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INTRODUCTION
PD is a progressive neurodegenerative disorder with an estimated prevalence of 1-3% among adults aged over 60 years of age and 4% over 80 years of age. The average age at onset is 58-62 years, with most cases occurring at 60-79 years of age (Kouli et al., 2018). In India, increasing life expectancy has raised age-related disorders, such as Parkinson’s disease, with a prevalence expected to grow by 50% by 2030. While levodopa with carbidopa is the standard therapy, prolonged treatment leads to reduced response and motor fluctuations, necessitating adjunct therapies such as safinamide (Ahmad Al-Sabbagh et al., 2024).
Safinamide (Xadago®) is an orally active, 50 mg and 100 mg are FDA approved strengths which is a selective (USFDA, 2017; Marika et al., 2024). and reversible MOA-B inhibitor, used as an adjunct treatment in PD (Blair et al., 2017).
Nasal route is expedited route over other routes of administration where the absorption limitations and drug targeting issues. The nasal route effectively bypasses the blood-brain barrier to deliver drugs to the Central Nervous System (CNS). In situ system converts solution to gel once administered in the body (Sabale et al., 2020). In situ gelling systems are popular because of their advantages over conventional drug delivery systems, including sustained drug release, reduced administration frequency, and improved patient compliance and comfort (Vigani et al., 2020).
Physiological barriers such as the Blood-Brain Barrier (BBB) and Blood-Cerebrospinal Fluid (CSF) barrier limit drug delivery for neurological disorders, including Parkinson’s disease. Nose-to-brain delivery offers a promising alternative by using the direct connection between the nasal cavity and the CNS through the olfactory and trigeminal pathways, enabling rapid brain transport while bypassing hepatic first-pass metabolism and reducing systemic exposure (Achar et al., 2021 and Drath et al., 2025).
Conventional intranasal liquids, however, suffer from rapid mucociliary clearance and short residence time. To overcome this, thermosensitive in situ gels have been developed. These systems remain fluid at room temperature but gel at nasal temperature, improving retention and enabling sustained release, thereby enhancing intranasal delivery efficiency (Pires et al., 2022). The aim of the study was to facilitate targeted delivery of safinamide to its therapeutic site of action (brain), thereby circumventing first-pass hepatic metabolism and reducing the need for concomitant adjuvant therapy.
MATERIALS AND METHODS
Materials
Safinamide was obtained from MSN Life Sciences, Poloxamer-188 and Oleic acid were obtained from Sigma-Aldrich, HPMC K 100 M samples obtained from BASF and Ethanol from Merck. All materials used in the research work were analytical grades.
Formulation of Safinamide in situ gel
A full factorial experimental design was employed to develop a safinamide-loaded nasal in situ gel. The drug was gradually dissolved in an ethanol-distilled water mixture (1:1, v/v) under continuous stirring. Poloxamer solution: Poloxamer 188 was slowly added in purified water (part I) to which oleic acid is added under continuous mixing. Polymeric solution: Part II of water (preheated at 40ºC) HPMC K100 M is added under continues stirring, cool down to room temperature. Polymeric solution is added to the poloxamer solution followed by drug solution incorporated into the polymeric dispersion, and the resulting formulation was stirred for 15 min using a stirrer to ensure homogeneity. Finally, the volume was adjusted to the required level using distilled water (Sherafudeen et al., 2015 and Agrawal et al., 2011).
Evaluation of Safinamide in situ Gel
Ternary Phase Diagram
Ternary phase diagram is a triangular graphical representation used to illustrate the phase behavior of three-component (ternary) systems in equilibrium (Dhoot et al., 2018). This helps visualize how the composition of a mixture affects gel formation.
Gelling time
Gelling time was defined as time required for the transformation of the sol into a gel. The gelation time was monitored by placing a small amount of solution in a test tube and adding a small amount of simulated nasal fluid, maintaining the temperature at 37ºC, and visually observing the gel (the test tube was placed horizontally to observe the state of the sample and examine the gelation) formation. The gelation onset time recorded as the gelling time in seconds (Ann Rose Augusthy et al., 2017).
Mucoadhesive strength
The force required to detach the in situ gel between the two nasal mucosae was determined using a specialized chemical balance. One nasal mucosa was tied to a glass surface with a rubber band, while the other was placed in an inverted position so that both mucosal surfaces faced each other (Paul et al., 2017). The prepared in situ gel was placed between the mucosa and left in contact for a few minutes. Different mucosae were used for each formulation. The weight was increased on the other pan until the mucosa detached. Mucoadhesive strength was expressed as force detachment per cm² of the mucosa area.
Measurement of transition temperature (T sol-gel)
The gelling temperature refers to the temperature at which the meniscus of the formulation does not move when the test tube is slanted at 90 ºC. The gelling temperature was determined by placing a test tube containing a sufficient quantity of the prepared solution in a water bath at 30ºC. The temperature of the water bath was increased slowly at a constant rate of 1ºC every 2 min (Agrawal et al., 2011).
Measurement of in situ gel Rheology
A Brookfield viscometer (DV-II+Pro, USA) was used to measure the viscosities of the prepared formulation. Spindle No. 64 was used for this purpose. The viscosity was recorded by rotating the spindle at 50 rpm (Ashutosh et al., 2014).
Determination of pH
1 mL of the prepared gel was transferred to a 10 mL volumetric flask, and the solution was diluted with distilled water. The pH of the resulting solution was determined using a digital pH meter that was previously calibrated using phosphate buffers at pH 4 and 7 (Garala et al., 2013).
Drug content
1 mL of the prepared formulation was dispersed in 10 mL of distilled water with occasional shaking. The resulting solution was filtered through a 0.45 µm filter paper (Girase et al., 2021; Rushikesh Ugale et al., 2025). The amount of safinamide mesylate in the formulation was determined spectrophotometrically at 225 nm.
Ex vivo permeation study
Fresh goat nasal mucosa was collected from the slaughterhouse. The skin hair was shaved, and the skin washed with normal saline. The skin was dried between two filter papers and used directly without storage for further analysis. The dried mucosa was placed between the donor and receptor compartments in the Franz diffusion cell, approximately 5 mL of solution was placed in the donor compartment, and the receptor compartment containing pH 6.8 buffer acted as the dissolution media. The study was conducted using a magnetic stirrer (50 rpm), and the temperature was maintained at 37±0.5ºC (Basu et al., 2012). At a predetermined time, a 2 mL sample was taken up to 6 hr, and drug permeation was analyzed using UV-visible spectrophotometry (225 nm).
Droplet size distribution
The droplet size distribution of the nasal spray was evaluated using laser diffraction (Malvern Spraytec). Laser diffraction measures the droplet size distribution based on the angular variation of light scattered by droplets passing through a laser beam. Larger droplets scatter light at small angles, whereas smaller droplets scatter light at wider angles. The scattered light pattern was analyzed using Mie theory to calculate the droplet size distribution. The formulations were analyzed in triplicate, and the droplet size distribution was expressed in terms of D10, D50, D90, and Span (Strien et al., 2024).
RESULTS
Ternary Phase System
The ternary phase system prepared with varying concentrations of poloxamer 188, oleic acid, and water (Table 1) was used to construct the phase diagram (Figure 1), which provides essential insights into the formulation variables governing the performance of nasal in situ gel systems. The red-highlighted region in the diagram corresponds to formulations that successfully formed thermoresponsive gels at physiological temperature, whereas the rest were either unstable or failed to gel appropriately at physiological temperature.
| Formulation | Poloxamer 188 (%) | Oleic acid (%) | Water (%) | Formation of a Successful Gel |
|---|---|---|---|---|
| TG1 | 15 | 5 | 80 | Yes |
| TG2 | 20 | 4 | 76 | Yes |
| TG3 | 25 | 5 | 70 | Yes |
| TG4 | 30 | 8 | 62 | Yes |
| TG5 | 25 | 16 | 59 | No |
| TG6 | 20 | 8 | 72 | Yes |
| TG7 | 18 | 11 | 71 | No |
| TG8 | 15 | 10 | 75 | Yes |
| TG9 | 10 | 15 | 75 | No |
| TG10 | 15 | 15 | 70 | No |
| TG11 | 20 | 16 | 64 | No |
| TG12 | 30 | 20 | 50 | No |
| TG13 | 40 | 5 | 55 | No |
In summary, the optimal gelation zone in the ternary diagram was associated with Poloxamer 188:15-30%, Oleic Acid: 2-8%, and water: 62-80%.
Experimental Design
A full factorial design was opted to evaluate the effects of independent formulation variables on gelling time, mucoadhesive strength, and permeability coefficient. Mathematical fitting and analysis were performed using a polynomial equation. Table 2 represents the different trails proposed by the software and tested responses.
| Factor A | Factor B | Response A | Response B | Response C | |
|---|---|---|---|---|---|
| Run | Poloxamer 188 | Oleic acid | Gelling time | Mucoadhesive strength | Permeability coefficient |
| % | % | s | mg/cm2 | cm/s | |
| S1 | 17.5 | 1.25 | 38.6 | 2109.4 | 0.00203 |
| S2 | 17.5 | 1.25 | 38.4 | 2008.8 | 0.00156 |
| S3 | 17.5 | 0.5 | 32.1 | 1895.1 | 0.00144 |
| S4 | 5 | 2 | 22.9 | 2674.1 | 0.00334 |
| S5 | 5 | 0.5 | 28.4 | 2783.8 | 0.00167 |
| S6 | 5 | 1.25 | 27.3 | 2613.9 | 0.00174 |
| S7 | 17.5 | 2 | 29.7 | 1785.3 | 0.00339 |
| S8 | 17.5 | 1.25 | 38.6 | 2004.7 | 0.00199 |
| S9 | 30 | 2 | 42.1 | 1561.1 | 0.00197 |
| S10 | 30 | 1.25 | 45.3 | 1603.7 | 0.00152 |
| S11 | 30 | 0.5 | 47.9 | 1658.6 | 0.0013 |
Optimization of Dependent Variables
Response A (Gelling Time Analysis)
Gelling time was predominantly influenced by Poloxamer 188 concentration, with formulations gelling between 22.9 to 47.9 sec. The quadratic model showed strong fit (p=0.0002, r²=0.984) and predictive adequacy which was represented using 3D response surface plot (Figure 2A) and Contour plot (Figure 2B).
Response B (Mucoadhesive Strength Analysis)
Mucoadhesive strength results ranged from 1561.1 to 2783.8 g/cm2. The quadratic model was highly significant (p: 0.0001 and r2: 0.972). 3D response surface plot (Figure 3A) and Contour plot (Figure 3B) indicate that the mucoadhesive strength is majorly impacted by Poloxamer 188 concentration.
Response C (Permeability Coefficient Analysis)
Permeability coefficient results of runs ranging from 0.0013 to 0.00339 cm/s. The quadratic model was statistically significant with (p value: 0.0023 and r²: 0.898). Figures 4A, 4B represents the 3D response plots and 2D contour plots respectively which indicates, poloxamer 188 and oleic acid showed significant individual effects, while their interaction was not significant.
The model's suitability for prediction and optimization of all responses was affirmed by the non-significant lack-of-fit, confirming its adequacy.
Optimization of Formulation
To optimize the composition of independent variables, target ranges of responses were freeze i.e., gelling time (35-45 sec), mucoadhesive strength (̴2000 mg/cm2) and permeation coefficient (̴ 0.002 cm/s). The objective is to formulate the composition which can result in targeted response results. With poloxamer 188 at 17.5% and Oleic acid at 1.25% the target results were achieved, the same obtained from overlay plot (Figure 5) and point predication table is represented (Table 3) and reproducibility was observed in trials S2 and S8 at same concentrations.
| Response | Predicted Mean | Predicted Median | Observed | Std Dev | SE Mean | 95% CI low for Mean | 95% CI high for Mean | 95% TI low for 99% Pop | 95% TI high for 99% Pop |
|---|---|---|---|---|---|---|---|---|---|
| Gelling time | 37.4483 | 37.4483 | 39.11 | 2.2073 | 0.916533 | 35.281 | 39.6155 | 25.4939 | 49.4027 |
| Mucoadhesive strength | 1990.76 | 1990.76 | 1935.38 | 88.1783 | 36.614 | 1904.18 | 2077.33 | 1513.2 | 2468.31 |
| Permeability coefficient | 0.00193448 | 0.00193448 | 0.00199 | 0.000273975 | 0.000113762 | 0.00166548 | 0.00220349 | 0.000450679 | 0.00341829 |
Transition Temperature (T sol-gel)
The gelling temperature of all the formulations were in the range of 32.4 to 40.6ºC. Concentration of poloxamer 188 predominately impacted the gelling temperature. Target Nasal gelation is 34-37ºC.
Rheological parameters of in situ gel
Viscosity increased significantly with increasing poloxamer 188 concentrations, which is reflected in the results (Table 4). Viscosity results of all the runs were in the range of 424. 2 cP to 727.9 cP.
| Run | Gelling temperature | Viscosity | pH | Drug content |
|---|---|---|---|---|
| Units | ºC | Cp | % | |
| S1 | 37.8 | 554.6±4.1 | 7.4±0.4 | 98.52±7.8 |
| S2 | 37.6 | 543.2±6.8 | 7.2±0.2 | 98.97±3.6 |
| S3 | 32.4 | 503.9±5.3 | 7.1±0.3 | 97.44±4.1 |
| S4 | 35.9 | 424.2±6.1 | 7.5±0.3 | 97.72±3.8 |
| S5 | 36.3 | 478.4±9.3 | 7.2±0.5 | 98.85±7.8 |
| S6 | 36.7 | 466.8±1.8 | 7.2±0.2 | 98.26±6.6 |
| S7 | 38.8 | 612.7±3.6 | 7.5±0.5 | 96.41±4.1 |
| S8 | 37.6 | 555.1±7.1 | 7.2±0.5 | 97.26±5.2 |
| S9 | 40.6 | 688.3±14.7 | 7.4±0.6 | 98.83±4.3 |
| S10 | 39.3 | 701.6±11.6 | 7.1±0.3 | 99.12±8.3 |
| S11 | 39.4 | 727.9±8.3 | 7.4±0.7 | 99.44±6.7 |
pH
The pH of the formulation was found to be between 7.1-7.5, which is optimal.
Drug Content
The drug content of all formulations was within the target specifications, i.e., 96.41-99.44% w/w.
Ex vivo permeation results
Ex vivo permeation of all the formulations was evaluated, and results are represented in Table 5 and Figure 6. The impact of dependent variables concentrations on the drug release was clearly demonstrated.
| Time (h) | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | S11 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 0.5 | 36.09±3.4 | 36.51±1.5 | 26.09±3.7 | 43.36±4.5 | 28.75±3.2 | 30.18±4.1 | 40.37±3.8 | 35.37±3.7 | 38.73±3.1 | 32.65±1.8 | 24.21±2.8 |
| 1 | 43.25±5.2 | 43.85±3.7 | 33.07±5.1 | 55.59±5.1 | 36.27±4.4 | 39.66±4.3 | 50.68±2.9 | 44.28±4.1 | 47.17±2.7 | 41.73±6.2 | 31.44±3.8 |
| 2 | 68.48±1.8 | 70.77±2.9 | 50.12±4.6 | 78.15±6.7 | 51.94±5.5 | 54.34±2.7 | 76.91±5.6 | 68.79±3.9 | 71.95±5.8 | 58.11±3.6 | 48.81±1.9 |
| 4 | 69.27±5.3 | 72.28±5.7 | 57.49±2.8 | 81.86±3.5 | 62.61±3.2 | 65.38±6.1 | 78.38±2.2 | 71.15±6.2 | 74.15±5.2 | 70.95±5.2 | 53.59±5.3 |
| 6 | 82.65±6.2 | 78.34±2.6 | 64.13±4.7 | 99.87±5.9 | 68.49±1.9 | 71.75±4.8 | 99.61±6.7 | 76.26±5.7 | 86.88±4.5 | 74.37±4.4 | 60.18±4.7 |
| 8 | 94.39±4.4 | 96.18±4.8 | 70.85±6.2 | 75.18±4.3 | 84.86±3.7 | 94.73±2.9 | 99.17±6.1 | 88.21±3.8 | 68.26±1.8 | ||
| 10 | 99.14±5.2 | 99.44±3.3 | 79.33±1.3 | 86.38±6.8 | 94.27±2.9 | 98.83±4.1 | 99.11±1.9 | 71.85±6.4 | |||
| 12 | 88.01±5.2 | 99.92±4.4 | 99.34±5.1 | 84.22±2.9 | |||||||
| 14 | 99.67±4.8 | 95.61±5.3 | |||||||||
| 16 | 99.84±3.6 |
Droplet Size Distribution
The droplet size distribution of the optimized formula is shown in Figure 7. The span of the formulation was 1.944, indicating a moderately broad droplet size distribution, suggesting acceptable uniformity of atomized droplets suitable for nasal administration, with minimal risk of excessive fine or oversized droplets.
DISCUSSION
Safinamide nasal in situ gel was successfully formulated using a simple solution method and optimized through a full factorial design (Design Expert software version 11.03) by varying Poloxamer 188 (5-30% w/w) and oleic acid (0.5-2% w/w) concentration. Initial concentration ranges of Poloxamer 188 and oleic acid were established based on the ternary phase diagram, ensuring selection within the gel-forming domain (Dhoot et al., 2018). The formulations were evaluated for critical quality attributes including gelling time, mucoadhesive strength, and permeation coefficient.
Gelling time was significantly influenced by Poloxamer 188 concentrations. Higher polymer concentrations, i.e., 30% w/w, exhibited prolonged gelation (more than 40 S) due to increased viscosity and formation of a denser micellar network, which delays sol-gel transition. Oleic acid concentration showed no significant influence on gelling time which was observed in S4 and S7 trials where the oleic acid concentration is 2% w/w whereas the observed gelling time was 22.9 S and 29.7 S respectively.
Mucoadhesive strength exhibited an inverse relationship with Poloxamer 188 concentration. Lower polymer concentrations (5% w/w) showed higher mucoadhesive strength (S4: 2674.1; S5: 2783.8), whereas higher concentrations (S9: 1561.1; S10: 1658.6) demonstrated reduced adhesion. This behavior may be attributed to increased flexibility and chain mobility of the polymer network at lower concentrations, allowing greater interpenetration with nasal mucin (Paul et al., 2017).
Permeation studies demonstrated that increasing the concentration of Poloxamer 188 significantly decreased drug permeation. Formulations with higher polymer content exhibited lower permeability coefficients (S9: 0.00197 cm/s; S10: 0.00152 cm/s), whereas those with lower concentrations showed enhanced drug transport (S4: 0.00334 cm/s; S6: 0.00339 cm/s). This reduction in permeation at elevated polymer levels is attributed to increased viscosity and the formation of a dense gel network that restricts drug diffusion. Oleic acid contributed to a modest enhancement in drug permeation owing to its penetration-enhancing properties, which facilitate diffusion by disrupting lipid bilayers. Formulations containing higher oleic acid levels (2% w/w), such as S4 and S7, exhibited increased permeability coefficients (0.00334 cm/s and 0.00339 cm/s, respectively). However, formulation S9, despite having elevated oleic acid content, showed reduced permeation (0.00197 cm/s) due to the dominant diffusion-retarding effect of the high Poloxamer 188 concentration.
The study further demonstrated that Poloxamer 188 and oleic acid significantly influence the physicochemical properties of the formulation, including gelation temperature, viscosity, and drug release behavior. Poloxamer 188 exhibited concentration-dependent thermoresponsive behavior, with higher concentrations (30% w/w) showing elevated gelation temperatures (≈39-41ºC), while lower concentrations (5-17.5% w/w) gelled near physiological temperature. Oleic acid reduced gelation temperature by interfering with micellar organization, with more pronounced effects at higher concentrations (Agrawal et al., 2011; Ashutosh et al., 2014).
Viscosity increased with increasing Poloxamer 188 concentration due to enhanced micellar entanglement, while oleic acid modulated rheology through hydrophobic interactions. All formulations are maintained in a pH range of 7.1-7.5, indicating suitability for nasal administration and formulation stability. Drug content ranged from 96.41-99.44%, confirming uniform drug distribution in all formulations.
Drug permeation followed a concentration-dependent pattern, where formulations with lower Poloxamer 188 content (S4-S7) showed faster initial release due to a looser gel network (6 H), whereas higher polymer concentrations (S9-S11) provided sustained drug release up to 16 hr. Oleic acid further enhanced drug permeation due to its lipid-disrupting properties (Basu et al., 2012).
To validate the results, two additional trials were conducted at the optimized concentration, and the outcomes demonstrated good reproducibility.
The mean droplet size of 47.8 µm and a span value of 1.944 indicate a narrow particle size distribution, reflecting good formulation homogeneity and controlled processing. A span value below 2 is generally considered indicative of uniform particle distribution suitable for nasal delivery systems.
CONCLUSION
The present study successfully developed and optimized a safinamide-loaded nasal in situ gel using a factorial design approach. Poloxamer 188 and oleic acid were identified as critical formulation variables governing the physicochemical properties and performance of the system. Poloxamer 188 predominantly influenced gelling behavior, viscosity, mucoadhesion, and drug permeation, exhibiting concentration-dependent thermoresponsive characteristics. Higher polymer concentrations resulted in increased viscosity, prolonged gelation time, reduced mucoadhesive strength, and sustained drug release due to the formation of a dense gel matrix. In contrast, lower polymer concentrations favored faster drug permeation and enhanced mucoadhesion.
Oleic acid acted as an effective permeation enhancer and gel modifier, improving drug transport across the membrane without significantly affecting pH or formulation stability. The optimized formulations demonstrated suitable pH values, high drug content uniformity, controlled drug release profiles, and desirable rheological properties. Particle size analysis further confirmed a narrow size distribution and good homogeneity, supporting improved dissolution and bioavailability. The present study was limited to in vitro and ex vivo evaluations using nasal mucosa as a surrogate for clinical conditions. Therefore, further investigations employing appropriate animal models are planned to comprehensively assess the in vivo performance of the optimized formulation.
Overall, the study establishes that a rational balance between Poloxamer 188 and oleic acid concentrations is essential for achieving optimal gelation, permeability, and drug release behavior. Optimized formulation of nasal in situ gel system shows strong potential as an efficient and patient-friendly platform for brain targeted delivery of Safinamide.