In vitro model for amyloid aggregation in regular insulin formulation and the use of polyphenols and vitamins as excipients for stabilization

Autor: Souza e Silva, Alessandra Carvalho de
Přispěvatelé: Rosado, Luiz Henrique Guerreiro, Rocha e Lima, Lu?s Maur?cio Trambaioli da, Oliveira, Renata Nunes, Rey, Nicol?s Adri?n
Jazyk: portugalština
Rok vydání: 2020
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFRRJ
Universidade Federal Rural do Rio de Janeiro (UFRRJ)
instacron:UFRRJ
Popis: Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2022-09-01T14:19:41Z No. of bitstreams: 1 2020 - Alessandra Carvalho de Souza e Silva.pdf: 3102717 bytes, checksum: 031602ba4a99aa481d6351f0a65032f4 (MD5) Made available in DSpace on 2022-09-01T14:19:41Z (GMT). No. of bitstreams: 1 2020 - Alessandra Carvalho de Souza e Silva.pdf: 3102717 bytes, checksum: 031602ba4a99aa481d6351f0a65032f4 (MD5) Previous issue date: 2020-01-31 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior Diabetes Mellitus (DM) is a disease characterized by the increased glucose blood concentration (hyperglycemia), which may result from defects in secretion or action of insulin hormone, produced by pancreatic islet beta cells. Insulin is responsible for metabolizing glucose for energy production, and when some dysfunction in this hormone production happens, it is necessary to control the glucose in the bloodstream, by an exogenous replacement of insulin. However, care should be taken, as amyloid aggregates can be formed, which results from molecular destructing of the protein, causing it to lose its biological activity. These amyloid aggregates in insulin are known in the literature, but are not very well characterized, besides there are no reports on amyloid tests directly in the biopharmaceutical formulation. It was developed a simple and reproducible model that mimics the transport and storage of insulin in unrefrigerated condition, to analyze the formation of amyloid aggregates within the formulation of commercial insulin. Insulin formulations were submitted to agitation conditions (175 rpm) and temperature of 37 ?C, and its activity was monitored by Tioflavin T fluorescence (ThT) and Congo Red spectrophotometry (CR), and the morphology of the aggregates was verified though the use of transmission electron microscopy (TEM). It was possible to observe that there is formation of amyloid aggregates in commercial insulin formulations, which may present a direct relationship with the stability and efficacy of this drug in its daily use. Thus, the described system can be used in the study of amyloid aggregation and in the search for molecules with antiamyloidogenic potential. From the proposed aggregation model, substances were evaluated as excipients to increase the stability of this biopharmaceutical. Results showed that polyphenols (curcumin, quercetin and rutin) had no effect, while the use of vitamins (ascorbic acid, retinol and ? ? tocopherol) had, it and retinol and ? ? tocopherol demonstrated a more pronounced effect that was confirmed by the 3 techniques applied. Therefore, these two substances presented potential for use as excipients in improving the stability of pharmaceutical formulations, which can generate in increasing the efficacy and safety of treatment using this drug O Diabetes Mellitus (DM) ? uma doen?a caracterizada pelo aumento da glicose no sangue (hiperglicemia), que pode decorrer de defeitos na secre??o ou na a??o do horm?nio insulina, que ? produzido pelas c?lulas beta das ilhotas pancre?ticas. A insulina ? respons?vel por metabolizar a glicose para a produ??o de energia, e quando ocorre alguma disfun??o na produ??o deste horm?nio, para que haja o controle de glicose na corrente sangu?nea ? necess?rio a realiza??o de uma reposi??o ex?gena da insulina. Todavia deve-se tomar cuidado, pois pode ocorrer forma??o de agregados amiloides na insulina, que decorre da desestrutura??o molecular da prote?na fazendo com que ela perca a sua atividade biol?gica. Estes agregados amiloides na insulina s?o conhecidos na literatura, por?m n?o s?o muito bem caracterizados, al?m de n?o haver relatos sobre testes de amiloides diretamente na formula??o do biof?rmaco. Visto isso foi desenvolvido um modelo simples e reprodut?vel que mimetiza o transporte e armazenamento de insulina em condi??es n?o refrigeradas, para an?lise da forma??o de agregados amiloides dentro da formula??o de insulina comercial. As formula??es de insulina foram submetidas as condi??es de agita??o (175 rpm) e temperatura de 37 ?C, com monitoramento atrav?s das t?cnicas de fluoresc?ncia de Tioflavina T (ThT) e espectrofotometria de Vermelho de Congo (VC), e a morfologia dos agregados foi verificada atrav?s do uso de microscopia eletr?nica de transmiss?o (MET). A partir destas t?cnicas foi poss?vel observar que h? forma??o de agregados amiloides em formula??es comerciais de insulina, o que pode apresentar uma rela??o direta com a estabilidade e efic?cia deste medicamento em seu uso cotidiano. Sendo assim, o sistema estudado pode ser utilizado no estudo de agrega??o amiloide e na busca por mol?culas com potencial antiamiloidog?nico. A partir do modelo de agrega??o proposto foram avaliadas subst?ncias como excipientes para o aumento da estabilidade deste biof?rmaco. Os resultados mostraram que os polifen?is (curcumina, quercetina e rutina) n?o tiveram efeito, enquanto que o uso das vitaminas (?cido asc?rbico, retinol e ?-tocoferol) tiveram, sendo que o retinol e o ?-tocoferol demonstraram um efeito mais pronunciado que foi confirmado nas 3 t?cnicas aplicadas. Por conseguinte, estas duas subst?ncias apresentaram potencial para o uso como excipientes na melhora da estabilidade de formula??es farmac?uticas, o que pode gerar no aumento da efic?cia e seguran?a do tratamento utilizando este medicamento
Databáze: OpenAIRE
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