伊来西胺对急性分离神经元电压依赖性钠通道作用的研究
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摘要
研究背景
癫痫(Epilepsy)是大脑神经元异常放电,导致反复发作的大脑功能障碍的一种慢性疾病。其发病率高,病程长,对家庭及社会的影响大。离子通道的活动是神经系统电兴奋性的基础。电压依赖性钠离子通道(Voltage-gated sodium channels, VGSCs)在脑兴奋过程中发挥重要的作用,主要负责神经元动作电位的产生与传导。VGSCs是由1个260 kD的α亚基和1个或多个33~36 kD的β亚基(β1-β4)组成的蛋白复合物,其中α亚基组成VGSCs的主要部分,是VGSCs的功能性亚基[1,2]。目前发现了九个α-亚基异构体,其中Nav1.4在骨骼肌中表达, Nav1.5在心脏表达,Nav1.7、1.8和1.9在感觉神经元中表达,Nav1.1,1.2、1.3和1.6主要分布在中枢神经系统(Central nervous system, CNS)[3,4]。VGSCs功能异常所导致的离子通道病是目前为止人类最常见的通道病,已被证实与癫痫、孤独症及偏头痛等多种神经系统疾病相关。世界卫生组织估计全球有超过5000万人正在承受着癫痫所带来的痛苦。
现有的第一、二代抗癫痫药物中,卡马西平、苯妥英钠、拉莫三嗪等,均通过作用于VGSCs,在一定程度上控制癫痫的发作。但是,由于它们作用机制广泛,长期大量服用控制患者发作的同时,都存在一定的毒副作用[7,8]。因此发展出特异性强,安全,耐受性好的抗癫痫药物符合广大癫痫患者的迫切需要。现阶段抗癫痫药物已开发至第三代,大多数开发中的第三代抗癫痫药物都能够特异性的作用于VGSCs的α亚基或β亚基,为VGSCs的阻断剂,它们特异性好,主要是通过抑制钠通道的功能,从而抑制钠电流,进而抑制神经元的异常放电,以达到治疗癫痫的目的[9,10]。其中近期通过美国食品药品管理局(Food and Drug Administration,FDA)验证允许上市的第一例第三代抗癫痫药物拉科酰胺,就是通过特异性作用于VGSCs的慢失活通道而起作用[11]。由于它们作用强,安全,耐受性好,第三代抗癫痫药物具有广泛的开发与应用前景。有文献指出引起部分性癫痫伴热性惊厥附加症(Partial epilepsywith febrile seizure plus,PEFS+)、全面性癫痫伴热性惊厥附加症(Generalized epilepsy with febrile seizure plus,GEFS+)、重症婴儿肌阵挛性癫痫(Severe myoclonic epilepsy in infancy, SMEI)等不同类型癫痫的原因分别与不同位置的VGSCs基因突变导致的钠通道蛋白功能的不同改变密切相关。电生理研究初步证实:细胞中表达钠通道突变改变了正常的钠电流,导致神经元兴奋性改变而致病,这为研究癫痫发作的机制提供了更多的依据。由于产生癫痫的原因复杂,针对癫痫发病机制选择对症药物,才能够实现个体化给药,达到最好的疗效。
近年来我国中药产业面临着巨大的发展机遇,国家政策规划要求不断丰富和完善中医药理论体系,加大对中药研发的投入力度,大力发展具备自主知识产权的优秀中药制剂。但相较于成分确定的单体化合物为主的西药,现阶段广大中药面临着成份复杂,作用机制不明确等问题,难以被规范、科学的现代医疗理念所认同,中药提取单体并阐明作用机制,最终获得国际学术界认可,是中药成药的发展趋势。
伊来西胺,又名3,4-亚甲基二氧肉桂哌啶,或名3-(3,4-亚甲基二氧苯基)丙烯酰哌啶,曾用名抗痫灵(AES),为胡椒嗪的衍生物之一,是一种新的抗癫痫药物。我国对伊来西胺有着完全的知识产权,它是获得国际公认的纯植物中药成果。基于动物模型的药理实验证明,其抗癫痫作用显著。临床结果表明:作用于整体小儿癫痫,伊来西胺与安慰剂比较无显着差异;但伊来西胺对强直-阵挛发作有显著疗效,强直-阵挛发作是癫痫发作最常见的类型。在伊来西胺有效及无效组中进行比较发现,血液中药物水平没有显著性差异。伊来西胺是一个较为安全的药物,药品原料日常可食用,儿童给予10毫克/公斤/天的大剂量治疗没有显示严重的副作用。临床认为,伊来西胺有可能改善病人的心理和认知状况。北大医院曾经临床应用于超过10万患者,结果表明伊来西胺有效率为95.6%,综合显效率83.3%。
伊来西胺成份单一,治疗效果良好,毒副作用轻微,但一直以来对其抗癫痫机制的研究甚少。通过临床观察曾经认为,伊来西胺可能的抗癫痫机制有:1,平衡细胞电荷,阻止异常放电。伊来西胺进入大脑分解后,直达脑神经病变部位,平衡细胞内外钾、钙离子电荷水平,消除细胞异常流动,达到阻止大脑异常放电的目的。2,稳定递质代谢,控制癫痫症状。伊来西胺可平衡传递大脑指令的神经递质代谢,有效阻断错误指令的传输,从而控制癫痫发作症状。3,修复神经细胞,恢复患者智力。伊来西胺还可以直接参与细胞合成,增强脑细胞携氧能力,修复脑神经损伤,逐步恢复指令的正常传导,从而恢复患者智力。但伊来西胺以上作用机制仅限于假说和推测,尚无明确结论,难以满足现代社会对安全、有效药物的要求。FDA就要求上市的新药必须拥有明确的分子水平的作用机制。受限于此,伊来西胺作为抗癫痫药物的影响非常有限,且影响仅限于国内——这也是大多数优秀中药的悲哀。
因此,从分子水平研究伊来西胺的作用机制符合现代医药的要求,是伊来西胺以及所有中药的推广和使用所不可或缺的。现今人们了解到神经元放电与离子通道、尤其是钠离子通道有关,癫痫发生与VGSCs的关系成为了当下研究的重点,VGSCs的功能改变导致癫痫易感,调节VGSCs的功能能够减少癫痫发作。因此伊来西胺对VGSCs的作用可能是其抗癫痫的机制之一。
研究目的和内容
在急性分离的FVB品系WT小鼠海马锥体神经元中加入不同浓度的伊来西胺,观察不同浓度伊来西胺对小鼠海马锥体神经元钠电流的影响,同时观察在不同钳制电压下给予伊来西胺对小鼠海马锥体神经元钠电流的影响。通过探讨不同的浓度与钳制电位下钠电流的电生理学变化,有助于进一步认识伊来西胺的抗癫痫作用的分子机理,规范其正确适用范围,探讨其使用剂量,使伊来西胺这一中国拥有完全知识产权的抗癫痫药物拥有更广泛的应用前景。给其他广大中药制剂提供一个可供参考的研究模式。
研究方法
1.饲养并繁殖FVB品系WT小鼠
FVB品系WT小鼠饲养在19～21℃自然光条件下,自由获取食物和水分。选取10-17天龄的WT鼠,一般选取雄性小鼠。
2.采用急性分离技术分离小鼠海马锥体神经元
腹腔注射1%戊巴比妥钠麻醉动物,快速断头取脑,分离出海马,切为350um厚的脑片,在人工脑脊液中孵育1小时,再加胰酶消化18分钟,用HSSB清洗后置入HSSB中孵育1小时,而后用Pasteur吸管吹打脑组织,使细胞游离,最后置入覆有0.05%多聚赖氨酸的载玻片上。使游离的海马锥体神经元贴壁后等待下一步膜片钳实验。
3.免疫荧光标记观察锥体神经元上VGSCs的分布
多聚甲醛灌注小鼠,冰冻切片,做锥体神经元细胞免疫组化,统计细胞数目,并孵育Nav1.1的抗体以及相应二抗,在荧光显微镜下观察锥体神经元上Nav1.1的表达。
4.采用全细胞记录膜片钳技术检测钠电流特征
使用全细胞记录膜片钳技术检测伊来西胺对持续性钠离子电流抑制的浓度依赖性和电压依赖性:分别测量出在钳制电压(Vh)为-70mV和-90mV时,伊来西胺的半数最大抑制浓度(IC50);并与其他抗癫痫药物(卡马西平与拉莫三嗪)的IC50值进行比较,了解伊来西胺的作用强度与作用的电压依赖性。测量治疗剂量下伊来西胺对VGSCs激活状态、失活状态及失活恢复状态等的影响,计算不同状态下的V1/2值、曲线斜率k、及时间常数τ,并用软件分析
结果,将这些结果与卡马西平、拉莫三嗪等抗癫痫药物作用于锥体神经元放电的特征比较,找出伊来西胺与卡马西平、拉莫三嗪等药物对VGSCs影响的异同;进一步了解治疗浓度下伊来西胺对VGSCs的作用机制,确认伊来西胺在治疗浓度下作用的主要目标。
5.统计学分析
计量资料以均数±标准差( X±S)表示,统计方法用SPSSl6.0 for Windows软件包进行统计分析,采用t检验。P<0.05为差异具有统计学意义
研究结果
1. FVB品系WT鼠海马神经元上Nav1.1蛋白显著表达
Nav1.1通道蛋白在海马区的神经元均有表达。
2.钳制电位变化时不同浓度拉莫三嗪对钠电流的影响
对全细胞记录状态下的WT鼠海马锥体神经元给予不同浓度的拉莫三嗪作用,记录给药前后钠电流的变化。计算得出钳制电位为-90mV时拉莫三嗪的IC50值为1200μM;钳制电位为-70mV时拉莫三嗪的IC50值为400μM。
3.钳制电位变化时不同浓度伊来西胺对钠电流的影响
对全细胞记录状态下的WT鼠海马锥体神经元给予不同浓度的伊来西胺作用,记录给药前后钠电流的变化。计算得出钳制电位为-90mV时伊来西胺的IC50值为5.2μM;钳制电位为-70mV时伊来西胺的IC50值为0.4μM。
4. -90mV时10μM伊来西胺给药前后海马钠通道电生理特性比较
4.1.钠通道的激活
WT鼠锥体神经元正常状态下V1/2与k值分别为(n=5):-36.63±0.22(mV),3.87±0.19;WT鼠锥体神经元给药后V1/2与k值分别为(n=5):-43.93±0.90(mV),4.22±0.70。差异有统计学意义(P<0.05)。
4.2.钠通道的失活
WT鼠锥体神经元正常状态下V1/2与k值分别为(n=5):-43.76±0.49(mV),7.03±0.43;WT鼠锥体神经元给药后V1/2与k值分别为(n=5):-61.74±0.65(mV),8.19±0.58。差异有统计学意义(P<0.05)。
4.3.钠通道的失活后恢复
WT鼠锥体神经元正常状态下恢复时间常数τ值为(n=5):2.05±0.42(ms);WT鼠锥体神经元给药后恢复时间常数τ值为(n=5):2.20±0.16(ms)。差异无统计学意义(P>0.05)。
4.4.钠通道激活与失活窗口变化
WT鼠锥体神经元正常状态下与给药后激活曲线与失活曲线窗口下面积比值为(n=5)1:0.53。可见明显缩小。
结论
1.本研究首次通过全细胞膜片钳技术研究伊来西胺对神经元钠通道的影响,电生理研究发现伊来西胺对钠电流有显著抑制作用,不同钳制电位下伊来西胺对钠通道作用强度有显著性差异:计算得出钳制电位为-90mV时伊来西胺的IC50值为5.2μM;钳制电位为-70mV时伊来西胺的IC50值为0.4μM。与拉莫三嗪相比,对神经元钠通道起作用所需的伊来西胺浓度更低。和拉莫三嗪类似,伊来西胺对神经元上钠通道的作用有着电压依赖性及浓度依赖性。
2.本研究首次发现伊来西胺对WT鼠锥体神经元上VGSCs的通道特性有影响:
2.1.伊来西胺能使WT鼠锥体神经元上VGSCs的激活曲线往超极化方向移动(左移);
2.2.伊来西胺能使WT鼠锥体神经元上VGSCs的失活曲线往超极化方向移动(左移);
2.3.伊来西胺对WT鼠锥体神经元上VGSCs的失活恢复曲线没有影响;
2.4.伊来西胺能使WT鼠锥体神经元上VGSCs的激活、失活曲线之间的窗口面积缩小;说明相比之下,伊来西胺更主要作用于VGSCs的失活通道。
3.本研究认为伊来西胺可能与锥体神经元上VGSCs蛋白作用,延长了锥体神经元上VGSCs的失活状态,从而抑制了持续去极化(电压依赖性),使锥体神经元兴奋性降低,为伊来西胺的抗癫痫作用的分子机制提供了依据。但对于伊来西胺显示出的良好耐受性和轻微副作用的原因需要进一步药代动力学研究;伊来西胺与旧的抗癫痫药物药物相互作用以及伊来西胺对不同原因导致的癫痫治疗效果及其分子机制也是下一步研究的重点。
Background
Epilepsy has long been recognized as a disorder of brain hyper-excitability. It’s a chronic disease leading by repeated episodes of brain dysfunction. Voltage-gated sodium channels (VGSCs) play an important role in the initiation and propagation of action potentials and are crucial regulators of neuronal excitability. However, it was been found that a loss-of-function mutation in human SCN1A gene causes severe infant myoclonic epilepsy. VGSCs are composed of a central, pore-formingα-subunit (VGSC-α) and one or moreβ-subunits (VGSC-β). Currently, nine isoforms of theα-subunit have been characterized, of which Nav1.4 is present in skeletal muscles, Nav1.5 in heart, Nav1.7, 1.8 and 1.9 in sensory neurons and Nav1.1, 1.2, 1.3 and 1.6 are mainly found in the central nervous system (CNS). The World Health Organization estimates that 50 million people worldwide suffer from epilepsy. Voltage-gated ion channels are key targets for AEDs that inhibit epileptic bursting, synchronization and seizure spread. Synaptic inhibition and excitation are mediated by neurotransmitter-regulated channels; these channels permit synchronization of neural ensembles and allow propagation of the abnormal discharge to local and distant sites. AEDs that modify excitatory and inhibitory neurotransmission therefore can also suppress bursting and, when they inhibit synaptic excitation, can have prominent effects on seizure spread.
The existing first-generation antiepileptic drugs (AEDs) (phenytoin, phenobarbitone, carbamazepine, valproic acid) were the main pharmacological targets for the treatment of epileptic disorder. However, the use of these AEDs is associated with severe adverse events (AEs), drug interactions and pharmacokinetic variations. The over-treatment of epilepsy results in serious AEs following unnecessary fast titration of AEDs. In addition, AED polytherapy during pregnancy may increase the risk of fetal malformations. Moreover, the problem of pharmacoresistance may be encountered, where reduced drug sensitivity is due to certain neurobiological changes in brain, thereby making treatment of epilepsy less efficient. It is believed that approximately 30-40% of individuals with epilepsy continue to suffer from uncontrolled seizures despite AED treatment. This observation further limits the use of first-generation antiepileptic drugs in the management of epilepsy. Therefore, the development of a specific, safely, tolerable AED in patients with epilepsy is urgent. Antiepileptic drugs have now been developed to the third generation, most of the third generation antiepileptic drugs can act on VGSCs specificαsubunit orβsubunit, as VGSCs blockers. They achieve the purpose of treatment of epilepsy by specificity inhibited the sodium channel function and the sodium current. The newer AEDs act on diverse and novel molecular targets. Nevertheless, inhibition of VGSCs continues to bean effective strategy for the development of third-generation AEDs. Many of the third-generation compounds are currently undergoing clinical evaluation, and some are even approved and marketed (e.g., lacosamide). The third-generation drugs offer various favorable properties such as broad-spectrum efficacy, and many have demonstrated efficacy mainly in refractory partial seizures. In general, the third-generation compounds have shown excellent tolerability and/or milder adverse effects, an improved pharmacokinetic profile and/ or lesser drug interactions as compared to the older AEDs.
In recent years, the development of Chinese medicine industry was confronted with enormous challenges and opportunities. National policy increase R&D investment of traditional Chinese medicine with independent intellectual property rights and asked for enrich and improve the theoretical system of Chinese medicine. However, compared with Western medicine, Chinese medicine faced a lot of problems such as complicated composition and unclear mechanism of action. The therapeutic effect was difficult to be approved by modern medicine. Clarify the mechanisms is the trend of Chinese medicine development.
Ilepcimide (ICM), 3, 4-methylene dioxy cinnamoyl piperidine, used to called antiepilepsirine (AES), is an effective Chinese antiepileptic drug. It was extracted from an effective traditional Chinese medicine prescription. Pharmacological experiments on animal models prove that its antiepileptic action is remarkable. The clinical results showed that there are no significant differences in clinical effects between ilepcimide and placebo in pediatric epilepsies as a whole, but ilepcimide is effective in tonic-clonic seizures, the most common type of seizure in the series. There are no significant differences in AED blood levels between the AES effective and ineffective groups. It is very safely, children been given large doses (10 mg/kg/day) demonstrate no serious side-effects. It is suggested that there is potential improvement in patient psychological and cognitive status. Clinical results also showed that the effective rate of ilepcimide was 95.6%, consolidated markedly effective rate was 83.3%.
Purpose
Whole-cell patch clamp analysis was used to characterize biophysical properties of acute isolation of pyramidal neurons in hippocampus with or without Ilepcimide. Although its clinical treatment is effective, its antiepileptic mechanism is unclear. We intend to find the antiepileptic mechanism of ilepcimide.
Methods
1. Experimental animals
Male WT mice (10-17 days old for whole-cell recording) of the FVB strain were used for the preparation of acute isolated hippocampus pyramidal neurons. All animals were maintained on a 12:12 h light/dark cycle with a constant room temperature, and with sufficient food and water.
2. Isolation of mice hippocampal pyramidal neurons with acute isolated skill
Intraperitoneal injects 1% sodium pentobarbital in WT mice. Took out the brain and isolated the hippocampus quickly. Cut the hippocampus to 350μM thick slices, incubated it in artificial cerebrospinal fluid bubbled with 95% O2/5% CO2 at 32℃for 1 hour. Then digest with 0.5mM trypsin in 100% O2 atmosphere at 37℃for 18 minutes. After that put the tissue in HSSB and bubbled with 100% O2 at 32℃for 1 hour. At last, percussion the tissue with Pasteur pipettes, and placed it in glass cover slips covered with 0.05% poly-D-lysine.
3. Immunohistochemistry
Mice were randomly obtained for immunofluorescence histochemistry to detect localization of Nav1.1 sections were blocked and incubated with primary antibody anti-Nav1.1 overnight at 4oC and with the secondary antibodies at room temperature for l hour. Fluorescence signals were detected with a microscope at excitation/emission wavelengths of 495/519 nm (FITC,green).
4. Whole-cell voltage-clamp recordings of acute isolated hippocampus pyramidal neurons
The cells on cover slips were transferred to a recording chamber and superfused at room temperature with extracellular solution containing (mM): CH3SO3Na 120, KC1 3, MgC12 1, TEACl 20,BaCl2 5, Glucose 10,CdCl2 0.03,4-aminopyridine 4, and 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES) 10, pH 7.4 adjusted with NaOH. The recording chamber volume was approximately 0.5 ml and the flow rate was 0.2 ml/min. Patch pipettes were pulled from borosilicate glass capillaries and were filled with an internal solution containing (mM): CH3SO3Cs 110,
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) 5, TEACl 20, MgCl2 5,CaCl2 0.5,Na2ATP 5 and HEPES 10, pH 7.4 adjusted with CsOH. Current recordings were obtained under whole-cell voltage-clamp conditions (EPC-10, HEKA Instruments) and were filtered at 5 kHz. The recording electrodes had resistances of 2.5-3.5 M?. Compensation circuitry was used to minimize series resistance errors and 80-90% of the series resistance could be compensated. In most cases where Na+ currents ranged between -0.5 nA and -5 nA, the voltage drop across the compensated series resistance was < 5 mV. Leakage currents were subtracted using a P/4 protocol. Membrane potentials quoted were not corrected for junction potentials (5±2.5 mV, n = 4). Na+ currents recorded from these cells always increased progressively by approximately 50~150% within the first 10-20 minutes of recordings at a holding potential (Vh) of -90 mV and then were stable for a further 20-40 minutes. Thus, drugs were applied only when the control currents had stabilized. Most compounds and agents were obtained from Sigma-Aldrich. Ilepicimide was synthesized by the Renji Pharma.Co, (Beijing, China). Ilepicimide and its stock solutions (10 mM) were prepared freshly with 100% alcohol and diluted with the perfusate to the desired concentrations for experiments.
5. Data acquisition and analysis
Data collection and analysis was performed using Patchmaster v2x35, EPC 10 (HEKA Elektronic) and Origin7.5E (MicroCal Software). Data are presented as the mean±SEM, unless specified. Student's t-test was used for statistical evaluation. Theoretical curves were fitted to the data using a least squares algorithm (for details see the figure legends).
Results
1. Localization of Nav1.1 in neurons
Nav1.1 channel protein is localization in the hippocampus, especially in pyramidal neurons.
2. Different concentrations of lamotrigine act on sodium current when holding potential change
IC50 of lamotrigine were 1200μM (-90mV) and 400μM (-70mV).
3. Different concentrations of ilepcimide act on sodium current when holding potential change
IC50 of ilepcimide were 5.2μM (-90mV) and 0.4μM (-70mV).
4. Sodium channel properties with or without 10μM ilepcimide at holding potential -90mV
4.1. Activation
WT mice pyramids neurons Voltage dependence of activation: V1/2 and k (slope factor) were(n=5): without ilepcimide: -36.63±0.22(mV),3.87±0.19; with ilepcimide: -43.93±0.90(mV),4.22±0.70 (P<0.05).
4.2. Inactivation
WT mice pyramids neurons Voltage dependence of inactivation: V1/2 and k (slope factor) were(n=5): without ilepcimide: -43.76±0.49(mV),7.03±0.43; with ilepcimide: -61.74±0.65(mV),8.19±0.58 (P<0.05).
4.3. Recovery from inactivation
WT mice pyramids neurons recovery from inactivation: the time constant was:without ilepcimide (n=5): 2.05±0.42(ms),with ilepcimide: 2.20±0.16(ms) (P>0.05).
4.4. The area under activation and inactivation curves
In WT mice pyramids neurons, the ratio of the area under the curve of activation and inactivation between without or with ilepcimide were (n=5): 1:0.53. The area was minimized.
Conclusion
1. This is the first time to use the whole cell recording technique to study the mechanism of ilepcimide. We found that ilepcimide significantly inhibited the sodium current. The IC50 of ilepcimide was 5.2μM at the holding potential (Vh) of -90mV compare with the IC50=0.4μM at the holding potential of -70mV. These actions were similar to those produced by the antiepileptic drug lamotrigine in various cell types.
2. This is the first time we found that ilepcimide at therapeutically relevant concentrations caused a hyperpolarising shift of the activation curve and the inactivation curve, but had no influence to recovery from inactivation curve. The ratio of the area under the curve of activation and inactivation also minimized. In contrast, ilepcimide play a major role in inactivation state of VGSCs channel.
3. Our study suggests that ilepcimide may combine with VGSCs proteins, decrease the excitability of pyramidal neurons by promoting the inactivation state of VGSCs. We provide a basic molecular mechanism of ilepcimide as an AED. We would focus on the reason why ilepcimide showed good tolerability and mild side effects in future research.
癫痫(Epilepsy)是大脑神经元异常放电,导致反复发作的大脑功能障碍的一种慢性疾病。其发病率高,病程长,对家庭及社会的影响大。离子通道的活动是神经系统电兴奋性的基础。电压依赖性钠离子通道(Voltage-gated sodium channels, VGSCs)在脑兴奋过程中发挥重要的作用,主要负责神经元动作电位的产生与传导。VGSCs是由1个260 kD的α亚基和1个或多个33~36 kD的β亚基(β1-β4)组成的蛋白复合物,其中α亚基组成VGSCs的主要部分,是VGSCs的功能性亚基[1,2]。目前发现了九个α-亚基异构体,其中Nav1.4在骨骼肌中表达, Nav1.5在心脏表达,Nav1.7、1.8和1.9在感觉神经元中表达,Nav1.1,1.2、1.3和1.6主要分布在中枢神经系统(Central nervous system, CNS)[3,4]。VGSCs功能异常所导致的离子通道病是目前为止人类最常见的通道病,已被证实与癫痫、孤独症及偏头痛等多种神经系统疾病相关。世界卫生组织估计全球有超过5000万人正在承受着癫痫所带来的痛苦。
现有的第一、二代抗癫痫药物中,卡马西平、苯妥英钠、拉莫三嗪等,均通过作用于VGSCs,在一定程度上控制癫痫的发作。但是,由于它们作用机制广泛,长期大量服用控制患者发作的同时,都存在一定的毒副作用[7,8]。因此发展出特异性强,安全,耐受性好的抗癫痫药物符合广大癫痫患者的迫切需要。现阶段抗癫痫药物已开发至第三代,大多数开发中的第三代抗癫痫药物都能够特异性的作用于VGSCs的α亚基或β亚基,为VGSCs的阻断剂,它们特异性好,主要是通过抑制钠通道的功能,从而抑制钠电流,进而抑制神经元的异常放电,以达到治疗癫痫的目的[9,10]。其中近期通过美国食品药品管理局(Food and Drug Administration,FDA)验证允许上市的第一例第三代抗癫痫药物拉科酰胺,就是通过特异性作用于VGSCs的慢失活通道而起作用[11]。由于它们作用强,安全,耐受性好,第三代抗癫痫药物具有广泛的开发与应用前景。有文献指出引起部分性癫痫伴热性惊厥附加症(Partial epilepsywith febrile seizure plus,PEFS+)、全面性癫痫伴热性惊厥附加症(Generalized epilepsy with febrile seizure plus,GEFS+)、重症婴儿肌阵挛性癫痫(Severe myoclonic epilepsy in infancy, SMEI)等不同类型癫痫的原因分别与不同位置的VGSCs基因突变导致的钠通道蛋白功能的不同改变密切相关。电生理研究初步证实:细胞中表达钠通道突变改变了正常的钠电流,导致神经元兴奋性改变而致病,这为研究癫痫发作的机制提供了更多的依据。由于产生癫痫的原因复杂,针对癫痫发病机制选择对症药物,才能够实现个体化给药,达到最好的疗效。
近年来我国中药产业面临着巨大的发展机遇,国家政策规划要求不断丰富和完善中医药理论体系,加大对中药研发的投入力度,大力发展具备自主知识产权的优秀中药制剂。但相较于成分确定的单体化合物为主的西药,现阶段广大中药面临着成份复杂,作用机制不明确等问题,难以被规范、科学的现代医疗理念所认同,中药提取单体并阐明作用机制,最终获得国际学术界认可,是中药成药的发展趋势。
伊来西胺,又名3,4-亚甲基二氧肉桂哌啶,或名3-(3,4-亚甲基二氧苯基)丙烯酰哌啶,曾用名抗痫灵(AES),为胡椒嗪的衍生物之一,是一种新的抗癫痫药物。我国对伊来西胺有着完全的知识产权,它是获得国际公认的纯植物中药成果。基于动物模型的药理实验证明,其抗癫痫作用显著。临床结果表明:作用于整体小儿癫痫,伊来西胺与安慰剂比较无显着差异;但伊来西胺对强直-阵挛发作有显著疗效,强直-阵挛发作是癫痫发作最常见的类型。在伊来西胺有效及无效组中进行比较发现,血液中药物水平没有显著性差异。伊来西胺是一个较为安全的药物,药品原料日常可食用,儿童给予10毫克/公斤/天的大剂量治疗没有显示严重的副作用。临床认为,伊来西胺有可能改善病人的心理和认知状况。北大医院曾经临床应用于超过10万患者,结果表明伊来西胺有效率为95.6%,综合显效率83.3%。
伊来西胺成份单一,治疗效果良好,毒副作用轻微,但一直以来对其抗癫痫机制的研究甚少。通过临床观察曾经认为,伊来西胺可能的抗癫痫机制有:1,平衡细胞电荷,阻止异常放电。伊来西胺进入大脑分解后,直达脑神经病变部位,平衡细胞内外钾、钙离子电荷水平,消除细胞异常流动,达到阻止大脑异常放电的目的。2,稳定递质代谢,控制癫痫症状。伊来西胺可平衡传递大脑指令的神经递质代谢,有效阻断错误指令的传输,从而控制癫痫发作症状。3,修复神经细胞,恢复患者智力。伊来西胺还可以直接参与细胞合成,增强脑细胞携氧能力,修复脑神经损伤,逐步恢复指令的正常传导,从而恢复患者智力。但伊来西胺以上作用机制仅限于假说和推测,尚无明确结论,难以满足现代社会对安全、有效药物的要求。FDA就要求上市的新药必须拥有明确的分子水平的作用机制。受限于此,伊来西胺作为抗癫痫药物的影响非常有限,且影响仅限于国内——这也是大多数优秀中药的悲哀。
因此,从分子水平研究伊来西胺的作用机制符合现代医药的要求,是伊来西胺以及所有中药的推广和使用所不可或缺的。现今人们了解到神经元放电与离子通道、尤其是钠离子通道有关,癫痫发生与VGSCs的关系成为了当下研究的重点,VGSCs的功能改变导致癫痫易感,调节VGSCs的功能能够减少癫痫发作。因此伊来西胺对VGSCs的作用可能是其抗癫痫的机制之一。
研究目的和内容
在急性分离的FVB品系WT小鼠海马锥体神经元中加入不同浓度的伊来西胺,观察不同浓度伊来西胺对小鼠海马锥体神经元钠电流的影响,同时观察在不同钳制电压下给予伊来西胺对小鼠海马锥体神经元钠电流的影响。通过探讨不同的浓度与钳制电位下钠电流的电生理学变化,有助于进一步认识伊来西胺的抗癫痫作用的分子机理,规范其正确适用范围,探讨其使用剂量,使伊来西胺这一中国拥有完全知识产权的抗癫痫药物拥有更广泛的应用前景。给其他广大中药制剂提供一个可供参考的研究模式。
研究方法
1.饲养并繁殖FVB品系WT小鼠
FVB品系WT小鼠饲养在19～21℃自然光条件下,自由获取食物和水分。选取10-17天龄的WT鼠,一般选取雄性小鼠。
2.采用急性分离技术分离小鼠海马锥体神经元
腹腔注射1%戊巴比妥钠麻醉动物,快速断头取脑,分离出海马,切为350um厚的脑片,在人工脑脊液中孵育1小时,再加胰酶消化18分钟,用HSSB清洗后置入HSSB中孵育1小时,而后用Pasteur吸管吹打脑组织,使细胞游离,最后置入覆有0.05%多聚赖氨酸的载玻片上。使游离的海马锥体神经元贴壁后等待下一步膜片钳实验。
3.免疫荧光标记观察锥体神经元上VGSCs的分布
多聚甲醛灌注小鼠,冰冻切片,做锥体神经元细胞免疫组化,统计细胞数目,并孵育Nav1.1的抗体以及相应二抗,在荧光显微镜下观察锥体神经元上Nav1.1的表达。
4.采用全细胞记录膜片钳技术检测钠电流特征
使用全细胞记录膜片钳技术检测伊来西胺对持续性钠离子电流抑制的浓度依赖性和电压依赖性:分别测量出在钳制电压(Vh)为-70mV和-90mV时,伊来西胺的半数最大抑制浓度(IC50);并与其他抗癫痫药物(卡马西平与拉莫三嗪)的IC50值进行比较,了解伊来西胺的作用强度与作用的电压依赖性。测量治疗剂量下伊来西胺对VGSCs激活状态、失活状态及失活恢复状态等的影响,计算不同状态下的V1/2值、曲线斜率k、及时间常数τ,并用软件分析
结果,将这些结果与卡马西平、拉莫三嗪等抗癫痫药物作用于锥体神经元放电的特征比较,找出伊来西胺与卡马西平、拉莫三嗪等药物对VGSCs影响的异同;进一步了解治疗浓度下伊来西胺对VGSCs的作用机制,确认伊来西胺在治疗浓度下作用的主要目标。
5.统计学分析
计量资料以均数±标准差( X±S)表示,统计方法用SPSSl6.0 for Windows软件包进行统计分析,采用t检验。P<0.05为差异具有统计学意义
研究结果
1. FVB品系WT鼠海马神经元上Nav1.1蛋白显著表达
Nav1.1通道蛋白在海马区的神经元均有表达。
2.钳制电位变化时不同浓度拉莫三嗪对钠电流的影响
对全细胞记录状态下的WT鼠海马锥体神经元给予不同浓度的拉莫三嗪作用,记录给药前后钠电流的变化。计算得出钳制电位为-90mV时拉莫三嗪的IC50值为1200μM;钳制电位为-70mV时拉莫三嗪的IC50值为400μM。
3.钳制电位变化时不同浓度伊来西胺对钠电流的影响
对全细胞记录状态下的WT鼠海马锥体神经元给予不同浓度的伊来西胺作用,记录给药前后钠电流的变化。计算得出钳制电位为-90mV时伊来西胺的IC50值为5.2μM;钳制电位为-70mV时伊来西胺的IC50值为0.4μM。
4. -90mV时10μM伊来西胺给药前后海马钠通道电生理特性比较
4.1.钠通道的激活
WT鼠锥体神经元正常状态下V1/2与k值分别为(n=5):-36.63±0.22(mV),3.87±0.19;WT鼠锥体神经元给药后V1/2与k值分别为(n=5):-43.93±0.90(mV),4.22±0.70。差异有统计学意义(P<0.05)。
4.2.钠通道的失活
WT鼠锥体神经元正常状态下V1/2与k值分别为(n=5):-43.76±0.49(mV),7.03±0.43;WT鼠锥体神经元给药后V1/2与k值分别为(n=5):-61.74±0.65(mV),8.19±0.58。差异有统计学意义(P<0.05)。
4.3.钠通道的失活后恢复
WT鼠锥体神经元正常状态下恢复时间常数τ值为(n=5):2.05±0.42(ms);WT鼠锥体神经元给药后恢复时间常数τ值为(n=5):2.20±0.16(ms)。差异无统计学意义(P>0.05)。
4.4.钠通道激活与失活窗口变化
WT鼠锥体神经元正常状态下与给药后激活曲线与失活曲线窗口下面积比值为(n=5)1:0.53。可见明显缩小。
结论
1.本研究首次通过全细胞膜片钳技术研究伊来西胺对神经元钠通道的影响,电生理研究发现伊来西胺对钠电流有显著抑制作用,不同钳制电位下伊来西胺对钠通道作用强度有显著性差异:计算得出钳制电位为-90mV时伊来西胺的IC50值为5.2μM;钳制电位为-70mV时伊来西胺的IC50值为0.4μM。与拉莫三嗪相比,对神经元钠通道起作用所需的伊来西胺浓度更低。和拉莫三嗪类似,伊来西胺对神经元上钠通道的作用有着电压依赖性及浓度依赖性。
2.本研究首次发现伊来西胺对WT鼠锥体神经元上VGSCs的通道特性有影响:
2.1.伊来西胺能使WT鼠锥体神经元上VGSCs的激活曲线往超极化方向移动(左移);
2.2.伊来西胺能使WT鼠锥体神经元上VGSCs的失活曲线往超极化方向移动(左移);
2.3.伊来西胺对WT鼠锥体神经元上VGSCs的失活恢复曲线没有影响;
2.4.伊来西胺能使WT鼠锥体神经元上VGSCs的激活、失活曲线之间的窗口面积缩小;说明相比之下,伊来西胺更主要作用于VGSCs的失活通道。
3.本研究认为伊来西胺可能与锥体神经元上VGSCs蛋白作用,延长了锥体神经元上VGSCs的失活状态,从而抑制了持续去极化(电压依赖性),使锥体神经元兴奋性降低,为伊来西胺的抗癫痫作用的分子机制提供了依据。但对于伊来西胺显示出的良好耐受性和轻微副作用的原因需要进一步药代动力学研究;伊来西胺与旧的抗癫痫药物药物相互作用以及伊来西胺对不同原因导致的癫痫治疗效果及其分子机制也是下一步研究的重点。
Background
Epilepsy has long been recognized as a disorder of brain hyper-excitability. It’s a chronic disease leading by repeated episodes of brain dysfunction. Voltage-gated sodium channels (VGSCs) play an important role in the initiation and propagation of action potentials and are crucial regulators of neuronal excitability. However, it was been found that a loss-of-function mutation in human SCN1A gene causes severe infant myoclonic epilepsy. VGSCs are composed of a central, pore-formingα-subunit (VGSC-α) and one or moreβ-subunits (VGSC-β). Currently, nine isoforms of theα-subunit have been characterized, of which Nav1.4 is present in skeletal muscles, Nav1.5 in heart, Nav1.7, 1.8 and 1.9 in sensory neurons and Nav1.1, 1.2, 1.3 and 1.6 are mainly found in the central nervous system (CNS). The World Health Organization estimates that 50 million people worldwide suffer from epilepsy. Voltage-gated ion channels are key targets for AEDs that inhibit epileptic bursting, synchronization and seizure spread. Synaptic inhibition and excitation are mediated by neurotransmitter-regulated channels; these channels permit synchronization of neural ensembles and allow propagation of the abnormal discharge to local and distant sites. AEDs that modify excitatory and inhibitory neurotransmission therefore can also suppress bursting and, when they inhibit synaptic excitation, can have prominent effects on seizure spread.
The existing first-generation antiepileptic drugs (AEDs) (phenytoin, phenobarbitone, carbamazepine, valproic acid) were the main pharmacological targets for the treatment of epileptic disorder. However, the use of these AEDs is associated with severe adverse events (AEs), drug interactions and pharmacokinetic variations. The over-treatment of epilepsy results in serious AEs following unnecessary fast titration of AEDs. In addition, AED polytherapy during pregnancy may increase the risk of fetal malformations. Moreover, the problem of pharmacoresistance may be encountered, where reduced drug sensitivity is due to certain neurobiological changes in brain, thereby making treatment of epilepsy less efficient. It is believed that approximately 30-40% of individuals with epilepsy continue to suffer from uncontrolled seizures despite AED treatment. This observation further limits the use of first-generation antiepileptic drugs in the management of epilepsy. Therefore, the development of a specific, safely, tolerable AED in patients with epilepsy is urgent. Antiepileptic drugs have now been developed to the third generation, most of the third generation antiepileptic drugs can act on VGSCs specificαsubunit orβsubunit, as VGSCs blockers. They achieve the purpose of treatment of epilepsy by specificity inhibited the sodium channel function and the sodium current. The newer AEDs act on diverse and novel molecular targets. Nevertheless, inhibition of VGSCs continues to bean effective strategy for the development of third-generation AEDs. Many of the third-generation compounds are currently undergoing clinical evaluation, and some are even approved and marketed (e.g., lacosamide). The third-generation drugs offer various favorable properties such as broad-spectrum efficacy, and many have demonstrated efficacy mainly in refractory partial seizures. In general, the third-generation compounds have shown excellent tolerability and/or milder adverse effects, an improved pharmacokinetic profile and/ or lesser drug interactions as compared to the older AEDs.
In recent years, the development of Chinese medicine industry was confronted with enormous challenges and opportunities. National policy increase R&D investment of traditional Chinese medicine with independent intellectual property rights and asked for enrich and improve the theoretical system of Chinese medicine. However, compared with Western medicine, Chinese medicine faced a lot of problems such as complicated composition and unclear mechanism of action. The therapeutic effect was difficult to be approved by modern medicine. Clarify the mechanisms is the trend of Chinese medicine development.
Ilepcimide (ICM), 3, 4-methylene dioxy cinnamoyl piperidine, used to called antiepilepsirine (AES), is an effective Chinese antiepileptic drug. It was extracted from an effective traditional Chinese medicine prescription. Pharmacological experiments on animal models prove that its antiepileptic action is remarkable. The clinical results showed that there are no significant differences in clinical effects between ilepcimide and placebo in pediatric epilepsies as a whole, but ilepcimide is effective in tonic-clonic seizures, the most common type of seizure in the series. There are no significant differences in AED blood levels between the AES effective and ineffective groups. It is very safely, children been given large doses (10 mg/kg/day) demonstrate no serious side-effects. It is suggested that there is potential improvement in patient psychological and cognitive status. Clinical results also showed that the effective rate of ilepcimide was 95.6%, consolidated markedly effective rate was 83.3%.
Purpose
Whole-cell patch clamp analysis was used to characterize biophysical properties of acute isolation of pyramidal neurons in hippocampus with or without Ilepcimide. Although its clinical treatment is effective, its antiepileptic mechanism is unclear. We intend to find the antiepileptic mechanism of ilepcimide.
Methods
1. Experimental animals
Male WT mice (10-17 days old for whole-cell recording) of the FVB strain were used for the preparation of acute isolated hippocampus pyramidal neurons. All animals were maintained on a 12:12 h light/dark cycle with a constant room temperature, and with sufficient food and water.
2. Isolation of mice hippocampal pyramidal neurons with acute isolated skill
Intraperitoneal injects 1% sodium pentobarbital in WT mice. Took out the brain and isolated the hippocampus quickly. Cut the hippocampus to 350μM thick slices, incubated it in artificial cerebrospinal fluid bubbled with 95% O2/5% CO2 at 32℃for 1 hour. Then digest with 0.5mM trypsin in 100% O2 atmosphere at 37℃for 18 minutes. After that put the tissue in HSSB and bubbled with 100% O2 at 32℃for 1 hour. At last, percussion the tissue with Pasteur pipettes, and placed it in glass cover slips covered with 0.05% poly-D-lysine.
3. Immunohistochemistry
Mice were randomly obtained for immunofluorescence histochemistry to detect localization of Nav1.1 sections were blocked and incubated with primary antibody anti-Nav1.1 overnight at 4oC and with the secondary antibodies at room temperature for l hour. Fluorescence signals were detected with a microscope at excitation/emission wavelengths of 495/519 nm (FITC,green).
4. Whole-cell voltage-clamp recordings of acute isolated hippocampus pyramidal neurons
The cells on cover slips were transferred to a recording chamber and superfused at room temperature with extracellular solution containing (mM): CH3SO3Na 120, KC1 3, MgC12 1, TEACl 20,BaCl2 5, Glucose 10,CdCl2 0.03,4-aminopyridine 4, and 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES) 10, pH 7.4 adjusted with NaOH. The recording chamber volume was approximately 0.5 ml and the flow rate was 0.2 ml/min. Patch pipettes were pulled from borosilicate glass capillaries and were filled with an internal solution containing (mM): CH3SO3Cs 110,
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) 5, TEACl 20, MgCl2 5,CaCl2 0.5,Na2ATP 5 and HEPES 10, pH 7.4 adjusted with CsOH. Current recordings were obtained under whole-cell voltage-clamp conditions (EPC-10, HEKA Instruments) and were filtered at 5 kHz. The recording electrodes had resistances of 2.5-3.5 M?. Compensation circuitry was used to minimize series resistance errors and 80-90% of the series resistance could be compensated. In most cases where Na+ currents ranged between -0.5 nA and -5 nA, the voltage drop across the compensated series resistance was < 5 mV. Leakage currents were subtracted using a P/4 protocol. Membrane potentials quoted were not corrected for junction potentials (5±2.5 mV, n = 4). Na+ currents recorded from these cells always increased progressively by approximately 50~150% within the first 10-20 minutes of recordings at a holding potential (Vh) of -90 mV and then were stable for a further 20-40 minutes. Thus, drugs were applied only when the control currents had stabilized. Most compounds and agents were obtained from Sigma-Aldrich. Ilepicimide was synthesized by the Renji Pharma.Co, (Beijing, China). Ilepicimide and its stock solutions (10 mM) were prepared freshly with 100% alcohol and diluted with the perfusate to the desired concentrations for experiments.
5. Data acquisition and analysis
Data collection and analysis was performed using Patchmaster v2x35, EPC 10 (HEKA Elektronic) and Origin7.5E (MicroCal Software). Data are presented as the mean±SEM, unless specified. Student's t-test was used for statistical evaluation. Theoretical curves were fitted to the data using a least squares algorithm (for details see the figure legends).
Results
1. Localization of Nav1.1 in neurons
Nav1.1 channel protein is localization in the hippocampus, especially in pyramidal neurons.
2. Different concentrations of lamotrigine act on sodium current when holding potential change
IC50 of lamotrigine were 1200μM (-90mV) and 400μM (-70mV).
3. Different concentrations of ilepcimide act on sodium current when holding potential change
IC50 of ilepcimide were 5.2μM (-90mV) and 0.4μM (-70mV).
4. Sodium channel properties with or without 10μM ilepcimide at holding potential -90mV
4.1. Activation
WT mice pyramids neurons Voltage dependence of activation: V1/2 and k (slope factor) were(n=5): without ilepcimide: -36.63±0.22(mV),3.87±0.19; with ilepcimide: -43.93±0.90(mV),4.22±0.70 (P<0.05).
4.2. Inactivation
WT mice pyramids neurons Voltage dependence of inactivation: V1/2 and k (slope factor) were(n=5): without ilepcimide: -43.76±0.49(mV),7.03±0.43; with ilepcimide: -61.74±0.65(mV),8.19±0.58 (P<0.05).
4.3. Recovery from inactivation
WT mice pyramids neurons recovery from inactivation: the time constant was:without ilepcimide (n=5): 2.05±0.42(ms),with ilepcimide: 2.20±0.16(ms) (P>0.05).
4.4. The area under activation and inactivation curves
In WT mice pyramids neurons, the ratio of the area under the curve of activation and inactivation between without or with ilepcimide were (n=5): 1:0.53. The area was minimized.
Conclusion
1. This is the first time to use the whole cell recording technique to study the mechanism of ilepcimide. We found that ilepcimide significantly inhibited the sodium current. The IC50 of ilepcimide was 5.2μM at the holding potential (Vh) of -90mV compare with the IC50=0.4μM at the holding potential of -70mV. These actions were similar to those produced by the antiepileptic drug lamotrigine in various cell types.
2. This is the first time we found that ilepcimide at therapeutically relevant concentrations caused a hyperpolarising shift of the activation curve and the inactivation curve, but had no influence to recovery from inactivation curve. The ratio of the area under the curve of activation and inactivation also minimized. In contrast, ilepcimide play a major role in inactivation state of VGSCs channel.
3. Our study suggests that ilepcimide may combine with VGSCs proteins, decrease the excitability of pyramidal neurons by promoting the inactivation state of VGSCs. We provide a basic molecular mechanism of ilepcimide as an AED. We would focus on the reason why ilepcimide showed good tolerability and mild side effects in future research.
引文
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2. Meisler MH, Kearney JA. Sodium channel mutations in epilepsy andother neurological disorders. J Clin Invest 2005, 115(8):2010-2017.
3. Shao D, Okuse K, Djamgoz MBA. Protein-protein interactions involving voltage-gated sodium channels: Posttranslational regulation, intracellular trafficking and functional expression. Int J Biochem Cell Biol 2009, 41(7): 1471-1481.
4. Ragsdale DS, Avoli M. Sodium channels as molecular targetsfor antiepileptic drugs. Brain Res Rev 1998; 26(1): 16-28.
5. Weiss LA, Escayg A, Kearney JA, Trudeau M, MacDonald BT, Mori M, Reichert J, Buxbaum JD, Meisler MH. Sodium channels SCN1A, SCN2A and SCN3A in familial autism. Molecular Psychiatry 2003, 8(2):186-194.
6. www.who.int/mediacenter/factsheets
7. Banu SH, Jahan M, Koli UK, Ferdousi S, Khan NZ, Neville B. Side effects of phenobarbital and carbamazepine in childhood epilepsy: randomised controlled trial. BMJ 2007, 334(7605):1207.
8. Tomson T, Dahl ML, Kimland E. Therapeutic monitoring of antiepileptic drugs for epilepsy. Cochrane Database Syst Rev 2007, 24 (1):CD002216.
9. Vohora D, Saraogi P, Yazdani MA, Bhowmik M, Khanam R, Pillai KK. Recent advances in adjunctive therapy for epilepsy: focus on sodium channel blockers as third-generation antiepileptic drugs Drugs Today (Barc) 2010, 46(4):265-277.
10. Massimo M, Giulia C, Giuseppe B, David SR, Massimo A. Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders. Lancet Neurol 2010, 9(4):413-424.
11. Escayg A, Goldin AL. Sodium channel SCN1A and epilepsy: mutations and mechanisms. Epilepsia 2010, 51(9):1650-1658.
12. Liao WP, Shi YW, Long YS, Zeng Y, Li T, Yu MJ, Su T, Deng P, Lei ZG, Xu SJ,Deng WY, Liu XR, Sun WW, Yi YH, Xu ZC, Duan S. Partial epilepsy with antecedent febrile seizures and seizure aggravation by antiepileptic drugs: associated with loss of function of Na(v) 1.1. Epilepsia 2010, 51(9):1669-1678.
13. Wang L, Zhao DY, Zhang ZH, Liu CS, Lin Q, Hu SX, Wu XR, Zuo QH, Zhang YY, Pei YQ, et al. Double-blind crossover controlled study on antiepilepsirine. Chin Med J (Engl) 1989, 102(2):79-85.
14. Wang L, Zhao D, Zhang Z, Zuo C, Zhang Y, Pei YQ, Lo YQ. Trial of antiepilepsirine (AES) in children with epilepsy. Brain Dev 1999, 21(1):36-40.
15. Wang L, Zhao DY, Zhang ZH, Liu CS, Lin Q, Hu SX, Wu XR, Zuo QH, Zhang YY, Pei YQ, et al. Double-blind crossover controlled study on antiepilepsirine. Chin Med J (Engl) 1989, 102(2):79-85.
16. Pei YQ. A review of pharmacology and clinical use of piperine and its derivatives. Epilepsia 1983, 24(2):177-182.
17. Yan QS, Mishra PK, Burger RL, Bettendorf AF, Jobe PC, Dailey JW. Evidence that carbamazepine and antiepilepsirine may produce a component of their anticonvulsant effects by activating serotonergic neurons in genetically epilepsy-prone rats. J Pharmacol Exp Ther 1992, 261(2):652-659.
18. Xie XM, Lancaster B, Peakman T, Garthwaite J. Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurons. Pflfigers Arch-Eur J Physiol 1995, 430: 437-446.
19. Karoly R, Lenkey N, Juhasz AO, Vizi ES, Mike A. Fast- or slow-inactivated state preference of Na+ channel inhibitors: a simulation and experimental study. PLoS Comput Biol 2010, 17; 6(6):e1000818.
20. Kuo CC, Lu L. Characterization of lamotrigine inhibition of Na+ channels in rat hippocampal neurons. Bri J Pharmacol. 1997; 121, 1231-1238.
21. Bialer M. New antiepileptic drugs that are second generation to existing antiepileptic drugs. Expert Opin Investig Drugs. 2006; 15(6): 637-647.
22. Stefan H, Feurstein TJ. Novel anticonvulsant drugs. Pharmac Ther 2007, 113(1): 165-183.
23. Kwan P, Brodie MJ. Refractory epilepsy: mechanism and solution. Expert Rev Neurothera 2006, 6(3): 397-406.
24. Pellock JM, Watemberg N. New antiepileptic drugs in children: present and children. Semin Pediatr Neurol 1997, 4(1): 9-18.
25. Luszcki JJ. Third generation antiepileptic drugs: mechanism of action, pharmacokinetics and interactions. Pharmac Rep 2009, 61(2): 197-1016.
26. Perucca E. Established antiepileptic drugs. Baillieres Clin Neurol 1996, 5(4): 693-722.
27. Herman ST, Pedley TA. New options for the treatment of epilepsy. JAMA 1998, 280(8): 693-694.
28. Sitges M, Guarneros A, Nekrassov V. Effects of carbamazepine, phenytoin, valproic acid, oxcarbazepine, lamotrigine, topiramate and vinpocetine on the presynaptic Ca2+ channel-mediated release of [3H]glutamate: comparison with the Na+ channel-mediated release. Neuropharmacology 2007, 53(7): 854-862.
29. Bialer M, Johannessen SI, Kupferberg HJ, Levy RH, Perucca E, Tomson T, SteveWhite H. Progress report on new antiepileptic drugs: A summary of the Ninth EILAT Conference (EILAT IX). Epilepsy Res 2009, 83(1): 1-43.
30. Rogawski MA. Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy Res 2006, 69(3): 273-294.
31. Clare JJ, Tate SN, Nobbs M, Romanos MA. Voltagegated sodium channels as therapeutic targets. Drug Discov Today 2000, 5(11): 506-20.
32. Bolcskei H, Tarnawa I, Kocsis P. Voltage gated sodium channel blockers.2001-2006: An overview. Med Chem Res 2008, 17: 356-368.
33. Tarnawa I, Bolcskei H, Kocsis P. Blockers of voltage gated sodium channels for the treatment of CNS diseases. Recent Pat CNS Drug Discov 2007, 2(1): 57-78.
34. Beyreuther BK, Freitag J, Heers C, Krebsfanger N, Scharfeneckar U, Stohr T. Lacosamide: A review of preclinical properties. CNS Drug Rev 2007, 13(1): 21-42.
35. Wehner T, Bauer S, Hamer HM. Six months of post marketing experience with adjunctive lacosamide in patients with pharmacoresistant focal epilepsy at atertiary epilepsy center in Germany. Epilepsy Behav 2009, 16(3): 423-425.
36. Errington AC, CoyneL, St?hr L, Selve N, Lees G. Seeking a mechanism of action for the novel anticonvulsant lacosamide. Neuropharmacology 2006, 50(8): 1016-1029.
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