Avaliação da eficácia e segurança de formulações tópicas contendo ativos moduladores das enzimas LOX
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2023
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A lysyl oxidase (LOX) e as lysyl oxidase like (LOXL), são enzimas participantes no processo de regeneração cutânea. A nanotecnologia permite formular sistemas de veiculação eficientes para aplicação tópica como os transportadores lipídicos nanoestruturados (NLCs). O presente estudo teve como objetivo explorar a aplicação de compostos moduladores das enzimas LOXL2 para o controlo da regeneração cutânea. Para tal, foi avaliado o impacto de compostos inibidores da LOXL2 identificados pelo grupo CBIOS (8?,9?-epoxycoleon-U-quinone - EPCU e Pimarane 7, 15-isopimaradien-19-ol - PN1) na viabilidade de queratinócitos humanos. Foi testado o impacto de PN1, EPCU e BAPN na viabilidade de queratinócitos humanos (células HaCaT), recorrendo ao método de Cristal Violeta. O IC50 de inibição da LOXL2 de EPCU é de 58,3 uM, e no presente estudo os queratinócitos submetidos a concentração de 50 nM sofreram uma redução significativa da sua viabilidade. Relativamente ao PN1, o seu IC50 de inibição da LOXL2 é de 104,4 uM, e a partir dos resultados obtidos neste estudo com a concentração máxima de 200 nM, o composto não impactua a viabilidade celular, sendo este promissor para estudos futuros. Adicionalmente, foi verificada a possibilidade de veiculação um composto inibidor das LOXL2 comercialmente disponível, o ?-aminopropionitrilo (BAPN) através de NLCs. Relativamente à caracterização das NLCs com e sem BAPN, o diâmetro hidrodinâmico (Dh) das NLCs sem BAPN foram menores do que das NLCs com ativo, o seu índice de polidispersão (PDI) não ultrapassou 0,3 em ambos os casos, e o potencial zeta situou-se entre -18 e -22 mV. As NLCs com BAPN apresentaram um valor de pH mais alto em relação às sem BAPN. Os testes de avaliação da estabilidade demonstraram que as formulações se mantiveram inalteradas. A segurança e eficácia das NLCs sem BAPN foi testada num grupo de voluntários humanos. No que diz respeito às medições de parâmetros cutâneos após aplicação tópica, os parâmetros perda transépidermica de água (PTEA), eritema e hidratação revelaram que as NLCs têm boa compatibilidade, mas o aumento da hidratação foi pequeno. O trabalho permitiu concluir que as concentrações máximas dos inibidores testados que não induzem alterações na viabilidade das células são respetivamente: 500 nM para BAPN, 20 nM para EPCU e 200 nM para PN1. O BAPN foi incorporado em NLC, mas por ser ionizado reduziu o seu potencial zeta que poderá condicionar a estabilidade ao longo do tempo. As NLCs podem ser consideradas compatíveis com a pele. Estes dados evidenciam o potencial destes compostos como possíveis moduladores do processo de cicatrização, que poderão ser veiculados por NLCs. Palavras-chave: células HaCat, BAPN, viabilidade celular, NLC, regeneração cutânea
Lysyl oxidase (LOX) and lysyl oxidase like (LOXL) are enzymes involved in the skin regeneration process. Nanotechnology makes it possible to formulate efficient delivery systems for topical application such as nanostructured lipid carriers (NLC). The present study aimed to explore the application of compounds that modulate LOXL2 enzymes to control skin regeneration. For this purpose, the impact of LOXL2 inhibitor compounds identified by the CBIOS group (8α,9α-epoxycoleon-U-quinone - EPCU and Pimarane 7, 15-isopimaradien-19-ol - PN1) on the viability of human keratinocytes was evaluated. The impact of PN1, EPCU and BAPN on the viability of human keratinocytes (HaCaT cells) was tested using the Crystal Violet method. The IC50 of LOXL2 inhibition of EPCU is 58.3 uM, and in the present study, keratinocytes subjected to a concentration of 50 nM suffered a significant reduction in their viability. Regarding PN1, its LOXL2 inhibition IC50 is 104.4 uM, and from the results obtained in this study, with a maximum concentration of 200 nM, the compound does not impact cell viability, which is promising for future studies. In addition, the carrier compatibility of a commercially available LOXL2 inhibitor compound, β-aminopropionitrile (BAPN) was verified by NLC. Regarding the characterization of nanoparticles (NPs) with and without BAPN, the hydrodynamic diameter (Dh) indicates that the NPs without BAPN were smaller, their polydispersion index (PDI) did not exceed 0.3 in both, and the zeta potential was situated between -18 and -22 mV. NPs with BAPN have a higher pH value compared to NPs without BAPN, and this value slightly increased with storage time. Stability evaluation tests demonstrated that the formulations remained unchanged. The safety and efficacy of NPs without BAPN was tested in a group of human volunteers. With regard to the measurements of skin parameters after topical application, the parameters transepidermal water loss (TEWL), erythema and hydration revealed that the NPs have good compatibility, but the increase in hydration was small. The work led to the conclusion that the maximum concentrations of the tested inhibitors that do not induce changes in cell viability are respectively: 500 nM for BAPN, 20 nM for EPCU and 200 nM for PN1. BAPN was successfully incorporated in NLC formulation, but because it is ionized it reduces its zeta potential, which may have an impact in stability over time. NPs can be considered compatible with the skin. These data show the potential of these compounds as possible modulators of the healing process, which may be conveyed by NPs. Keywords: HaCat cells, BAPN, cell viability, NLC, skin regeneration
Lysyl oxidase (LOX) and lysyl oxidase like (LOXL) are enzymes involved in the skin regeneration process. Nanotechnology makes it possible to formulate efficient delivery systems for topical application such as nanostructured lipid carriers (NLC). The present study aimed to explore the application of compounds that modulate LOXL2 enzymes to control skin regeneration. For this purpose, the impact of LOXL2 inhibitor compounds identified by the CBIOS group (8α,9α-epoxycoleon-U-quinone - EPCU and Pimarane 7, 15-isopimaradien-19-ol - PN1) on the viability of human keratinocytes was evaluated. The impact of PN1, EPCU and BAPN on the viability of human keratinocytes (HaCaT cells) was tested using the Crystal Violet method. The IC50 of LOXL2 inhibition of EPCU is 58.3 uM, and in the present study, keratinocytes subjected to a concentration of 50 nM suffered a significant reduction in their viability. Regarding PN1, its LOXL2 inhibition IC50 is 104.4 uM, and from the results obtained in this study, with a maximum concentration of 200 nM, the compound does not impact cell viability, which is promising for future studies. In addition, the carrier compatibility of a commercially available LOXL2 inhibitor compound, β-aminopropionitrile (BAPN) was verified by NLC. Regarding the characterization of nanoparticles (NPs) with and without BAPN, the hydrodynamic diameter (Dh) indicates that the NPs without BAPN were smaller, their polydispersion index (PDI) did not exceed 0.3 in both, and the zeta potential was situated between -18 and -22 mV. NPs with BAPN have a higher pH value compared to NPs without BAPN, and this value slightly increased with storage time. Stability evaluation tests demonstrated that the formulations remained unchanged. The safety and efficacy of NPs without BAPN was tested in a group of human volunteers. With regard to the measurements of skin parameters after topical application, the parameters transepidermal water loss (TEWL), erythema and hydration revealed that the NPs have good compatibility, but the increase in hydration was small. The work led to the conclusion that the maximum concentrations of the tested inhibitors that do not induce changes in cell viability are respectively: 500 nM for BAPN, 20 nM for EPCU and 200 nM for PN1. BAPN was successfully incorporated in NLC formulation, but because it is ionized it reduces its zeta potential, which may have an impact in stability over time. NPs can be considered compatible with the skin. These data show the potential of these compounds as possible modulators of the healing process, which may be conveyed by NPs. Keywords: HaCat cells, BAPN, cell viability, NLC, skin regeneration
Descrição
Orientação: Nuno Ricardo de Almeida Saraiva ; coorientação: Catarina Fialho Rosado
Palavras-chave
MESTRADO INTEGRADO EM CIÊNCIAS FARMACÊUTICAS, CIÊNCIAS FARMACÊUTICAS, PHARMACEUTICAL SCIENCES, ENZIMAS, ENZYMES, PELE, CUTANEOUS ADMINISTRATION, FORMAS FARMACÊUTICAS, PHARMACEUTICAL FORMS