Development of pharmaceutical formulation for topical administration: responsiveness of human melanoma cell lines

Author(s)

Marina Pereira Dias

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-03-30

Examining board members

Carmen Veríssima Ferreira; Silvya Stuchi Maria Engler; Adair Vieira Junior

• Advisor: Karin Juliane Pelizzaro Rocha; Carmen Veríssima Ferreira

Abstract

Drug resistance is still a challenge in the treatment of cancer and it has received great attention in the area of melanoma treatment. The discovery of B-RAF inhibitors, such as Vemurafenib, has brought a breakthrough in the treatment of this type of tumor. However, some patients develop resistance to this drug after 6 months of treatment. Therefore, the discovery of new strategies for the treatment of melanoma becomes relevant. In this context, based on the premise that irradiated riboflavin presents antimelanoma activity in in vitro and in vivo models, the main objectives of this study were to develop and characterize pharmaceutical formulations for topical administration of riboflavin (RF), to evaluate the responsiveness of normal human keratinocytes and melanoma cell lines to the treatment with the formulations and to investigate the influence of the treatment with the formulations on the melanoma biology. Four formulations based on Pluronic® (F1, F2, F3 and F4) were developed, which showed high efficiency of incorporation of RF and micellar diameter average lesser than 32nm, which make them excellent for topical administration. The bimodal distribution of the micelles population, at 25 °C, changed to a unimodal distribution at 32.5 °C, and with the incorporation of RF. In addition, the exploratory calorimetry analysis indicates an increase in ?H values, indicating that an interaction of the RF is occurring with the micellar core. The formulations had a gel temperature (T gel) lower than 25°C. In the context of the biological action, among the four formulations, the formulations F1 and F2 containing riboflavin incorporated, followed by irradiation, displayed low cytotoxic effect on human keratinocytes. However, they were able to decrease the viability of melanoma cell lines. Interestingly, both formulations tended to induce apoptosis of the SKMel-103 cells more efficiently, after 24 h of treatment. Another important finding was the fact that both formulations inhibited the cell cycle progression, as indicated by the increased expression of the p21 protein. F3 formulation, without riboflavin, presented a toxic effect on the tumor cell lines, especially for SKMel- 29 PLX - R (Vemurafenib resistant melanoma). From a molecular point of view, the treatment of this cell line with F3 promoted inhibition of autophagy, evidenced by the accumulation of p62 protein, and inhibition of AKT kinase. Our findings reinforce the antimelanoma action of RF and indicate the applicability of the F1 and F2 formulations containing incorporated riboflavin in the treatment of this cancer (AU)

• FAPESP's process: 15/01138-1 - Irradiated riboflavin in human melanoma: responsiveness of different cell lines and development of pharmaceutical formulation for topical use
• Grantee: Marina Pereira Dias
• Support Opportunities: Scholarships in Brazil - Master