intense crystalline peaks. Pure AmB showed two peaks
characteristics in the range 14 and 21°, which are agreement with the reported
data in the literature (18). H-AmB, M-AmB and Fungizone (before freeze-drying)
demonstrated crystalline peaks similares in the range 25, 31˚ and a new intense
peak at 45°. NaDC showed one peak characteristic in the range 15° in agreement
with the reported data in the literature (22).
Phase transitions are commonly observed during manufacturing of
pharmaceutical products. For example, removal of a solvent is a substantial part
25
of many pharmaceutical manufacturing processes, such as freeze-drying, spray-
drying, or drying after wet granulation, resulting in changes of both chemical and
phase compositions of a system (23). Lyophilization is widely used for
pharmaceuticals to improve the stability and long term storage of labile drugs.
This is important in pharmaceutical manufacturing technology by allowing
drying of heat-sensitive drugs and biological at low temperature under
conditions that allow removal of water by sublimation, or a change of phase from
solid to vapor without passing through the liquid phase(24, 25). These processes
can change the crystal structures of some drugs by the variation of pressure and
temperature. This stress tends to modify the structure of the crystal, making the
amorphous state of the samples.. This can explain why the physical-chemistry
characteristics of Fungizone are not altered before and after lyophilization. The
literature also reports that the lyophilization process does not change the
biological properties of the superaggregates (9)
NaDC is a white powder used as a supportive solubilizing agent in
pharmaceutical formulation. D’Archivio et al. (26) suggested the possibility that
NaDC was stabilized by the formation of a tetrahydrate helix structure of NaDC
(Figure 10) with the four water molecules held rigid by the formation of ion–ion,
ion–dipole interactions and hydrogen bonding(26-28)
26
Figure 10. Sodium Desoxycolate Molecular structure. (Adapted)
Studies with characterization of the sodium deoxycholate performed by
A.R. Campanelli et al. (20) have shown that varying some thermodynamic
parameters such as ionic strength, pH, temperature and pressure, it is possible to
increase the size of the NaDC micelles aggregates in order to obtain a gel NaDC
compound helical macromolecules. Heating treatment promotes the formation of
micelles, which are connected to AmB molecule by hydrophobic interactions and
hydrogen bonding between the hydroxyl groups. These interactions play an
important role in aggregation, thus they can lead to the formation of AmB
superaggregates, which may explain the theory of formation of this system.
NaDC in aqueous solution produces fibers that give rise to a default
detailed X-ray diffraction with intense peaks as shown (Figure 11). This pattern
can be interpreted in terms of a hexagonal lattice in which the steroid molecules
are probably arranged in helices. The fibers become hexagonal crystal from
NaDC by crystallization from water or a mixture of water and acetone (20).
Diffraction pattern of each molecule is related to the crystal structure
characteristic. Since the intensity distributions of the X-ray NaDC in aqueous
27
solution, and the crystals fibers are similar as reported in literature (22), it can
be assumed that the micelles aggregates are also helical. This new molecular
rearrangement explains the displacement of the diffraction peaks relative to the
standard AmB, which showed a similar profile and reproducible to Fungizone
(before freeze-drying), M-AmB, H-AmB (Figure 11). These displacement peaks
may be resultant from that new crystalline phase.
Figure 11. X-ray diffraction patterns of H-AmB, M-AmB, Fungizon, AmB Pure,
NaDC
2.4. Chemical structure characterization
The 1H NMR spectrum of AmB, were identified by characteristic signals of
simpletos of hydrogen and methyl groups (CH3). The drug with chemical shift of
δ 0.5 to 1.5 as well as the characteristic signs of olefinic protons (C = C) from δ
6.0 to 6.5.
In the 13C NMR spectrum of M-AmB was possible to identify two
characteristic signals of unprotected carbonyl carbons (C=O) and representing
28
carboxylic acid ester with displacement δ 176.71 and 208.57 respectively,
oxygenated carbons (CO) with a displacement δ between 40.06 and 71.08 δ and
secured signals over the spectrum are related to the methylene (CH2) and methyl
(CH3) structure. In the spectrum of RMN 13C H-AmB was possible to identify an
unprotected signal characteristics of carbons carbonyl (C=O) representing
carboxylic acid with displacement δ 176.71; oxygenated carbons (C-O) with a
displacement δ between 40.06 and 71.08 δ secured signals over the spectrum
are related to the methylene (CH2) and methyl (CH3) structure.
The results of 1H-NMR referring M-AmB and H-AmB showed no
differences in the characteristic peaks simpletos concerning to methyl groups
(CH3) drug with chemical shift of δ 0.5 to 1.5 as well as characteristic signals of
olefinic protons (C=C) between δ 6.0 to 6.5 as described in the literature (17).
However, when comparing the 13C NMR spectra of M-AmB and H-AmB observed
the absence of carbons and a carbonyl grouping at δ 208.57, the results suggest
that the heating process of M-AmB at 70°C encourages greater interaction
between the drug molecules and NaDC. This interaction enables the system to
reorganize chemically and form a component to have less toxicity(3, 9). The
results of the spectrum of H-AmB presented sodium deoxycholate and
deoxycholic acid (29) as showing in (Table 2 and 3), differently from M-AmB.
The deoxycholic acid is formed after the heating process and maybe the balance
between sodium deoxycholate and deoxycholic acid be responsible by the
decreasing toxicity.
29
DA H-AmB (AmB+NaDC)
Position δH δC, δH δC,
1 0.99 35.55
35.66
2 1.62 30.21 1.38 30.21 3 3.50 72.61 1.50 71.04 4 1.67 39.28 - - 5 1.41 39.63 - - 6 2.02 34.64 - - 7 3.87 69.62 3.40 69.94
8 1.58 36.05 - - 9 - 33.18 1.97 33.21
10 - 35.40 - - 11 1.31 20.99 - - 12 2.00 40.13 - -
13 - 43.05 - - 14 - 50.87 - - 15 1.12 24.04 - - 16 1.31 28.47 1.38 28.60 17 - 56.23 - - 18 0.67 11.86 - -
19 0.92 22.91 - - 20 1.43 41.79 1.38 41.62 21 0.93 18.63 1.14 18.54 22 1.72 33.18 - - 23 2.10 35.35 1.38 35.27 24 - 185.78 - -
Table 2. Comparison between the chemical shifts of deoxycholic acid (DA) and Amphotecin B + sodium deoxycholate (AmB + NaDC).
30
NaDC H-AmB (AmB-NaDC)
Position δH δC, δH δC,
1 0.98 30.04 1.50 30.21 2 1.62 33.19 1.97 33.21 3 3.64 72.59 - - 4 1.49 35.97 - - 5 1.41 42.62 - -
6 1.86 26.76 1.14 26.11 7 1.44 27.65 1.78 27.25 8 1.49 36.51 - - 9 - 34.30 1.97 -
10 - 34.55 - - 11 1.57 28.74 1.38 28.60 12 4.06 74.67 - - 13 - 46.94 - - 14 - 48.93 - - 15 1.09 24.30 - - 16 1.27 28.17 1.38 28.60
17 - 47.77 - - 18 0.71 12.96 0.62 12.50 19 0.92 23.32 1.02 23.55 20 1.40 36.16 1.66 36.27 21 0.95 17.51 0.93 17.10 22 1.71 35.50 1.38 35.66 23 2.10 35.35 1.38 35.27
24 - 185.70 - 176.92
Table 3. Comparison between the chemical shifts of sodium deoxycholic (NaDC) and Amphotecin B + sodium deoxycholate (AmB + NaDC).
31
3. Experimental Section
3.1. Preparations of the AmB formulations
Stock solution of heated and unheated AmB micellar system (Cristália,
Itapira, Brazil) (5x10-3 M (5.000mg. L-1)) was prepared by adding bi-distillated
water (10 mL) into a vial, and it was vortex shaking until complete dissolution.
Into each vial there was AmB (50 mg), sodium deoxycholate (≈ 41 mg), dibasic
sodium phosphate (10 mg) and monobasic sodium phosphate (0.9 mg).
The H-AmB was prepared by stock solution under controlled heated (70
˚C) for 20 min in a thermostatic bath without stirring.
The AmB Pure was obtained for INDOFINE Chemical company, Inc. New
Jersey, USA. Micelar system before freeze-drying (M-AmB), was analyzed for
comparison. Frozen M-AmB and H-AmB were obtained by freezing at -80 ˚C
(Brunswick Innova® Ultra-Low U525 freezer) for 24 hr. were obtained by drying
M-AmB and H-AmB under reduced pressure (0.0018 mbar - Christ Alpha 1-2 LD,
Germany) at -65˚C for 24 h.
3.2. Physicochemical characterization
3.2.1.Superaggregates size analysis
M-AmB stock solution (5 mg. L-1) was diluted to 50 mg. L-1 and heated at
70°C for 30 min in Dynamic Light Scattering (DLS) and the size distribution was
32
evaluated every 5 min on the same equipment (ZetasizerNanoZS, Malvern
Instruments Ltd, Worcestershire, UK).
3.2.2.Thermal analysis
Differential scanning calorimetry (DSC) measurements were run on a DSC
60-H Cell (Shimadzu Corporation, Kyoto Japan), calibrated with indium and zinc.
DSC analyses were performed for pure AmB, M-AmB, H-AmB, NaDC and the
micellar system after the freeze-drying over the temperature range of 25 to
400˚C, with a heating rate of 10˚C min-1. The samples were encapsulated in
hermetically sealed aluminum pans, and the pan weights were kept constant.
Thermogravimetry (TG) and Differential Thermal Analysis (DTA) have
been performed on a TGA-60 thermobalance (Shimadzu Corporation, Kyoto
Japan), with a heating ramp of 10˚C min-1 to 600˚C in nitrogen atmosphere. TG
using a dynamic method by varying the heating rate of 5˚C min-1, 10˚C min-1,
20˚C min-1, and 30˚C min-1 rated pure AmB, M-AmB and H-AmB samples the
profile decomposition.
3.2.3. X-ray diffraction (XRD)
X-ray diffraction (XRD) patterns were recorded using a powder X-ray
diffractometer (MiniFlex II, Rigaku Corporation, Japan), using Ni-filtered, Cu Kα
radiation, a voltage of 30 kV and a 15 mÅ current. The scanning rate employed
was 5˚min-1 over the 5˚ - 80˚ 2θ range. XDR patterns of pure AmB, M-AmB, H-
33
AmB, and the micellar system after the freeze-drying and main constituent as
sodium deoxycholate were recorded.
3.3. Chemical structure organization
Nuclear Magnetic Resonance Hydrogen spectra (1H NMR) and carbon-13
(NMR C13) to be determined of the chemical structure of M-AmB and H-AmB, the
single and two-dimensional, were obtained on Bruker spectrometer Avance
DRX-500 model. Frequencies were applied to 500.13 MHz (1H) and 125.75
MHz (13C) under a magnetic field of 11.7 T with 5 mm dual probe direct
detection. Samples were dissolved in chloroform (CDCl3) deuterated (0.6 ml).
Chemical shifts δ are expressed in parts per million (ppm) and referenced in
the case of hydrogen spectra, the peaks of the protons belonging to the non-
deuterated molecules residual chloroform (δ7.27 deuterated, in carbon-13
spectra, the central peaks of the carbon-13 chloroform referenced chemical
shifts (δ 77.23).
Multiplicities of the signals hydrogen in NMR 1H spectra are indicated
according to convention: s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), q (quartet), m (multiplet). In experiments dimensional 1H and 13C the
following values were established for the acquisition parameters, respectively,
spectral widths of 24 and 260 ppm range for relaxation 1 s the width 90˚ of 9.60
µs (0 dB) and 10.90 µs (-3 dB) for 1H and 13C respectively. For every one-
dimensional experiments were used for the acquisition points 65356 and 32768
for processing, while for two-dimensional experiments were 2048 x 256 dots
used for data acquisition matrix and 2048 x 1024 points for processing. Linear
prediction processing for 2D, using 80 coefficients, is used when necessary.
34
Number of transients around either 8 to 1H in 16384, to 13C, for one-dimensional
experiments and 2n (n ≥ 2) for two-dimensional, depending on the design and
amount of sample available.
Microprograms used for the acquisition of data: 1H (zg) 13 C-CPD
(zgpg) 13 C-DEPT 135 (dept135). Technical DEPT (Distortionless Enhancement
by Polarization Transfer), with a nutation angle of 135 , CH and CH3 amplitudes
as opposed to CH2, and 90 ,only CH, was used to determine the hydrogenation of
carbon in standard NMR 13C, described according to the convention: C (non-
hydrogenated carbon), C (carbon metínico), CH2 (methylene carbon or
metilidênico) and CH3 (methyl carbon). Non-hydrogenated carbons were
characterized by spectral subtraction DEPT 135 BB spectrum.
4. Conclusions
The size of superaggregation is possible for administration by
intravenous route and the time manufactured of the new system may influence
the process of formation of superaggregates, demonstrating that 20 minutes are
fundamental for its formation. The heating process at 70˚ C was the method that
presented ideal because it does not alter the thermal behavior of the micellar
system, demonstrating to be a suitable alternative. The thermal properties and
stability of the drug is of great importance, once these properties determine the
clinical utility of the compound.
The major cause of formation of the superaggregates can be assigned of
the helical structure of NaDC that involved of the drug and after the heating
35
process occur the increasing of size of the new system. The NMR data show that
after the heating process occurs the new organization structure of molecules,
and this new structure probably is responsible for this decreasing in toxicity.
Therefore, these results indicate that the heating process alters the
organizational structure of the micelles, what may result in less toxicity, in an
improving of thermal stability and activity maintenance.
Acknowledgments
The authors wish to thank CNPQ for the financial support and Cristália for the generous gift of Fungizone® samples.
Author Contributions
C.S., M.S., L.M. and R.A. carried out the experimental work, B.S., F.R., S. and S.E. designed, supervised the work and prepared the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
11
Supplementary Materials
Table 1.Thermal Properties for the samples
Sample First event Second event Third event Fourth event
DSC
AmB 25-107˚C (89.2˚C) [∆H=-65.4Jg-1] 132.3-182.3˚C (173˚C ) [∆H=-30.6Jg-1] 188.6-204.7˚C (198.3˚C) [∆H=-36.4Jg-1] Not Presented
Fungizone 42-103˚C (68.9˚C) [∆H=-90.2Jg-1] 162.3-180.8˚C (168˚C) [∆H=-33.1Jg-1] Not Presented Not Presented
M-AmB 43.2-99˚C (69.1˚C) [∆H=-89.2Jg-1] 150.4-180˚C (167˚C) [∆H=-29.1Jg-1] Not Presented Not Presented
H-AmB 42.8-97˚C (74.3˚C) [∆H=-86.8Jg-1] 152.4-181˚C (167˚C) [∆H=-30.2Jg-1] Not Presented Not Presented
NaDC 112-131˚C (121˚C) [∆H=-0.68Jg-1] 173-178˚C (178˚C) [∆H=+0.01Jg-1] 198-205˚C (202˚C) [∆H=+0.10Jg-1] 233-239˚C (235˚C) [∆H=-0.01Jg-1]
Sample First event Second event Third event Fourth event
TG
AmB 25.8-108˚C [Tmax=49˚C (∆m=-7.7%)] 176.1-204˚C [Tmax=181˚C (∆m=-12%)] 295-337˚C [Tmax=319˚C (∆m=-50.7%)] Not Presented
Fungizone 48-102.3˚C [Tmax=60˚C (∆m=10%)] 154-184˚C [Tmax=170˚C (∆m=-5.2)] 370-453˚C [Tmax=395˚C (∆m=-39%)] Not Presented
M-AmB 46-98˚C [Tmax=49˚C (∆m=-7.4%)] 160-170˚C [Tmax=163˚C (∆m=-8.4%)] 378-464˚C [Tmax=411˚C (∆m=-45%)] Not Presented
H-AmB 37.5-95˚C [Tmax=63˚C (∆m=9.6%)] 166-181˚C [Tmax=169˚C (∆m=-8.3%)] 372-466˚C [Tmax=406˚C (∆m=-45%)] Not Presented
NaDC 46-80˚C {Tmax= 60˚C (∆m=-15%)] 428-477˚C [Tmax=450˚C (∆m=-65%)] Not Presented Not Presented
11
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