A preclinical study found that safinamide shows potent anti-myotonic effects

A preclinical study found that safinamide shows potent anti-myotonic effects in a mouse model of Myotonia Congenita, more effectively than the reference drug mexiletine

1. Preclinical study of the anti myotonic efficacy of safinamide in the myotonic mouse model Presented by Ayan Hossain Course: M. Pharm(Pharmaceutical chemistry) Year-1 st , Sem-1 st

2. Contents Introduction Rationale Novelty Material and Methods Results Limitations References

3. Introduction Myotonia is a neuromuscular disorder characterized by delayed muscle relaxation after contraction, leading to stiffness, cramps, and difficulty in movement. Current treatments are limited and often associated with side effects, creating a need for safer and more effective therapies. . 1

4. Rationale 1 2 3 4 5 6 Current treatments like mexiletine or carbamazepine have limited efficacy, tolerability issues, and side effects . Safinamide, approved for Parkinson’s disease, has a well-established safety record. Its dual mechanism (sodium channel blockade + modulation of glutamate release) may reduce muscle hyperexcitability in myotonia. Existing drugs show poor efficacy & safety concerns. 2

5. Novelty 1 2 3 4 5 6 Safinamide has never been studied for myotonia; its known use is in Parkinson’s disease. First to explore the dual pharmacological action (sodium channel blockade + glutamate modulation) against muscle hyperexcitability. Provides a drug repurposing approach — safer, faster, and more cost-effective than developing a new drug. Potential to expand the therapeutic scope of safinamide beyond neurological disorders. 3

6. Animal Care: • Myotonic (ADR) mice bred in the lab from Jackson Lab stock • Wild-type & heterozygous identified by genotyping (PCR on tail DNA) • Standard housing conditions: - Temperature: 20–24 °C - 12:12 light–dark cycle - Standard diet (7 g/day) & water ad libitum • Total 15 ADR mice (male & female) used for experiments. Materials and Method 4

7. Chemicals and Drugs: Safinamide methanesulphonate salt was supplied by Zambon SpA . Mexiletine hydrochloride was purchased from Sigma Aldrich. For in vitro (muscle bath) experiments: Both drugs dissolved in Ringer’s saline solution. For in vivo (mouse injection) experiments : Both drugs dissolved in 0.9% NaCl (normal saline). Control (vehicle group): Mice injected with the same volume of 0.9% NaCl only. Injection volume kept below 200 µL. 5

8. In Vitro Muscle Studies: Anaesthesia & dissection : Anesthetize mouse (ketamine/xylazine 100/10 mg/kg, i.p. ). Dissect the extensor digitorum longus (EDL) muscle carefully. Euthanize with anesthetic overdose immediately after dissection. Recording setup : Place EDL in 25 mL chamber with Ringer solution at ~30 °C: (mM) 148 NaCl, 4.5 KCl , 2.0 CaCl ₂, 1.0 MgCl ₂, 12.0 NaHCO ₃, 0.44 NaH₂PO ₄, 5.5 glucose. Bubble 95% O₂/5% CO₂ to keep pH 7.2–7.3. Prevent movement: Incubate muscle 45 min with 50 µM BTS (in DMSO) to stop unwanted contractions. Two-microelectrode current-clamp: Glass microelectrodes: Recording Electrode(3mMKCL), Current injecting electrode( 2mM k Citrate). Insert ~50 µm apart into the same fiber. Goal: Measure muscle fiber excitability and action potentials; test safinamide vs mexiletine 6

9. In vivo Studies Procedure: Record baseline TRR (before drug). Inject drug (safinamide, mexiletine, or saline control) i.p. Measure TRR at 20, 60, 90, 120 min after injection. Each time point: 5 trials → average. Doses tested: Safinamide: 1, 3, 10 mg/kg ; Mexiletine: 1, 3, 10 mg/kg, Control: 0.9% NaCl (≤200 µL). 5 mice per dose, Drug effect lasts only a few hours → reversible, Washout period of a few days between tests. Max 3 experiments per mouse. Goal: See if the drug reduces myotonia in live mice . 7

10. OPEN FIELD TEST Result: 🧪 Effects of Safinamide vs Mexiletine on Muscle Excitability ⚡ Problem in ADR Mice: Chloride channel dysfunction → hyperexcitable muscles Signs : 🔄 Spontaneous discharges ( SpD ) 🔁 Abnormal repetitive firing ⏱ After-discharges ( AfD ) → delayed relaxation (myotonia) 💊 Drug Effects (in vitro): Safinamide (10 μM ) 📉 Reduced abnormal spikes by ~48% ⚖️ Similar to healthy mice 8

11. Mexiletine (10 μM ) 📉 Reduced spikes only by ~24% Potency : Safinamide ≈ 3× stronger than mexiletine 📊 After-discharges ( AfD ): Safinamide (10 μ M) ↓ 85% Mexiletine (30 μ M) ↓ 81% ✅ Safinamide slightly better in reducing SpD too 9

12. IMMUNO-HISTOLOGICAL ANALYSIS 🐭 In Vivo Anti-Myotonic Effects (ADR Mouse Model) 🧪 Test Used: Righting Reflex Test (TRR) Time taken by mouse to stand up from back position. ADR mice: 2.6 sec (delayed, myotonic). Healthy (WT): <0.5 sec. 💊 Drug Effects on TRR: Safinamide At 3 & 10 mg/kg → strong reduction in TRR (20–120 min ) At 1 mg/kg → significant effect (20 & 60 min) ⏳ Long-lasting effect up to 2 hours 10

13. Mexiletine: At 10 mg/kg → effective, but always less potent than safinamide At 3 mg/kg → effect only at 60 & 90 min At 1 mg/kg → ❌ ineffective 11

14. Fig. 3 Excitability parameters measured in EDL muscle of Wild-Type (WT) and myotonic ADR mice. a. Maximum number of action potentials (N Spikes) elicited by a fixed depolarizing current pulse. b. Maximum number of after discharges (AD) measured in muscle fibers. In WT, this number is zero. Columns represent the mean SEM of 4–20 muscle fibers from nine ADR mice and five WT mice. Statistical differences were calculated using one-way ANOVA followed by Bonferroni's t-test. *Significantly different with respect to WT (P < 0.05 or less); #Significantly different with respect to ADR (P < 0.05 or less); xSignificantly different with respect to safinamide 10 μM (P < 0.05). 12

15. STATISTICAL ANALYSIS Data were shown as mean SEM (standard error of the mean) from n experiments and statistical analysis was performed using Student's t-test or two-way ANOVA followed by Bonferroni's t-test, as reported in the Results section. compare TRR values in all drug conditions at each time point (vehicle and three doses of safinamide and mexiletine) by using analysis of variance (ANOVA) followed by Bonferroni's t-test. mexiletine and safinamide effect was compared at the same dose using unpaired Student's t-test. 13

16. Limitations: 1 2 3 4 5 6 Animal model only – Results in mice may not fully match human disease. Genetic difference – ADR mouse model mimics myotonia, but not all human symptoms. Short-term study – Long-term safety and effectiveness were not checked. No human variability – Factors like age, sex, and other diseases not tested. Dose translation – Mouse doses may not be the same as safe/effective human doses. Mechanism not fully explored – Exact molecular action of safinamide in myotonia needs more study. 14

17. Conclusion Safinamide showed positive anti myotonic effects in the myotonic mouse model. It helped reduce muscle stiffness and improved movement. The results suggest safinamide could be a potential alternative to mexiletine. More studies, especially in humans, are needed to confirm safety and effectiveness. 15

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