The antiarrhythmic sodium-channel blocker mexiletine is used to treat patients with myotonia. However, around 30% of patients do not benefit from mexiletine due to poor tolerability or suboptimal response. Safinamide is an add-on therapy to levodopa for Parkinson's disease. In addition to MAOB inhibition, safinamide inhibits neuronal sodium channels, conferring anticonvulsant activity in models of epilepsy. Here, we investigated the effects of safinamide on skeletal muscle hNav1.4 sodium channels and in models of myotonia, in-vitro and in-vivo. Using patch-clamp, we showed that safinamide reversibly inhibited sodium currents in HEK293T cells transfected with hNav1.4. At the holding potential (hp) of -120 mV, the half-maximum inhibitory concentrations (IC50) were 160 and 33 μM at stimulation frequencies of 0.1 and 10 Hz, respectively. The calculated affinity constants of safinamide were dependent on channel state: 420 μM for closed channels and 9 μM for fast-inactivated channels. The p.F1586C mutation in hNav1.4 greatly impaired safinamide inhibition, suggesting that the drug binds to the local anesthetic receptor site in the channel pore. In a condition mimicking myotonia, i.e. hp. of -90 mV and 50-Hz stimulation, safinamide inhibited INa with an IC50 of 6 μM, being two-fold more potent than mexiletine. Using the two-intracellular microelectrodes current-clamp method, action potential firing was recorded in vitro in rat skeletal muscle fibers in presence of the chloride channel blocker, 9-anthracene carboxylic acid (9-AC), to increase excitability. Safinamide counteracted muscle fiber hyperexcitability with an IC50 of 13 μM. In vivo, oral safinamide was tested in the rat model of myotonia. In this model, intraperitoneal injection of 9-AC greatly increased the time of righting reflex (TRR) due to development of muscle stiffness. Safinamide counteracted 9-AC induced TRR increase with an ED50 of 1.2 mg/kg, which is 7 times lower than that previously determined for mexiletine. In conclusion, safinamide is a potent voltage and frequency dependent blocker of skeletal muscle sodium channels. Accordingly, the drug was able to counteract abnormal muscle hyperexcitability induced by 9-AC, both in vitro and in vivo. Thus, this study suggests that safinamide may have potential in treating myotonia and warrants further preclinical and human studies to fully evaluate this possibility.

中文翻译：

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沙芬酰胺治疗非营养性肌强直的潜力：在体外和体内抑制骨骼肌电压门控性钠通道和骨骼肌过度兴奋性。

抗心律不齐的钠通道阻滞剂美西律用于治疗肌强直患者。但是，由于耐受性差或反应欠佳，约30％的患者无法从美西律中获益。沙芬酰胺是左旋多巴的帕金森氏病附加疗法。除MAOB抑制外，沙芬酰胺还抑制神经元钠通道，在癫痫模型中赋予抗惊厥活性。在这里，我们研究了沙芬酰胺对骨骼肌hNav1.4钠通道以及肌强直，体外和体内模型的影响。使用膜片钳，我们显示沙非酰胺可逆地抑制了hNav1.4转染的HEK293T细胞中的钠电流。在-120 mV的保持电位（hp）下，在0.1和10 Hz的刺激频率下，半最大抑制浓度（IC50）分别为160和33μM。沙非酰胺的亲和常数的计算取决于通道状态：封闭通道为420μM，快速灭活通道为9μM。hNav1.4中的p.F1586C突变大大削弱了沙芬酰胺的抑制作用，表明该药物与通道孔中的局部麻醉受体结合。在模仿肌强直的情况下，即hp。在-90 mV和50 Hz刺激下，沙芬酰胺抑制INa的IC50为6μM，效力比美西律高两倍。使用两细胞微电极电流钳方法，在氯离子通道阻滞剂9-蒽羧酸（9-AC）存在的情况下，在大鼠骨骼肌纤维中记录了动作电位放电，以增加兴奋性。沙芬酰胺以13μM的IC50抵消了肌纤维的过度兴奋性。体内，在肌强直大鼠模型中测试了口服沙芬酰胺。在该模型中，由于肌肉僵硬，腹膜内注射9-AC大大增加了正向反射（TRR）时间。沙芬酰胺以9 mg / kg的ED50抵消了9-AC诱导的TRR的增加，这是以前确定的美西律的7倍。总之，沙芬酰胺是一种有效的电压和频率依赖性骨骼肌钠通道阻滞剂。因此，该药物能够在体外和体内抵消由9-AC诱导的异常肌肉过度兴奋。因此，这项研究表明沙芬酰胺可能具有治疗肌强直的潜力，因此有必要进行进一步的临床前和人体研究以充分评估这种可能性。腹膜内注射9-AC由于肌肉僵硬而大大延长了翻身反射（TRR）时间。沙芬酰胺以9 mg / kg的ED50抵消了9-AC诱导的TRR的增加，这是以前确定的美西律的7倍。总之，沙芬酰胺是一种有效的电压和频率依赖性骨骼肌钠通道阻滞剂。因此，该药物能够在体外和体内抵消由9-AC诱导的异常肌肉过度兴奋。因此，这项研究表明沙芬酰胺可能具有治疗肌强直的潜力，因此有必要进行进一步的临床前和人体研究以充分评估这种可能性。腹膜内注射9-AC由于肌肉僵硬而大大延长了翻身反射（TRR）时间。沙芬酰胺以9 mg / kg的ED50抵消了9-AC诱导的TRR的增加，这是以前确定的美西律的7倍。总之，沙芬酰胺是一种有效的电压和频率依赖性骨骼肌钠通道阻滞剂。因此，该药物能够在体外和体内抵消由9-AC诱导的异常的肌肉过度兴奋。因此，这项研究表明沙芬酰胺可能具有治疗肌强直的潜力，因此有必要进行进一步的临床前和人体研究以充分评估这种可能性。这比以前确定的美西律低7倍。总之，沙芬酰胺是一种有效的电压和频率依赖性骨骼肌钠通道阻滞剂。因此，该药物能够在体外和体内抵消由9-AC诱导的异常肌肉过度兴奋。因此，这项研究表明沙芬酰胺可能具有治疗肌强直的潜力，因此有必要进行进一步的临床前和人体研究以充分评估这种可能性。这比以前确定的美西律低7倍。总之，沙芬酰胺是一种有效的电压和频率依赖性骨骼肌钠通道阻滞剂。因此，该药物能够在体外和体内抵消由9-AC诱导的异常肌肉过度兴奋。因此，这项研究表明沙芬酰胺可能具有治疗肌强直的潜力，因此有必要进行进一步的临床前和人体研究以充分评估这种可能性。