Intranasal Dantrolene Nanoparticles for Treatment of Amyotrophic Lateral Sclerosis as a Disease-Modifying Drug

Abstract

Calcium dysregulation, caused by pathological activation of ryanodine receptors, contributes to motor neuron degeneration, motor dysfunction, and muscle weakness in SOD1-G93A transgenic amyotrophic lateral sclerosis (ALS) mice. This study investigates the therapeutic efficacy of intranasally administered dantrolene nanoparticles, a ryanodine receptor antagonist, on motor neuron function, muscle strength, spinal cord degeneration, and survival outcomes. Male and female C57BL/6SJLF1 non-transgenic control and SOD1-G93A ALS transgenic mice were assigned to one of three experimental groups: 1) NO TX: No treatment control; 2) IN-DAN: Intranasal administration of dantrolene in the Ryanodex formulation vehicle (RFV), at a dosage of 5mg/kg, administered daily from ages 90-120 days; 3) IN-VEH: Intranasal administration of RFV alone (as a vehicle control), following the same dosing schedule as the IN-DAN condition. Body weight and general motor function were monitored weekly, with survival recorded daily throughout the treatment period. At the treatment conclusion, neurological function was comprehensively evaluated using a standardized neurological scoring system. Motor coordination and balance were assessed using the balance beam test (beam widths of 12 mm and 6 mm) and the rotarod test. Muscle strength was evaluated by measuring grip force using the Kondziela inverted screen test. After behavioral testing, spinal cord tissues were collected for analysis. The levels of neurofilament light chain (NFL), a skeletal neuron protein, and spinal cord weight were determined to measure spinal cord degeneration. Compared to non-transgenic control mice, SOD1-G93A mice exhibited significantly elevated neurological scores, indicating severely impaired neurological function. This deterioration was robustly and significantly ameliorated by IN-DAN treatment by 90% (P<0.0001). Similarly, ALS mice demonstrated impairments in motor coordination and balance on the beam balance test, with dramatic and significant increases in crossing time and the number of foot slips. These impairments were greatly and significantly mitigated by IN-DAN treatment, by 78% in crossing time (P<0.0001) and 84% in the number of slips (P<0.0001) on the 12 mm-wide beam, but not by the vehicle control. ALS mice demonstrated progressive body weight loss as well, which was similarly reversed by IN-DAN treatment, but not by the vehicle control. Muscle strength, as measured by grip force, was significantly reduced in ALS mice but robustly preserved IN-DAN treatment, which prevented the decrease by 213% (P<0.0001), while the vehicle control had no effect. Spinal cord weight was significantly reduced in ALS mice, a trend reversed by intranasal dantrolene nanoparticle treatment, but not by the vehicle control. Survival analysis revealed that 100% of control mice survived through the 30-day treatment period (up to 120 days of age), while survival in untreated or vehicle-treated ALS mice dropped to 67%. In contrast, ALS mice treated with intranasal dantrolene nanoparticles demonstrated a significantly improved survival rate of 89%. Thus, intranasal dantrolene nanoparticle treatment significantly and robustly improved neurological outcomes in SOD1-G93A ALS mice, inhibiting neurological impairment, motor dysfunction, balance deficits, and muscle weakness. These improvements were associated with a marked inhibition of spinal cord weight loss. Additionally, dantrolene treatment reversed body weight loss and significantly improved survival probability in ALS mice.

Figure 1.. Intranasal dantrolene nanoparticles robustly and significantly improve overall neurological function in SOD1-G93A mice.

Overall neurological scores were evaluated at the conclusion of a 30-day treatment period with intranasal dantrolene nanoparticles (IN-DAN), vehicle control (IN-VEH), and no treatment (NO TX) in SOD1-G93A transgenic ALS mice at 120 days of age. Higher neurological scores indicate more severe neurological function impairment. N=12 (NO TX), 10 (IN-VEH), and 16 (IN-DAN). Data are presented as means ± 95% CI and were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test (MCT). P<0.05 was considered statistically significant.

Figure 2.. Intranasal dantrolene nanoparticles robustly and significantly inhibit impairments in motor coordination and movement balance in SOD1-G93A ALS mice.

Overall motor coordination and balance were evaluated at the end of a 30-day treatment period (No treatment (NO TX), intranasal vehicle (IN-VEH) or intranasal dantrolene nanoparticles (IN-DAN) at 120 days of age, using the beam balance test at two beam widths: 12 mm (A, B) and 6 mm (C, D). Longer beam crossing time (A, C) and increased number of foot slips (B, D) indicate greater impairment of motor coordination and movement balance. N = 12 (NO TX), 10 (IN-VEH), and 12 (IN-DAN) for the non-transgenic control mice and 12 (NO TX), 10 (IN-VEH), and 16 (IN-DAN) for the transgenic SOD1-G93A ALS mice. Data are presented as means ± 95% CI and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT). P<0.05 was statistically significant.

Figure 3.. Intranasal dantrolene nanoparticles significantly inhibit body weight loss and muscle weakness in SOD1-G93A transgenic ALS mice.

A. Body weight changes were monitored weekly (W) throughout the 4-week treatment (TX) period. B. Muscle strength, assessed via grip force, was evaluated at the end of the 30-day treatment period, including the no treatment (NO TX), intranasal vehicle (IN-VEH) or intranasal dantrolene nanoparticles (IN-DAN) condition groups, using the Kondziela inverted screen test. A longer latency to fall from the inverted screen indicates greater muscle strength. N=12 (NO TX), 11 (IN-VEH), 12 (IN-DAN) for non-transgenic control mice. N=12 (No TX), 10 (IN-VEH), and 16 (IN-DAN) for the transgenic SOD1G93A transgenic ALS mice (B). Data are presented as means ± 95% Cl and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT). P<0.05 was considered statistically significant (B).

Figure 4.. Intranasal dantrolene nanoparticles significantly inhibit spinal cord degeneration in SOD1-G93A ALS mice.

Spinal cord degeneration was evaluated following 30 days of treatment with no treatment (NO TX), intranasal vehicle control (IN-VEH) or intranasal dantrolene nanoparticles (IN-DAN), at 135 days of age, by measuring levels of the neuronal skeletal protein neurofilament light chain (NFL) and spinal cord weight. (A) Representative Immunoblot illustrating changes in NFL protein level in the lumbar and sacral spinal cord (~L1 to S5). (B) Quantification and statistical analysis of NFL protein expression in the same region. N = 8 per group in both control and SOD1-G93A mice. (C) Fresh weight of lumbar and sacral spinal cord tissue collected for Western blot (~L1 to S5). N=10 per group in control mice, N=5 (NO TX), 9 (IN-VEH) and 7 (IN-DAN) in SOD1-G93A transgenic mice. D. Representative images of 4% paraformaldehyde-fixed cervical and thoracic spinal cords (~C1 to T12). E. Weight measurements of the cervical and spinal cord segments (~C1 to T12). N = 5 per experimental group. Data are presented as means ± 95% Cl and were analyzed using two-way ANOVA, followed by Tukey’s multiple comparison test (MCT). (B, C, E). P<0.05 was considered statistically significant.

Figure 5.. Intranasal dantrolene nanoparticle treatment robustly prolongs survival probability in SOD1-G93A transgenic ALS mice.

The probability of survival in SOD1-G93A transgenic ALS mice was monitored daily over a 30-day treatment period with intranasal dantrolene nanoparticle (IN-DAN), vehicle control (IN-VEH), or no treatment (NO TX), beginning at 90 days of age and ending at 120 days. At treatment onset, N = 18 (NO TX), 15 (IN-VEH), and 18 (IN-DAN) for SOD1-G93A mice. Kaplan-Meier survival analysis was performed to evaluate survival probability. At the end of the treatment period, the survival rate for all non-transgenic control mice was 100% (data not shown). In contrast, among SOD1-G93A mice: No TX: 67% survival (12/18), IN-VEH: 67% survival (10/15), IN-DAN: 89% survival (16/18).