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Nanoformulations of triazene analogues: in vitro stability and activity against human melanoma
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Resumo(s)
De entre os diversos tipos de cancros de pele, o melanoma maligno surge como a principal causa de morte. Fisiopatologicamente é caracterizado por uma multiplicação descontrolada de melanócitos. Actualmente, os fármacos disponíveis para o tratamento do melanoma apresentam um elevado número de efeitos secundários como consequência da sua falta de especificidade para as células cancerígenas. De forma a promover uma entrega específica destes fármacos no local de acção têm sido realizadas diversas estratégias constituindo a síntese de pró-fármacos uma das abordagens desenvolvidas.
Tal como em outros tipos de tumores, também no melanoma se encontram alteradas as diferentes vias bioquímicas. No caso específico do melanoma, observa-se um aumento da enzima tirosinase nos melanócitos afectados. Esta descoberta levou ao desenvolvimento de inúmeras investigações nesta área, assentes num conceito definido como Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT). Esta estratégia aproveita a vantagem da sobre expressão da tirosinase em células de melanoma utilizando assim esta enzima como um alvo para a activação dos pro-fármacos.
Hoje em dia, os agentes quimioterapêuticos utilizados no tratamento do melanoma contam com a utilização de diferentes agentes alquilantes como a dacarbazina. Este fármaco tem na sua estrutura uma função triazeno. Tendo como modelo o grupo triazeno e o aumento dos níveis de tirosinase nas células malignas, um grande número de pró-fármacos de triazenos tem vindo a ser desenvolvidos.
Neste trabalho procedemos à síntese de um pró-fármaco de triazeno através da junção de um monometiltriazeno com um substrato da tirosinase, análogo da tirosina, o ácido 5-(4-hidroxifenil)pentanóico. A aplicabilidade deste pró-fármaco enquanto candidato para estratégias MDEPT constituiu o principal objectivo deste trabalho de investigação.
Foram ainda desenvolvidas diferentes formulações lipossomais como sistemas de transporte do referido triazeno permitindo deste modo uma maior solubilização deste, sem necessidade de recorrer a solventes orgânicos, promovendo ainda um direccionamento específico do fármaco para seu local de acção particularmente quando administrado in vivo.
As formulações foram caracterizadas quanto às suas propriedades físico-químicas e a sua estabilidade em plasma foi determinada e comparada com o pró-fármaco na forma livre. Observou-se um aumento da estabilidade do pró-fármaco após a sua incorporação em lipossomas sendo este valor dependente da composição lipídica.
Finalmente, uma das formulações foi utilizada para a realização de estudos in vitro em células humanas de melanoma de forma a avaliar a sua citotoxicidade e comparar este parâmetro na forma livre e na forma lipossomal.
Malignant melanoma represents the main cause of death among skin cancers. It is characterized by an abnormal growth of melanocytes. Nowadays, therapeutic agents used for melanoma treatment have a considerable number of side-effects due to their lack of specificity. Many efforts have been made to overcome these issues and in order to achieve a selective drug delivery into tumor site, series of prodrugs have been synthesized. In melanoma, like in other cancers, many biochemical pathways are modified. In this specific case, the increase of melanogenesis suggests an over expression of the enzyme responsible for that process. In malignant melanocytes an over expression of the enzyme tyrosinase was detected and that led to an increase of research works based on a concept called Melanocyte-directed Enzyme Prodrug Therapy (MDEPT). This strategy takes advantage of tyrosinase overexpression in melanoma cells using this enzyme as a target for prodrug activation. Currently, chemotherapy for melanoma treatment uses a variety of alkylating agents, such as Dacarbazine. Based on its structure with a triazene function and exploiting the tyrosinase over expression in melanoma cells, a wide range of triazene prodrugs (TPD) have been developed. Herein, a new triazene prodrug was synthesized by coupling a monomethyltriazene to a tyrosinase substrate analogue of tyrosine, the 5-(4-hydroxyphenyl)pentanoic acid. The applicability of this new synthesized triazene prodrug as an MDEPT candidate constituted the main objective of this research work. Moreover, different liposomal formulations of the referred molecule were developed, since these lipid systems would be able to solubilize triazene prodrug without the need of toxic solvents and efficiently deliver them into malignant cells, particularly after in vivo administration. Physicochemical properties of liposomal formulations were fully characterized and their stability in biological media was compared with the respective free triazene prodrug. An increase on the stability of the prodrug after its incorporation in liposomes was observed, being this effect lipid composition dependent. One of the formulations was chosen to perform an in vitro assay with an human melanoma cell line and to determine the cytotoxicity of this agent in a free and incorporated form.
Malignant melanoma represents the main cause of death among skin cancers. It is characterized by an abnormal growth of melanocytes. Nowadays, therapeutic agents used for melanoma treatment have a considerable number of side-effects due to their lack of specificity. Many efforts have been made to overcome these issues and in order to achieve a selective drug delivery into tumor site, series of prodrugs have been synthesized. In melanoma, like in other cancers, many biochemical pathways are modified. In this specific case, the increase of melanogenesis suggests an over expression of the enzyme responsible for that process. In malignant melanocytes an over expression of the enzyme tyrosinase was detected and that led to an increase of research works based on a concept called Melanocyte-directed Enzyme Prodrug Therapy (MDEPT). This strategy takes advantage of tyrosinase overexpression in melanoma cells using this enzyme as a target for prodrug activation. Currently, chemotherapy for melanoma treatment uses a variety of alkylating agents, such as Dacarbazine. Based on its structure with a triazene function and exploiting the tyrosinase over expression in melanoma cells, a wide range of triazene prodrugs (TPD) have been developed. Herein, a new triazene prodrug was synthesized by coupling a monomethyltriazene to a tyrosinase substrate analogue of tyrosine, the 5-(4-hydroxyphenyl)pentanoic acid. The applicability of this new synthesized triazene prodrug as an MDEPT candidate constituted the main objective of this research work. Moreover, different liposomal formulations of the referred molecule were developed, since these lipid systems would be able to solubilize triazene prodrug without the need of toxic solvents and efficiently deliver them into malignant cells, particularly after in vivo administration. Physicochemical properties of liposomal formulations were fully characterized and their stability in biological media was compared with the respective free triazene prodrug. An increase on the stability of the prodrug after its incorporation in liposomes was observed, being this effect lipid composition dependent. One of the formulations was chosen to perform an in vitro assay with an human melanoma cell line and to determine the cytotoxicity of this agent in a free and incorporated form.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013
Palavras-chave
Mestrado Integrado - 2013