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- Intranasal administration of Simvastatin for NeuroprotectionPublication . Sousa, Francisco Gama; Santos, Adriana Oliveira dos; Pires, Patrícia Sofia Cabral; Meirinho, Sara AlexandraIschemic stroke is one of the most impactful chronic diseases in the world and a leading cause of disability and death. Due to the lack of therapeutic options, and the limitations of those currently available, it is crucial to find new approaches to prevent and treat ischemic stroke events. This study aimed to exploit simvastatin neuroprotective effects by developing a formulation capable of delivering it to the brain through nasal administration. For that, different formulation strategies of micro and nanoemulsions were pursued, to finally select the most promising to be compared in in vivo studies. To achieve this, an initial screening was conducted to optimize formulation composition, maximizing drug loading capacity while maintaining target nanometric and homogeneous droplet size and neutral to positive zeta potential. Evaluation of shortterm physical stability (drug precipitation upon storage at 4 °C up to 30 days) was combined with the initial screening in order to determine the formulations with the best potential to be further studied. The safety of the selected formulations was tested by hemolysis assays using human blood. An assay for simvastatin quantification was validated using liquid chromatography coupled with UV detection, and long-term physical and chemical stability tests were conducted at three different storage temperatures, 4 °C, 25 °C and 40 °C for 4 months. Preliminary in vitro drug release profiles were characterized using horizontal Ussing Chambers. The nano and microemulsions with the highest potential were also characterized in terms of their osmolality and viscosity, and nanoemulsion’s viscosity was further optimized. Two lead formulations (a microemulsion and a nanoemulsion) were intranasally administered to Wistar rats and compared at 3 time points (30, 120, and 360 min) regarding brain concentrations of both prodrug (simvastatin) and active form (tenivastatin). A second in vivo test was conducted at 4 time points (15, 30, 60, and 360 min) to obtain more data regarding the previously tested micro and nanoemulsions, to determine the impact of nanoemulsion’s viscosity and surface charge on simvastatin’s brain concentrations, and to compare these formulations to a tenivastatin solution. After the different optimizations and tests, a promising nanoemulsion strategy with cetalkonium chloride at 0.5% (w/w) was attained, exhibiting great potential in the in vivo studies. With drug strengths of 5.66% or 7.41% (w/w), this nanoemulsion under refrigeration had extremely low polydispersity index (0.069 and 0.074, respectively), small droplet sizes (117.6 nm and 116.4 nm, respectively) and a zeta potential of +25 mV for the lower drug strength. In terms of short-term physical stability, the nanoemulsion with higher simvastatin concentration only achieved stability for 3 days, but drug precipitation was not observed with the lowest concentration. Assuming the more frequently used simvastatin dose applied in ischemic stroke animal models (20 mg/Kg), and the respective two-fold dose (40 mg/Kg), the formulation proved to be safe regarding its hemolytic potential. The lower drug strength nanoemulsion (5.66%) achieved physical stability for a period of 4 months while stored at 4 °C. Simvastatin's chemical stability when using the nanoemulsion was also prolonged when stored at 4 °C. After osmolality characterization, the nanoemulsion obtained hyperosmotic values, which were considered safer than hypoosmotic values for nasal mucosa. However, to be within the safety range of 300 and 700 mOsm/Kg, the nanoemulsion would need to suffer a dilution between 1.59 and 2.66-fold. In a preliminary drug release test, the nanoemulsion displayed a Higuchi Model release profile. The baseline viscosity of the nanoemulsion at 25 °C was 24.6 mPa·s and increased to 186.2 mPa·s using PVP at 0.25% (w/w). In vivo, the nanoemulsion with cetalkonium chloride at 0.5% achieved the highest simvastatin brain concentration after 30 minutes in both the first (153 ng/mL) and second test (131 ng/mL). The obtained simvastatin brain concentrations might demonstrate some potential to be neuroprotective but, surprisingly, no tenivastatin was detected during the in vivo assays. In conclusion, a promising nanoemulsion with cetalkonium chloride 0.5% was obtained, enabling the possible use of simvastatin’s neuroprotective features in ischemic stroke using intranasal delivery. Further investigation needs to be done regarding the correlation between pharmacokinetic studies and functional studies using ischemic stroke models, to fully elucidate the role of simvastatin/tenivastatin and to improve drug applicability in cerebrovascular disease.