Classical Antiepileptic Drugs Research Articles

Unnatural Amino Acid Mutagenesis Reveals the Critical Role of Hydrogen Bonding for Binding of Retigabine in the Pore of KCNQ Channels

Retigabine is the prototypical member of a novel class of anti-epileptic drugs that act by potentiating neuronal KCNQ2/3 channels. Retigabine interacts directly with the pore of KCNQ2/3 channels, requiring a conserved Trp, present in both KCNQ2 and KCNQ3, but not KCNQ1. Application of 100 µM retigabine to KCNQ3 channels causes a leftward 40 mV shift of the G-V curve that can be strongly ameliorated with either Phe or Leu substitution of Trp 265, with minor effects on channel gating. We have used nonsense suppression to subtly alter the properties of this Trp residue (265) in homomeric KCNQ3[A315T] channels to probe the chemical features of retigabine binding in more detail. A tri-fluorinated Trp derivative (3,4,5-F-Trp), which reduces cation-pi binding ability of this side chain caused a 20 mV left-shift in the gating properties of KCNQ3, but these channels were still highly sensitive to retigabine, suggesting that a cation-pi interaction is not essential for retigabine interactions with KCNQ3. In contrast, Ind (an isosteric Trp derivative that lacks the indole nitrogen atom required for hydrogen bonding ability of Trp) substitution completely abolished he effects of retigabine with minimal effects on KCNQ3 gating. These observations demonstrate that retigabine binds KCNQ3 channels through an energetically significant H-bond interaction via the indole nitrogen of Trp 265. These findings stringently constrain models of retigabine binding to KCNQ channels, and will guide the rational development of improved retigabine derivatives.

P 248. Cortical inhibition by Retigabine in epilepsy patients: Assessment by transcranial magnetic stimulation

Introduction Transcranial magnetic stimulation (TMS) has been used to investigate the mechanism of action of several drugs. Sodium-channel blocking drugs elevate the threshold, while drugs that act on GABAergic transmission improve intracortical inhibition or prolong the silent period. While these basic studies are designed to investigate physiology and pharmacology, TMS can be suitable as biomarker in individual patients. A prospective study in newly diagnosed epilepsy demonstrated that TMS responses measured shortly after the application of an antiepileptic drug can predict long-term seizure control. ∗ Retigabine is a first-in-class antiepileptic drug that acts on potassium channels. Indirectly, changes in GABAergic synaptic transmission may contribute to its efficacy. In-vivo studies of the physiological effect of Retigabine on motor cortex excitability in patients with epilepsy could deepen our understanding of the involved mechanisms. The results of such a study may be compared with the literature on TMS-effects of classical anti-epileptic and other drugs acting on the central nervous system. Objectives The study aims to investigate the effect of Retigabine on cortical excitability measured by TMS in patients with partial onset epilepsy. If there is a change in the TMS variables after starting Retigabine, we would like to see whether this correlates with the therapeutic effect. Materials and methods This is an observational neurophysiological/neuropharmacological study. Fifteen patients with uncontrolled partial onset epilepsy, with or without secondary generalization that have an indication to initiate adjuvant treatment with Retigabine will participate. Doses of conconcurrent medication are kept constant during the study. TMS is performed before starting Retigabine, on 600 mg/day (post1), on 900/dag (post2) or after any other different maintenance dose is reached (post3). In each session resting motor threshold, short interval (2 and 5 ms) intracortical inhibition, intracortical facilitation (10 and 15 ms), long interval intracortical inhibition (250 and 300 ms) en silent period are measured. Results and conclusion At the conference preliminary results and conclusions will be presented.

Withdrawal of anti-epileptic medications during video EEG monitoring does not alter ECG parameters or HRV

To assess if anti-epileptic drug (AED) withdrawal/cessation results in changes in ECG parameters and/or measures of heart rate variability (HRV), in patients having VEEG monitoring, that might affect susceptibility to sudden death in epilepsy. In this study, we included 36 patients with medically refractory epilepsy undergoing continuous video-EEG-ECG monitoring in hospital for pre-surgical assessment. We recorded and analysed multiple 10-min epochs of 2-lead ECG during periods when the subjects were awake and in REM and non-REM sleep, both on admission, and after the subjects had been partially or completely weaned from their AEDs. We compared ECG parameters and measures of HRV from these recorded epochs before and after AED reduction/cessation, with each patient acting as their own comparator. Epochs measured during awake, REM, and non-REM periods were analysed separately. In addition, we analysed a subgroup of patients who had been withdrawn from Na+-channel blocking medications specifically, to analyse the effect of this particular class of AEDs in isolation. Upon AED withdrawal, we observed a small increase in heart rate and shortening of the QT interval, when subjects were awake, but no other changes in ECG parameters were detected, nor did we find changes in any measure of HRV. In addition, no significant changes were found during sleep. Similar results were found in the analysis of the subgroup of patients withdrawn from Na+-channel blocking AEDs. Our study does not support a prominent role for AEDs, and withdrawal/cessation of AEDs, in deranging cardiac physiology during video EEG monitoring in medically refractory epilepsy patients undergoing video EEG monitoring.

Imidazole Incorporated Semicarbazone Derivatives as a New Class of Anticonvulsants: Design, Synthesis and In-Vivo Screening

A series of novel imidazole incorporated semicarbazones was synthesized using an appropriate synthetic route and characterized by spectral analysis (IR, 1H NMR, 13C NMR and Mass). The anticonvulsant activity of the synthesized compounds was determined using doses of 30, 100, and 300 mg kg-1 against maximal electroshock seizure (MES), subcutaneous pentylenetetrazole (scPTZ) induced seizure and minimal neurotoxicity test. Six compounds exhibited protection in both models and 2-(1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethylidene)-N-p-tolylsemicarbazone emerged as the most active compound of the series without any neurotoxicity and significant CNS depressant effect. Liver enzyme estimations (SGOT, SGPT, Alkaline phosphatase) of the compound also showed no significant change in the enzymes levels. Moreover, it caused 80% elevation of γ-amino butyric acid (GABA) levels in the whole mice brain, thus indicating that it could be a promising candidate in designing of a potent anticonvulsant drug.

Effects of N-(morpholinomethyl)- p-isopropoxyphenylsuccinimide on the protective action of different classical antiepileptic drugs against maximal electroshock-induced tonic seizures in mice

BackgroundThe aim of this study was to determine the effects of N-(morpholinomethyl)-p-isopropoxy-phenylsuccinimide (MMIPPS) on the protective action of four classical antiepileptic drugs (AEDs: carbamazepine [CBZ], phenobarbital [PB], phenytoin [PHT] and valproate [VPA]) against maximal electroshock (MES)-induced seizures in mice. MethodsTonic hind limb extension (seizure activity) was evoked in adult male albino Swiss mice by a current (sine-wave, 25mA, 500V, 50Hz, 0.2s stimulus duration) delivered via auricular electrodes. Total brain concentrations of AEDs were measured to determine the characteristics of interaction between MMIPPS and classical AEDs in the mouse MES model. ResultsMMIPPS administered intraperitoneally (ip) at 100mg/kg significantly elevated the threshold for electroconvulsions in mice (p<0.01). MMIPPS at doses of 25 and 50mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, MMIPPS (50mg/kg) significantly enhanced the anticonvulsant activity of PB and VPA(p<0.05), but not that of CBZ or PHT, in the MES test in mice. Pharmacokinetic studies revealed that MMIPPS (50mg/kg) did not alter total brain concentrations of PB, but significantly elevated total brain concentrations of VPA in mice (p<0.05). ConclusionsThe enhanced anticonvulsant action of PB byMMIPPS in themouseMESmodel and lack of any pharmacokinetic interaction between drugs make the combination of MMIPPS with PB of pivotal importance for further experimental and clinical studies. Pharmacokinetic increase in total brain VPAconcentration seems to be responsible for the enhanced anticonvulsant action of VPAby MMIPPS in the mouse MES model. The combinations of MMIPPS with CBZ and PHT are neutral from a preclinical viewpoint.

Carbamazepine and Multiple Myeloma: Possible Interaction

To the Editor Epilepsy is the most common chronic neurological disease and patients are treated by various classes of antiepileptic drugs [1]. In addition to their acute side effects, there are long-term adverse effects of antiepileptic drugs [2]. Hemopoietic neoplasias such as lymphoma, multiple myeloma, and some solid cancers including lung, liver, pancreas, and gastrointestinal cancers are the malignant disorders most frequently discussed in relation to use of antiepileptic drugs [3,4]. The carcinogenic effect of carbamazepine is very limited. However, there are some case reports and series containing a limited number of cases of hypogammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), and multiple myeloma [3,4,5,6,7,8,9,10,11]. We present here a 54-year-old woman with multiple myeloma with long exposure to carbamazepine. Vertebral fracture was detected and vertebroplasty was done urgently. There was no evidence of neurologic deficit. She had a history of 400 mg carbamazepine usage for more than 20 years due to idiopathic epilepsy. Laboratory examination showed total protein/albumin of 8.85/3.41 g/dL and calcium of 10.77 mg/dL (8.4-9.2); she had macrocytic anemia. Bence-Jones protein in the urine was not demonstrated. Protein electrophoresis showed an M-peak (Figure 1) with an elevated serum level of immunoglobulin G (IgG) of 2740 mg/dL. Immunoelectrophoresis revealed M-protein composed with a kappa chain. Bone marrow aspiration and biopsy showed 90% plasma cell infiltration and CD38 and kappa were positive. Cytogenetic analysis of bone marrow showed 17 p deletion (+), 13 q deletion (+), t (11;14), t (4;14). Histopathological examination of a vertebroplasty specimen revealed plasma infiltration. Carbamazepine usage was stopped and treatment was continued with valproic acid. A regimen containing bortezomib (1.3 g/m2), dexamethasone (40 mg), and zoledronic acid was prescribed. After 4 courses of chemotherapy, blood and bone marrow exams were normal. Stem cell transplantation was planned for consolidation. Carbamazepine-related multiple myeloma has been reported in a few case reports. It has been suggested that carbamazepine may cause the IgG type of M-protein multiple myeloma, MGUS, and hypogammaglobulinemia [5,6,9,11,12]. Our patient was younger than is usual for multiple myeloma. We can speculate that the exposure to an extrinsic factor may be associated with younger age in this case. The relationships among the duration of use of antiepileptic drugs, cumulative dose of antiepileptic drugs, and development of multiple myeloma are not known completely. In our case, multiple myeloma developed after 20 years of carbamazepine use. In conclusion, carbamazepine may be a potential drug in the development of multiple myeloma. For this reason, periodical evaluation of serum levels of immunoglobulin is necessary in patients receiving carbamazepine. Figure 1 Abnormal serum protein electrophoresis pattern in a patient with multiple myeloma. Note the large spike in the early gamma region. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

Effect of acute and chronic tianeptine on the action of classical antiepileptics in the mouse maximal electroshock model

BackgroundThe aim of the study was to analyze the influence of acute and chronic treatment with tianeptine, an antidepressant selectively accelerating presynaptic serotonin reuptake, on the protective activity of classical antiepileptic drugs in the maximal electroshock test in mice. MethodsElectroconvulsions were produced by means of an alternating current (50Hz, 25mA, 0.2 s) delivered via ear-clip electrodes. Motor impairment and long-term memory deficits in animals were quantified in the chimney test and in the passive-avoidance task, respectively. Brain concentrations of antiepileptic drugs were measured by fluorescence polarization immunoassay. ResultsAcute and chronic treatment with tianeptine (25–50mg/kg) did not affect the electroconvulsive threshold. Furthermore, tianeptine applied in both acute and chronic protocols enhanced the anticonvulsant action of valproate and carbamazepine, but not that of phenytoin. Neither acute nor chronic tianeptine changed the brain concentrations of valproate, carbamazepine or phenytoin. On the other hand, both single and chronic administration of tianeptine diminished the brain concentration of phenobarbital. In spite of this pharmacokinetic interaction, the antidepressant enhanced the antielectroshock action of phenobarbital. In terms of adverse effects, acute/chronic tianeptine (50mg/kg) and its combinations with classic antiepileptic drugs did not impair motor performance or long-term memory in mice. ConclusionThe obtained results justify the conclusion that tianeptine may be beneficial in the treatment of depressive disorders in the course of epilepsy.

Perampanel

Perampanel is a novel antiepileptic drug (AED) used as adjunctive therapy in adolescents and adults with partial-onset seizures (with or without secondarily generalized seizures). It is a selective, noncompetitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors on post-synaptic neurons, and is the first in this new class of AED known as AMPA receptor antagonists. In three randomized, double-blind, placebo-controlled, phase III trials in adolescent and adult patients with refractory partial-onset seizures, once-daily administration of perampanel 4, 8 and 12 mg/day (6-week titration phase followed by 13-week maintenance phase), as adjunctive therapy with one to three AEDs, was statistically superior to adjunctive placebo in achieving the key efficacy endpoints of the median percentage change from baseline in seizure frequency and/or the proportion of patients with a ≥50 % reduction in seizure frequency relative to baseline. Adverse events were usually mild or moderate in severity and the most frequent treatment-emergent events reported among perampanel recipients were CNS-related, such as dizziness, somnolence, headache and fatigue. Interim data from a large extension study (16-week blinded conversion period followed by open-label maintenance phase), which enrolled patients who completed the phase III trials, showed a similar group response for the reduction in seizure frequency over at least 1 year of adjunctive treatment with perampanel. Perampanel was generally well tolerated over the longer-term in extension studies, with no unexpected adverse events reported. On the basis of its overall clinical profile and unique mechanism of action, perampanel is a useful adjunctive treatment option in patients with refractory partial-onset seizures.

Trazodone reduces the anticonvulsant action of certain classical antiepileptics in the mouse maximal electroshock model

BackgroundThe aim of the study was to examine effects of an acute and chronic treatment with trazodone, a serotonin antagonist and reuptake inhibitor (SARI), on the protective activity of four classical antiepileptic drugs provided in the maximal electroshock test in mice. MethodsElectroconvulsions were produced in mice by means of an alternating current (50Hz, 25mA, 0.2s) and delivered via earclip electrodes. Motor impairment in animals were assessed in the chimney test, and long-term memory deficits were quantified in the passive-avoidance task. Brain concentrations of antiepileptic drugs were analyzed by fluorescence polarization immunoassay. ResultsThe obtained results showed that a single administration of trazodone (up to 40mg/kg) did not influence the electroconvulsive threshold. In contrast, chronic treatment with the antidepressant (40mg/kg) significantly increased this parameter. Furthermore, both single and chronic administration of trazodone reduced the anticonvulsant effect of phenytoin and carbamazepine against the maximal electroshock. However, the antidepressant remained without effect on the anticonvulsant action of valproate and phenobarbital. Some interactions between trazodone and antiepileptic drugs may have a pharmacodynamic background. Both, acute and chronic treatment with the antidepressant diminished the brain concentration of phenytoin. Chronic trazodone lowered the brain levels of carbamazepine and phenobarbital. Moreover, acute and chronic trazodone increased the valproate concentration in the brain. As regards undesired effects, acute and chronic trazodone (40mg/kg), alone and in combination with phenytoin, significantly impaired long-term memory in tested animals, evaluated in the passive avoidance task. Acute trazodone (40mg/kg) alone and combined with phenytoin produced also significant motor deficits in mice, as measured in the chimney test. ConclusionThe obtained results allow to conclude that trazodone is not a good candidate for an antidepressant drug in epileptic patients.

Effect of ACEA—a selective cannabinoid CB1 receptor agonist on the protective action of different antiepileptic drugs in the mouse pentylenetetrazole-induced seizure model

Endogenous cannabinoid ligands and cannabinoid CB1 receptor agonists have been shown to exert anticonvulsant effects in various experimental models of epilepsy. The purpose of this study was to determine the effects of arachidonyl-2'-chloroethylamide (ACEA-a highly selective cannabinoid CB1 receptor agonist) on the protective action of clonazepam, ethosuximide, phenobarbital, and valproate against pentylenetetrazole (PTZ)-induced clonic seizures in mice. To ascertain any pharmacokinetic contribution of ACEA to the observed interactions between tested drugs, free (non-protein bound) plasma and total brain concentrations of the antiepileptic drugs were estimated. Additionally, acute adverse-effect profiles of the combination of ACEA and different classical antiepileptic drugs (clonazepam, ethosuximide, phenobarbital and valproate) with respect to motor performance, long-term memory and skeletal muscular strength were measured. Results indicated that ACEA (10mg/kg, i.p.) co-administered with phenylmethylsulfonyl fluoride (PMSF-a substance protecting ACEA against degradation by the fatty-acid hydrolase; 30mg/kg, i.p.) significantly potentiated the anticonvulsant activity of ethosuximide, phenobarbital and valproate in the mouse PTZ-induced clonic seizure model by reducing their median effective doses (ED(50) values) from 122.8mg/kg to 71.7mg/kg (P<0.01; for ethosuximide), from 13.77mg/kg to 5.26mg/kg (P<0.05; for phenobarbital), and from 142.7mg/kg to 87.3mg/kg (P<0.05; for valproate), respectively. In contrast, ACEA (10mg/kg, i.p.) in combination with PMSF (30mg/kg, i.p.) had no impact on the protective action of clonazepam against PTZ-induced seizures in mice. However, ACEA (10mg/kg)+PMSF (30mg/kg) considerably increased free plasma and total brain concentrations of ethosuximide and valproate in mice suggesting a pharmacokinetic nature of interaction between drugs. In contrast, free plasma and total brain concentrations of clonazepam and phenobarbital remained unchanged after ACEA+PMSF administration and thus, indicating pharmacodynamic interactions. Moreover, none of the examined combinations of ACEA (10mg/kg, i.p.)+PMSF (30mg/kg, i.p.) with clonazepam, ethosuximide, phenobarbital, and valproate (at their ED(50) values from the PTZ-induced seizure test) affected motor coordination in the chimney test, long-term memory in the passive avoidance task, and muscular strength in the grip-strength test in mice, indicating no possible acute adverse effects in animals. In conclusion, pharmacodynamic enhancement of the anticonvulsant potency of phenobarbital by ACEA+PMSF is worthy of recommendation for further clinical settings. Pharmacokinetic interactions of ACEA+PMSF with ethosuximide and valproate seem to be responsible for a significant suppression of PTZ-induced seizures in mice. The combination of ACEA+PMSF with clonazepam seems to be neutral from a preclinical viewpoint.

Influence of 5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione on the anticonvulsant action of 4 classical antiepileptic drugs in the mouse maximal electroshock-induced seizure model

BackgroundThe aim of this study was to determine the effects of 5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP10) on the protective action of 4 classical antiepileptic drugs – carbamazepine, phenobarbital, phenytoin and valproate – against maximal electroshock-induced seizures in mice. MethodsTonic hind limb extension (seizure activity) was evoked in adult male albino Swiss mice by an electric current (sine-wave, 25mA, 500V, 50Hz, 0.2s stimulus duration) delivered via auricular electrodes. Acute adverse-effect profiles with respect to motor performance, long-term memory and skeletal muscular strength were measured, together with total brain antiepileptic drug concentrations. ResultsTP10 administered intraperitoneally at 10mg/kg significantly elevated the threshold for electroconvulsions in mice. TP10 at doses of 2.5 and 5mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, TP10 (5mg/kg) significantly enhanced the anticonvulsant activity of valproate, but not that of carbamazepine, phenobarbital or phenytoin in the maximal electroshock seizure test in mice. Pharmacokinetic experiments revealed that TP10 significantly elevated total brain concentrations of valproate in mice. ConclusionThe enhanced anticonvulsant action of valproate by TP10 in the mouse maximal electroshock-induced seizure model was associated with a pharmacokinetic increase in total brain valproate concentrations in mice. The combinations of TP10 with carbamazepine, phenobarbital and phenytoin were neutral from a preclinical viewpoint.

Effect of 4-(4-bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione on the anticonvulsant action of different classical antiepileptic drugs in the mouse maximal electroshock-induced seizure model

The aim of this study was to determine the effects of 4-(4-bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP4—a new S-triazole derivative possessing anticonvulsant properties in preclinical studies) on the protective action of four different classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) against maximal electroshock-induced seizures in mice.Results indicate that TP4 administered intraperitoneally at doses of 75 and 100mg/kg significantly elevated the threshold for electroconvulsions in mice. TP4 at doses of 12.5, 25, 37.5 and 50mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, TP4 (50mg/kg) significantly enhanced the anticonvulsant activity of carbamazepine, phenobarbital and valproate, but not that of phenytoin in the maximal electroshock seizure test in mice. TP4 at 25mg/kg significantly potentiated the anticonvulsant action of carbamazepine, but not that of phenobarbital, phenytoin and valproate in the mouse maximal electroshock-induced seizure model. Pharmacokinetic experiments revealed that TP4 significantly elevated total brain concentrations of carbamazepine and valproate, having no impact on total brain concentrations of phenobarbital in mice.In conclusion, the enhanced anticonvulsant action of phenobarbital by TP4 was probably pharmacodynamic in nature and, therefore, the combination of TP4 with phenobarbital is worthy of consideration while extrapolating the results from this study into clinical settings. The enhanced anticonvulsant action of carbamazepine and valproate by TP4 in the mouse maximal electroshock-induced seizure model was associated with pharmacokinetic increases in total brain concentrations of the antiepileptic drugs in mice. The combination of TP4 with phenytoin was neutral from a preclinical point of view.

Newer antiepileptic drugs in the treatment of status epilepticus: Impact on prognosis

Newer antiepileptic drugs (AEDs) are increasingly prescribed and seem to have a comparable efficacy as the classical AEDs; however, their impact on status epilepticus (SE) prognosis has received little attention. In our prospective SE database (2006–2010), we assessed the use of older versus newer AEDs (levetiracetam, pregabalin, topiramate, lacosamide) over time and its relationship to outcome (return to clinical baseline conditions, new handicap, or death). Newer AEDs were used more often toward the end of the study period (42% of episodes versus 30%). After adjustment for SE etiology, SE severity score, and number of compounds needed to terminate SE, newer AEDs were independently related to a reduced likelihood of return to baseline (p<0.001) but not to increased mortality. These findings seem in line with recent findings on refractory epilepsy. Also, in view of the higher price of the newer AEDs, well-designed, prospective assessments analyzing the impact of newer AEDs on efficacy and tolerability in patients with SE appear mandatory.

Influence of N-hydroxymethyl-p-isopropoxyphenylsuccinimide on the anticonvulsant action of different classical antiepileptic drugs in the mouse maximal electroshock-induced seizure model

Experimental epileptology is mainly focused on searching for some active compounds suppressing seizures that could become efficacious antiepileptic drugs. Accumulating evidence indicates that succinimide derivatives would be good candidates for novel antiepileptic drugs. Therefore, the aim of this study was to determine the effects of N-hydroxymethyl-p-isopropoxyphenyl-succinimide (HMIPPS) on the protective action of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) in the maximal electroshock-induced seizure test in mice. Tonic hind limb extension (seizure activity) was evoked in adult male albino Swiss mice by a current (sine-wave, 25 mA, 500 V, 50 Hz, 0.2s stimulus duration) delivered via auricular electrodes. Acute adverse-effect profiles with respect to motor performance, long-term memory and skeletal muscular strength were measured along with total brain antiepileptic drug concentrations. Results indicate that HMIPPS administered intraperitoneally at 100mg/kg significantly elevated the threshold for electroconvulsions in mice (P<0.05). HMIPPS at doses of 12.5, 25 and 50mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, HMIPPS (50mg/kg) significantly enhanced the anticonvulsant activity of phenobarbital and valproate in the mouse maximal electroshock-induced seizure model by reducing their median effective doses (ED(50) values) from 23.25mg/kg to 16.82 mg/kg (P<0.01; for phenobarbital) and from 259.3mg/kg to 189.7 mg/kg (P<0.001; for valproate), respectively. In contrast, HMIPPS (50mg/kg) had no impact on the protective action of carbamazepine or phenytoin in the maximal electroshock seizure test in mice. HMIPPS (25mg/kg) significantly potentiated the anticonvulsant action of valproate by reducing its ED(50) value from 259.3mg/kg to 210.6 mg/kg (P>0.05), but not that of phenobarbital, phenytoin and carbamazepine in the mouse maximal electroshock-induced seizure model. Pharmacokinetic experiments revealed that HMIPPS did not alter total brain concentrations of phenobarbital or valproate in mice. Moreover, none of the examined combinations of HMIPPS (50mg/kg) with carbamazepine, phenobarbital, phenytoin and valproate (at their ED(50) values from the maximal electroshock-induced seizure test) affected motor coordination in the chimney test, long-term memory in the passive avoidance task, and muscular strength in the grip-strength test in mice, indicating no possible acute adverse effects in animals. In conclusion, the enhanced anticonvulsant action of phenobarbital and valproate by HMIPPS in the mouse maximal electroshock-induced seizure model, lack of pharmacokinetic interactions and no potential acute adverse effects make the combinations of HMIPPS with phenobarbital and valproate worthy of consideration for further experimental and clinical studies. The combinations of HMIPPS with carbamazepine and phenytoin are neutral from a preclinical viewpoint.

1‐[(2‐Arylthiazol‐4‐yl)methyl]azoles as a New Class of Anticonvulsants: Design, Synthesis, In vivo Screening, and In silico Drug‐like Properties

A series of novel thiazole incorporated (arylalkyl)azoles were synthesized and screened for their anticonvulsant properties using maximal electroshock and pentylenetetrazole models in mice. Among target compounds, 1-[(2-(4-chlorophenyl)thiazol-4-yl)methyl]-1H-imidazole (compound 4b), 1-[(2-phenylthiazol-4-yl)methyl]-1H-1,2,4-tria-zole (8a), and its 4-chlorophenyl analog (compound 8b) were able to display noticeable anticonvulsant activity in both pentylenetetrazole and maximal electroshock tests with percentage protection range of 33-100%. A computational study was carried out for prediction of pharmacokinetics properties and drug-likeness. The structure-activity relationship and in silico drug relevant properties (molecular weight, topological polar surface area, clog P, hydrogen bond donors, hydrogen bond acceptors, and log BB) confirmed that the compounds were within the range set by Lipinski's rule-of-five, and possessing favorable physicochemical properties for acting as CNS-drugs, making them potentially promising agents for epilepsy therapy.

Primary Amino Acid Derivatives: Substitution of the 4′-N′-Benzylamide Site in (R)-N′-Benzyl 2-Amino-3-methylbutanamide, (R)-N′-Benzyl 2-Amino-3,3-dimethylbutanamide, and (R)-N′-Benzyl 2-Amino-3-methoxypropionamide Provides Potent Anticonvulsants with Pain-Attenuating Properties

Recently, we reported that select N'-benzyl 2-substituted 2-amino acetamides (primary amino acid derivatives (PAADs)) exhibited pronounced activities in established whole animal anticonvulsant (i.e., maximal electroshock seizure (MES)) and neuropathic pain (i.e., formalin) models. The anticonvulsant activities of C(2)-hydrocarbon N'-benzyl 2-amino acetamides (MES ED(50) = 13-21 mg/kg) exceeded those of phenobarbital (ED(50) = 22 mg/kg). Two additional studies defining the structure-activity relationship of PAADs are presented in this issue of the journal. In this study, we demonstrated that the anticonvulsant activities of (R)-N'-benzyl 2-amino-3-methylbutanamide and (R)-N'-benzyl 2-amino-3,3-dimethylbutanamide were sensitive to substituents at the 4'-N'-benzylamide site; electron-withdrawing groups retained activity, electron-donating groups led to a loss of activity, and incorporating either a 3-fluorobenzyloxy or 3-fluorophenoxymethyl group using a rationally designed multiple ligand approach improved activity. Additionally, we showed that substituents at the 4'-N'-benzylamide site of (R)-N'-benzyl 2-amino-3-methoxypropionamide also improved anticonvulsant activity, with the 3-fluorophenoxymethyl group providing the largest (∼4-fold) increase in activity (ED(50) = 8.9 mg/kg), a value that surpassed phenytoin (ED(50) = 9.5 mg/kg). Collectively, the pharmacological findings provided new information that C(2)-hydrocarbon PAADs represent a novel class of anticonvulsants.

Effect of acutely and chronically administered venlafaxine on the anticonvulsant action of classical antiepileptic drugs in the mouse maximal electroshock model

The influence of acute and chronic treatments with intraperitoneal venlafaxine, a selective serotonin/norepinephrine reuptake inhibitor, on the anticonvulsant activity of selected antiepileptic drugs was studied in the maximal electroshock test in mice. Venlafaxine (12.5 and 25mg/kg), given either acutely or chronically, significantly increased the electroconvulsive threshold. Moreover, both acute and chronic venlafaxine, applied at the highest subprotective dose of 6.25mg/kg, enhanced the anticonvulsant effect of valproate, without affecting the protective action of carbamazepine, phenobarbital and phenytoin. The antidepressant did not affect brain concentration of valproate, indicating that the interaction between the two drugs seems pharmacodynamic in nature. Despite the lack of effect on the antielectroshock action of the remaining antiepileptics, acute venlafaxine increased the brain concentration of phenobarbital, while chronic venlafaxine reduced the brain level of phenytoin. In terms of adverse effects, acute/chronic venlafaxine and antiepileptic drugs alone, as well as their combinations, did not produce significant motor or long-term memory deficits in mice. Summing up, it seems that venlafaxine may be considered as a safe drug for the clinical use in patients with epilepsy and depressive disorders.

Effects of WIN 55,212-2 mesylate (a synthetic cannabinoid) on the protective action of clonazepam, ethosuximide, phenobarbital and valproate against pentylenetetrazole-induced clonic seizures in mice

The aim of this study was to determine the effect of WIN 55,212-2 mesylate (WIN - a non-selective cannabinoid CB1 and CB2 receptor agonist) on the protective action of four classical antiepileptic drugs (AEDs: clonazepam [CZP], ethosuximide [ETS], phenobarbital [PB], and valproate [VPA]) in the mouse pentylenetetrazole (PTZ)-induced clonic seizure model. WIN (15 mg/kg, i.p.) significantly enhanced the anticonvulsant action of ETS, PB and VPA, but not that of CZP against PTZ-induced clonic seizures. The ED(50) values of ETS, PB and VPA were reduced from 148.0, 13.9 and 137.1mg/kg to 104.0, 8.3 and 85.6 mg/kg, respectively (P<0.05). WIN (5 and 10mg/kg, i.p.) had no impact on the anticonvulsant action of all studied AEDs against PTZ-induced clonic seizures. WIN (15 mg/kg, i.p.) significantly elevated total brain concentrations of ETS and VPA, but not those of CZP and PB in mice. Moreover, WIN combined with CZP, ETS, PB and VPA significantly impaired motor performance, long-term memory and muscular strength in mice subjected to the chimney, passive avoidance and grip-strength tests, respectively. Pharmacodynamic enhancement of the anticonvulsant action of PB by WIN against PTZ-induced clonic seizures is favorable from a preclinical viewpoint. Advantageous effects of WIN in combination with ETS and VPA against PTZ-induced seizures were pharmacokinetic in nature. However, WIN combined with CZP, ETS, PB and VPA impaired motor coordination and long-term memory as well as reduced skeletal muscular strength in the experimental animals.

Interactions between ACE inhibitors and classical antiepileptic drugs in the mouse maximal electroshock seizures

This study evaluated the effect of two angiotensin-converting enzyme (ACE) inhibitors, enalapril and cilazapril, commonly used antihypertensive drugs, on the protective efficacy of the classical antiepileptics — carbamazepine (CBZ), phenytoin (PHT), valproate (VPA) and phenobarbital (PB). For this purpose, we used the maximal electroshock seizure (MES) test in mice. Additionally, adverse effects of combined treatment with ACE inhibitors and antiepileptic drugs in the passive avoidance task and chimney test were assessed. All drugs were administered intraperitoneally. Neither enalapril (10, 20 and 30mg/kg) nor cilazapril (5, 10 and 20mg/kg) affected the threshold for electroconvulsions. Enalapril (30mg/kg) but not cilazapril (20mg/kg), enhanced the protective action of VPA, decreasing its ED50 value from 249.5 to 164.9mg/kg (p<0.01). Free plasma (non-protein-bound) and total brain concentrations of VPA were not significantly influenced by enalapril. Therefore, the observed interaction could be pharmacodynamic in nature. The combinations of ACE inhibitors with other antiepileptics (CBZ, PHT, and PB) were ineffective in that their ED50 values against MES were not significantly changed. Enalapril and cilazapril remained ineffective as regards memory retention in the passive avoidance task or motor performance in the chimney test. The current study suggests that there are no negative interactions between the studied ACE inhibitors and classical antiepileptic drugs. Enalapril was even documented to enhance the anticonvulsant activity of VPA.

General conclusions

The first meeting on Immunity and Inflammation in Epilepsy overviewed and discussed the current knowledge in this field of experimental and clinical research with the principal aim of identifying areas of further investigations to address the still unresolved fundamental aspects. The interdisciplinary group of basic science and clinical investigators proposed preliminary answers to the identified crucial questions, as summarized in Table 1. The possible causes of brain inflammation were discussed in the frame of different epilepsy etiologies. The role of recurrent seizures per se as a cause of persistent inflammation, beyond a detectable structural insult, was highlighted. However, the identification of contributing factors to the inflammatory process and their clinical relevance are still largely unknown. The contribution of brain inflammation to hyperexcitability underlying seizures is well delineated in experimental models, although the role of inflammatory mediators in epileptogenesis requires additional studies. Aspects awaiting further investigations include: (1) the discovery and characterization of novel inflammatory molecules and pathways in multiple experimental models; (2) elucidating whether and how the inflammatory pathways so far identified converge into common molecular targets; (3) the validation of inflammatory processes described in the experimental setting in surgically resected epileptogenic tissue from epilepsies of different etiology; and (4) development of strategies to target these inflammatory factors in order to inhibit seizures and/or prevent epileptogenesis. Major challenges include exploring cellular, molecular, and genetic mechanisms that may contribute to seizures and comorbidities in immune-mediated epilepsies, and establishing disease-specific animal models, as has been done for tuberous sclerosis complex or Dravet syndrome. The elucidation of the specific bystander (association) or truly causative role of autoantibodies present in several epileptic syndromes and autoimmune disorders may also improve management (diagnosis, treatment, and long-term disease monitoring) of patients with these conditions. International disease-specific consortia will help to identify and recruit numbers of patients with the still very rare autoimmune epilepsy syndromes, sufficient to fulfil the criteria for well-designed clinical trials. Systematic collection of clinical information and biologic samples that take into account the possible immune or inflammatory aspects of the epileptic condition, the development of noninvasive techniques to image brain inflammation, and the identification of biomarkers of brain inflammation in cerebrospinal fluid (CSF) and blood will greatly improve the identification of patients who may best benefit from a specific immunosuppressant or antiinflammatory treatment. Proof-of-concept pharmacologic evidence in experimental models may contribute to the discovery of new compounds, or to the identification of drugs already used for other clinical indications (e.g., in chronic inflammatory diseases), which may be considered for prospective clinical trials for treating seizures in autoimmune-mediated epilepsies, and more in general for the treatment of seizures that are not controlled by classical antiepileptic drugs (AEDs) in nonimmune types of epilepsies. Future meetings and multidisciplinary collaborations between basic science and clinical research with translational purposes will reinforce the efforts of gaining further insights into the complex role of immunity and inflammation in epilepsy, and hopefully will encourage the development of new treatments with disease-modifying therapeutic potential. A. Vezzani and S. Rüegg thank the scientific committee, the speakers, and the discussants. They are especially grateful to the Commission of European Affair of ILAE; the American Epilepsy Society; UCB Pharma, Basel, Switzerland; Sanofi-Aventis, Milano, Italy; and Questcor Union City, CA, U.S.A. for their support, which was instrumental for the realization of this meeting. We thank Philip Schwarzkroin and Simon Shorvon for providing us the opportunity to publish the proceedings of this meeting in Epilepsia.