Estudo da genotoxicidade e citotoxicidade da indometacina nanoencapsulada in vitro
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Data
2016-02-16
Autores
Froder, Juliano Gabriel [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os anti-inflamatórios não esteroides (NSAIDs) estão entre os medicamentos mais amplamente utilizados no mundo inteiro. Isso se deve à medicina curativa implantada principalmente no ocidente. Com frequência os NSAIDs são usados para aliviar queixas musculoesqueléticas, artrite, bursite, dor de dentes, dismenorreia, dor de estados pós-parto e na dor de metástases de câncer no osso, sendo todas essas enfermidades associadas ao aumento da síntese de prostaglandinas. Os efeitos farmacológicos dos NSAIDs são devidos à inibição da ciclooxigenase (COX) e a subsequente diminuição da síntese de prostaglandinas (PGs), levando a uma diminuição da inflamação, dor e febre. Essa diminuição de PGs leva a uma larga escala de efeitos colaterais que são associados à NSAIDs, incluindo complicações gastrointestinais (CGI), problemas cardiovasculares, toxicidade renal, hipertensão e retenção de líquidos. Devido a essas características dos NSAIDs, justificam-se estudos envolvendo a utilização de outros meios para a entrega oral de fármacos anti-inflamatórios. Uma opção muito pesquisada, que vem crescendo de maneira significativa é o uso de nanopartículas, com potencial para constituir uma nova geração de “sistemas de entrega de drogas”. Nanopartículas apresentam uma série de características, tais como: detectar, monitorar e tratar doenças; proteger o fármaco de ácidos estomacais e enzimas do trato gastrointestinal; e proteger o estômago e intestino do próprio fármaco. A indometacina é um poderoso agente NSAID, utilizado para tratamento de artrite e osteoartrite. Como inibidora não seletiva da ciclooxigenase, principalmente no caso de inibição da ciclooxigenase-2 (COX-2), que é muito relacionada às lesões da mucosa gástrica. Por esta razão, é de grande importância que a indometacina seja objeto de estudo para novos métodos e formas de distribuição desse fármaco. Considerando que não há dados na literatura sobre a análise da biossegurança do uso da Indometacina Nanoencapsulada (NI) sobre o material genético e sua toxicidade em células de mamíferos, este estudo tem por objetivo avaliar a viabilidade celular deste medicamento nanoencapsulado pelo teste de coloração com o azul de Tripan, e sua citotoxicidade pelo ensaio do MTT, e sua genotoxicidade por intermédio do ensaio cometa e teste do micronúcleo com bloqueio na citocinese. Todos os ensaios foram feitos com o uso de culturas de linfócitos humanos de sangue periférico (células não metabolizadoras) e de células do hepatoma humano (HepG2) (células metabolizadoras) (exceto o MTT). Os ensaios dos testes de viabilidade foram realizados com tempo de exposição à substância teste por 24 horas, com as seguintes concentrações: 5, 10, 50, 125, 250, 500 e 1250 μg/ml. Os resultados obtidos permitiram a seleção de seis concentrações (5, 10, 50, 125, 250 e 500 μg/ml) 11 onde foi observado uma viabilidade celular ≥ 80%. Também foi possível realizar o ensaio de citotoxicidade utilizando as células HepG2, com as mesmas concentrações e pelo mesmo período de tempo usado no teste de exclusão azul de tripan. Os resultados foram bastante semelhantes resultados com o azul de tripan, onde apenas a concentração de 1250 μg/ml obteve uma viabilidade menor que 80%. Para o teste de genotoxicidade as células foram tratadas com as concentrações que obtiveram uma viabilidade maior que 80%, por um período de 24 horas para o teste do micronúcleo e por 4 horas para o teste do cometa. Os resultados obtidos mostraram que apenas a concentração máxima nas células metabolizadoras (HepG2), no teste do micronúcleo, foi observado um aumento estatisticamente significativo de células micronucleadas quando comparado ao controle negativo. Nas outras concentrações testadas, a NI não causou efeito genotóxico e/ou mutagênico significativo em ambas as linhagens celulares. Os resultados obtidos indicam que o nanoencapsulamento da indometacina pode ser um processo bastante promissor no sentido de desenvolver uma forma de administração deste medicamento, menos nociva a mucosa gástrica dos pacientes. Por outro lado, os resultados também apontam para a necessidade de realização de mais estudos relacionados com o potencial genotóxico de concentrações mais altas da NI, bem como de sua metabolização.
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the drugs most widely used worldwide, mainly by curative medicine that is implanted. Commonly NSAIDs are used to relieve muscle-skeletal complaints, arthritis, bursitis, toothache, dysmenorrhea, postpartum states of pain and pain of cancer metastases in the bone. Such diseases and complications are associated with increased prostaglandin synthesis. Pharmacological effects of NSAIDs are due to inhibition of cyclooxygenase (COX) and subsequent decrease of prostaglandin synthesis (PGs), causing a decrease in inflammation, pain and fever. PGs decrease leads to a wide range of side effects that are associated with NSAIDs, including gastrointestinal complications (CGI), cardiovascular disorders, renal toxicity, hypertension and water retention. Because of these characteristics of NSAIDs are justified studies involving the use of other means for oral delivery. A solution that is very researched, and has been growing significantly is the use of nanoparticles with potential to constitute a new generation of “drug delivery systems”. Nanoparticles have many characteristics, such as: detect, monitor and treat diseases; protecting the drug from stomach acids and enzymes in the gastrointestinal tract; and protect the stomach and intestines of the drug itself. Indomethacin is a potent NSAIDs agent used to treat arthritis and osteoarthritis. It has the ability to inhibit not selectively cyclooxygenase, particularly cyclooxygenase – 2 (COX-2) that is closely associated with gastric mucosal injury. For this reason, it is important that the Nanocoated Indomethacin (NI) is the subject of this study, to ensure the safety of new methods and forms of distribution of this type of drugs. There are no data in the literature on the analysis of biosafety using Nanocoated Indomethacin of the genetic material and its toxicity in mammalian cells. This study aims to assess cell viability of this medicine nanocoated by Trypan blue exclusion test, and cytotoxicity by MTT assay, and genotoxicity through the comet assay and micronucleus test with block in cytokinesis. All assays were performed using cultures of human lymphocytes from peripheral blood (not metabolizing cells) and human hepatoma cells (HepG2) (metabolizing cells) (except MTT assay). Tests of viability were performed by treatment of 24 hours with the following concentrations: 5, 10, 50, 125, 250, 500 and 1250 μg/ml. Results obtained enabled the selection of six concentrations (5, 10, 50, 125, 250 and 500 μg/ml) which was observed cell viability ≥ 80%. Was possible to perform cytotoxicity assay using the HepG2 cells with same concentrations and same period of treatment as trypan blue exclusion test. Results were quite similar with trypan blue, only the concentration of 1250 μg/ml achieved < 80% viability. In genotoxicity assay cells were 13 treated with the following concentrations: 5, 10, 50, 125, 250 and 500 μg/ml, for a period of 24 hours for micronucleus test and 4 hours for the comet assay. Results showed that only the maximum concentration in the metabolizing cells (HepG2), on micronucleus test, there was a statistically significant increase in micronucleated cells compared to the negative control. Other tested concentrations, NI did not cause any genotoxic and/or mutagenic effect, significantly, in both cell lines. The results indicate that Nanocoated Indomethacin can be a very promising method to develop an administration form of the drug, less harmful to the gastric mucosa of patients. However, the results also point to the need for further studies related to the genotoxic potential of higher concentrations of NI as well as your metabolism.
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the drugs most widely used worldwide, mainly by curative medicine that is implanted. Commonly NSAIDs are used to relieve muscle-skeletal complaints, arthritis, bursitis, toothache, dysmenorrhea, postpartum states of pain and pain of cancer metastases in the bone. Such diseases and complications are associated with increased prostaglandin synthesis. Pharmacological effects of NSAIDs are due to inhibition of cyclooxygenase (COX) and subsequent decrease of prostaglandin synthesis (PGs), causing a decrease in inflammation, pain and fever. PGs decrease leads to a wide range of side effects that are associated with NSAIDs, including gastrointestinal complications (CGI), cardiovascular disorders, renal toxicity, hypertension and water retention. Because of these characteristics of NSAIDs are justified studies involving the use of other means for oral delivery. A solution that is very researched, and has been growing significantly is the use of nanoparticles with potential to constitute a new generation of “drug delivery systems”. Nanoparticles have many characteristics, such as: detect, monitor and treat diseases; protecting the drug from stomach acids and enzymes in the gastrointestinal tract; and protect the stomach and intestines of the drug itself. Indomethacin is a potent NSAIDs agent used to treat arthritis and osteoarthritis. It has the ability to inhibit not selectively cyclooxygenase, particularly cyclooxygenase – 2 (COX-2) that is closely associated with gastric mucosal injury. For this reason, it is important that the Nanocoated Indomethacin (NI) is the subject of this study, to ensure the safety of new methods and forms of distribution of this type of drugs. There are no data in the literature on the analysis of biosafety using Nanocoated Indomethacin of the genetic material and its toxicity in mammalian cells. This study aims to assess cell viability of this medicine nanocoated by Trypan blue exclusion test, and cytotoxicity by MTT assay, and genotoxicity through the comet assay and micronucleus test with block in cytokinesis. All assays were performed using cultures of human lymphocytes from peripheral blood (not metabolizing cells) and human hepatoma cells (HepG2) (metabolizing cells) (except MTT assay). Tests of viability were performed by treatment of 24 hours with the following concentrations: 5, 10, 50, 125, 250, 500 and 1250 μg/ml. Results obtained enabled the selection of six concentrations (5, 10, 50, 125, 250 and 500 μg/ml) which was observed cell viability ≥ 80%. Was possible to perform cytotoxicity assay using the HepG2 cells with same concentrations and same period of treatment as trypan blue exclusion test. Results were quite similar with trypan blue, only the concentration of 1250 μg/ml achieved < 80% viability. In genotoxicity assay cells were 13 treated with the following concentrations: 5, 10, 50, 125, 250 and 500 μg/ml, for a period of 24 hours for micronucleus test and 4 hours for the comet assay. Results showed that only the maximum concentration in the metabolizing cells (HepG2), on micronucleus test, there was a statistically significant increase in micronucleated cells compared to the negative control. Other tested concentrations, NI did not cause any genotoxic and/or mutagenic effect, significantly, in both cell lines. The results indicate that Nanocoated Indomethacin can be a very promising method to develop an administration form of the drug, less harmful to the gastric mucosa of patients. However, the results also point to the need for further studies related to the genotoxic potential of higher concentrations of NI as well as your metabolism.
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Palavras-chave
Genotoxicidade, HepG2, Linfócitos humanos, Indometacina nanoencapsulada