Molecular and histopathological evaluation of nanostructured formulations of immunomodulator P-MAPA effects versus BCG immunotherapy in non-muscle invasive bladder cancer (NMIBC) progression

Abstract

The low efficacy of treatment and high rates of non-muscle invasive bladder cancer (NMIBC) recurrence may be related to low effects of these therapies on the mechanisms of tissue repair, angiogenesis and reactive oxygen species (ROS). Furthermore, current drugs have low residence time and low penetration in the urothelium, contributing to the reduction of therapeutic response. Nowadays, the most effective therapy to NMIBC is the Bacillus Calmette-Guerin (BCG) immunotherapy, which causes side effects of different intensities from irritative symptoms to serious systemic reaction, which contributes to treatment interruption besides increasing cancer index recurrence after treatment, up to 31%. Thus, the development of new molecules or improving of drugs that can increase penetration and residence time of drugs in the urothelium, with consequent reduction of intravesical instillation, rate of recurrence and progression of tumor and side effects are very relevant. In this scenario, we highlight the immunomodulator P-MAPA, which for its great versatility and minimal cytotoxicity opens a new perspective to treatment of some cancers types, including NMIBC. Thus, the aim of this study is to characterize and to compare the molecular and histopathological effects of P-MAPA formulations micro and nanostructured with BCG immunotherapy in the treatment of NMIBC progression induced in rats and to establish the effects of these therapeutic strategies in factors inducers and repairers of cell damage, angiogenesis and ROS. For this, first of all, the P-MAPA microcrystals will be nanonized and stabilized with chitosan, to increase the residence time of P-MAPA and its penetration in the urothelium, reducing thereby the number of infusions and increasing the effectiveness of this immunomodulator. These nanocrystals will be characterized in function of morphology by microscopic techniques, polymorphism and crystallinity by DSC and X-Ray diffraction, respectively, and diameter and surface charge by dynamic light scattering. Furthermore, biological activity of nanocrystals will be compared with the activity of microcrystals through of in vitro evaluation of Toll-Like Receptors (TLR) 2 and 4 stimulation, using HEK293 cells. To compare the antitumor activity of micro and nanocrystals of P-MAPA with BCG immunotherapy, Fischer 344 female rats will be used. These animals will be submitted to NMIBC chemical induction by instillation of 1.5 mg / kg of N-methyl-N-nitrosourea (MNU) every 15 days, a total of 4 doses. Two weeks after the last dose of MNU, the animals will be divided into 8 experimental groups, namely: 2 control groups for BC (1 Positive - Group 1 and 1 Negative - Group 2), 4 groups will be treated for 6 consecutive weeks with BCG (40 mg, Group 3), P-MAPA micro (5 mg / kg, Group 4), P-MAPA nano (5 mg / kg, Group 5) and P-MAPA nano with half the dose (2.5 mg / kg, Group 6); 2 groups will be treated for 3 weeks with BCG and continuing by more 3 weeks with only P-MAPA (5 mg / kg) micro (Group 7) and nano (Group 8). After 16 weeks of treatment, histopathological, molecular and toxicological evaluation of all groups will be performed. In this project, it is expected to obtain a formulation of P-MAPA nanometric in order to increase its efficiency, its penetration and its residence time in the urothelium obtaining, thereby, a more effective therapy with fewer side effects. (AU)