Interaction Of The Antiepileptic Drug Ilepcimide With Na+ Channels In Mice Hippocampal Neuronse

BackgroundEpilepsy 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%.PurposeWhole-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.Methods1. Experimental animalsMale 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 skillIntraperitoneal 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. ImmunohistochemistryMice 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 neuronsThe 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 analysisData 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).Results1. Localization of Nav1.1 in neuronsNav1.1 channel protein is localization in the hippocampus, especially in pyramidal neurons.2. Different concentrations of lamotrigine act on sodium current when holding potential changeIC50 of lamotrigine were 1200μM (-90mV) and 400μM (-70mV).3. Different concentrations of ilepcimide act on sodium current when holding potential changeIC50 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. ActivationWT 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. InactivationWT 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 inactivationWT 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 curvesIn 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.Conclusion1. 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.