Pentylenetetrazole Seizure Models Research Articles

Discovery of New 3-(Benzo[b]Thiophen-2-yl)Pyrrolidine-2,5-Dione Derivatives as Potent Antiseizure and Antinociceptive Agents-In Vitro and In Vivo Evaluation.

Background/Objectives: To address the unmet clinical needs in the treatment of epilepsy and pain, the continued development of more effective and safer anticonvulsants and analgesics is still necessary. Therefore, herein we report synthesis and antiseizure/antinociceptive evaluation of a focused series of 3-(benzo[b]thiophen-2-yl)pyrrolidine-2,5-dione derivatives. Methods: The anticonvulsant properties were investigated in acute models of seizures, namely the maximal electroshock (MES), the 6 Hz (32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure models, whereas analgesic activity was tested in the model of a tonic pain/formalin test and oxaliplatin-induced neuropathic pain (in CD-1-mice, i.p.). In addition, a number of in vitro assays were performed, aiming at the evaluation of the drug-like properties of the compounds disclosed herein. Results: We identified 33 as a lead compound with the most promising antiseizure properties, i.e., ED50 (MES) = 27.4 mg/kg and ED50 (6 Hz, 32 mA) = 30.8 mg/kg. Furthermore, 33 at a dose of 100 mg/kg significantly prolonged the latency time to the first seizure episode in the scPTZ model and at high doses did not impaire coordination of mice in the rotarod test (TD50 > 200 mg/kg). Apart from broad antiseizure protection, 33 demonstrated a significant analgesic effect in the formalin test (45 mg/kg, i.p.), and effectively alleviated allodynia in the oxaliplatin-induced neuropathic pain model (30 and 45 mg/kg). The binding assays suggest that the most plausible mechanism of action relies on interaction with the neuronal voltage-sensitive sodium channel (site 2). Furthermore, the drug-like potential of 33 supports favorable in vitro results, i.e., no hepatocytotoxicity and neurocytotoxicity at a high concentration of 100 μM, as well as a lack of mutagenicity at a concentration as high as 500 μM. Conclusions: Compound 33 identified in the current studies is proposed to be an interesting candidate for further preclinical development as therapy for epilepsy and neuropathic pain.

DESIGN, SYNTHESIS, IN SILICO STUDIES, AND PHARMACOLOGICAL EVALUATION OF 5-ARYL-4-(CHLOROACETYLAMINO)-3-MERCAPTO-1,2,4-TRIAZOLE DERIVATIVES AS ANTICONVULSANT AGENTS

Objective: In this study, we reported the synthesis of a novel series of 5-aryl-4(chloroacetylamino)-3-mercapto-1, 2,4-triazoles. Methods: These compounds were synthesized to screen for anticonvulsant effects in a Maximal Electroshock Seizure (MES) model and a Subcutaneous Pentylenetetrazole (sc‐PTZ) seizure model in rats. Furthermore, molecular docking studies with gamma-aminobutyric acid and in silico ADME prediction were carried out to determine interactions of these compounds with Benzodiazepine (BZD) receptors and their similarity with standard drugs. The rotarod test was used to evaluate neurotoxicity. Results: 08 out of 40 compounds exhibited neurotoxicity at the maximum tested dose. Most of the compounds showed anti‐MES effects without any signs of neurological deficit. All the tested compounds significantly reduced seizures induced by PTZ compared to the control group. Carbamazepine and phenytoin were used as positive controls for anticonvulsant effects. Compounds 3d, 3h (a diphenylamine derivative of 5-aryl-4(chloroacetylamino)-3-mercapto-1,2,4-triazole), and 4a (a piperidinyl derivative of 5-aryl-4(chloroacetylamino)-3-mercapto-1,2,4-triazole) exhibited greater safety than phenytoin and carbamazepine in terms of neurotoxicity. The docking scores for the identified compounds 3d, 3h and 4a was 6.5133; 6.6558 and 5.6524, respectively. Nearly all the compounds (90%) demonstrated decreased locomotor activity. Conclusion: It is gratifying that the compounds with higher hydrophobicity showed better performance in the seizure models. Many triazole derivatives holding a suitable aryl or alkyl group gave a better anticonvulsant activity in their analogs.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

In vitro and in vivo anti-seizure activity of hydromethanolic extract and fractions of Pterolobium stellatum

Ethnopharmacological relevanceIn Ethiopia, the whole plant juice of Pterolobium stellatum is used to treat seizures and epilepsy. Aim of the studyTo investigate the antiseizure activity of hydromethanolic crude extract and fractions collected from leaves of P. stellatum using both in vitro, and in vivo seizure models in mice. Materials and methodsFresh leaves of P. stellatum were collected from Awash Melka, Addis Ababa, Ethiopia. An 80% crude methanol extract was further fractionated to produce petroleum ether, chloroform, butanol, and aqueous fractions. Anti-seizure activity of the crude extract and fractions (petroleum ether, chloroform, butanol, and water) were assessed at a concentration of 0.7 mg/ml using the in vitro 0 Mg2+ model of seizures in mouse brain slices prepared from 14- to 21-day-old C57BL/6 mice. The maximal electroshock seizure (MES) model and the pentylenetetrazol (PTZ) seizure model for seizures were performed on male BALB/c mice using 400 mg/kg and 800 mg/kg of crude 80% methanol extract, as well as the four fractions described above. Diazepam and phenytoin were used as positive controls for PTZ and MES test respectively. ResultsAddition of 0.7 mg/ml of crude 80% methanol extract of P. stellatum prevented the onset of SLEs in most brain slices in the 0 Mg2+in vitro model of seizures, with similar efficacy to diazepam (3 μM). The same extract at 400 and 800 mg/kg was efficacious in reducing the hindlimb extension time in the MES model and delaying the onset of myoclonic convulsions in the PTZ model, although not to the same extent as phenytoin (10 mg/kg) or diazepam (5 mg/kg). The chloroform and water fractions of the crude extract also showed significant anti-seizure activity across all three models whilst the non-polar petroleum ether and butanol fractions did not. The UPLC-MS analysis indicated the presence of gallic acid, ellagic acid, kaempferol, myricitrin, isoquercitrin and quercitirin in the crude extract. Gallic acid and ellagic acid were observed in chloroform fraction and in the water fraction ellagic acid, kaempferol, myricitrin and isoquercitrin were detected. ConclusionThe crude hydromethanolic extract of P. stellatum has significant anti-seizure activity. The chloroform and aqueous fractions have antiseizure activity. The extracts have previously identified compounds with anticonvulsant activity which indicates the antiseizure potential of the plant.

Acute And Chronic Learning Impairment Following Seizures In Larval Zebrafish

Early‐life seizures (ELS) can disrupt brain development, often leading to cognitive impairments in humans and rodents. Here, we investigated whether seizures in larval zebrafish (Danio rerio) lead to acute and long‐term learning deficits. Based on ELS studies in rodents, we hypothesized that seizures in larval zebrafish would impair their performance in declarative memory tasks. To test this, we induced seizures in larval zebrafish using pentylenetetrazole (PTZ), which causes behavioral and electrical seizures in zebrafish. We modified the existing PTZ seizure model, exposing zebrafish to PTZ daily from 5‐7 days post‐fertilization (dpf). We then compared later‐life learning through short‐term memory tasks that lacked external punishment or motivation and focused on conscious recognition and interest in novel stimuli. To test learning and memory acutely after seizures, we used the visual lateralization novel object recognition (VLNOR) test, based on the novel object recognition test frequently used in rodents. Zebrafish show asymmetries in the use of left (LES) and right eye systems (RES), and will initially use LES to observe novel objects and RES to view familiar objects. We found that, when tested for recall of a previously novel object one hour after initially viewing it, 12‐14 dpf unhandled controls (UC group) reached a statistically significant majority of RES approaches by the end of the 8‐minute recall test (p=0.048). The PTZ group did not, and trended towards a significant decrease from 50% in the first 2 minute bin of the recall test (p=0.054). Taken together, these data indicate that the PTZ fish were not able to recall a previously viewed object, and when the object was reintroduced 1 hour later, responded as if the object were novel. We next used a y‐maze task to assess memory 3 months after seizures. In this task, fish had to recall the novel arm of the maze 90 minutes after being removed from the maze. The PTZ group spent significantly less time in the novel arm of the y‐maze during the recall test compared to their UC clutchmates (mean % time in novel arm +/‐ SE = 44.0+/‐3.2 for UC, 29.7+/‐2.1 for PTZ; p=0.002 by t‐test). Furthermore, our preliminary molecular studies show distinct expression profiles of synaptic plasticity genes between PTZ and UC groups by qPCR. Taken together, these results imply that inducing seizures in larval zebrafish leads to both acute and chronic learning and memory impairments. These findings expand the behavioral characterization of the larval zebrafish seizure model, strengthening the power of this model for researching the cognitive consequences of ELS.

Neural Activity Correlates With Behavior Effects of Anti-Seizure Drugs Efficacy Using the Zebrafish Pentylenetetrazol Seizure Model.

Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs and treatment methods. Therefore, a critical need exists to efficiently screen anti-seizure drugs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel anti-seizure compounds. However, previous research using this model was variable due to differing experimental methods. Here, we present a method that was optimized to improve reliability and reproducibility in our laboratory using this PTZ model to develop a more robust screening of anti-seizure drugs comparing behavior and neural activity. Our behavior assay, spanning 90 min using 10 mM PTZ on 7 days post fertilization zebrafish, provides a broad window to observe anti-seizure drug efficacy. To compare our method with previously published data, we tested carbamazepine, lamotrigine, and topiramate, which have been tested in previous PTZ zebrafish assays. In addition, we assessed the candidate anti-seizure compound GS967, which has not been previously tested in the zebrafish seizure model. We examined the efficacy of anti-seizure drugs by acute administration concurrent with PTZ application and by pretreatment prior to exposure with PTZ. Pretreatment permitted us to examine potential neuroprotection and determine whether treatment time affects anti-seizure drugs’ responses. As independent validation of anti-seizure drugs’ effects, we evaluated whether the anti-seizure drug efficacy in the behavioral assay correlated with neural activity measurements, using electroencephalogram (EEG) and calcium signaling using GCaMP. There was no significant difference in the reduction of PTZ-induced seizure behavior activity between the pretreatment groups and acute treatment groups. Acute treatment with anti-seizure drugs in the EEG and GCaMP assays from 15 to 30 min post-anti-seizure drug exposure revealed consistent results between behavioral, EEG, and GCaMP assays for two of the three anti-seizure drugs. Lamotrigine only reduced neural activity (EEG and GCaMP assays). Carbamazepine, topiramate, and GS967 reduced activity in all three assays. The findings show that EEG and GCaMP assays largely correlate with the behavior findings, helping us connect physiological and behavior responses to anti-seizure drug and better assess anti-seizure drug efficacy.

From the North Sea to Drug Repurposing, the Antiseizure Activity of Halimide and Plinabulin.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin’s antiseizure action.

COMPARATIVE STUDY OF ANTICONVULSANT EFFECT OF THE LEAVES OF SAPINDUS EMARGINATUS AND ACORUS CALAMUS IN EXPERIMENTALLY INDUCED ANIMAL MODELS OF EPILEPSY

Objective: To compare anticonvulsant activity of methanol extracts of Sapindus emarginatus (MESE) and Acorus calamus (MEAC) in experimental seizure models in mice.
 Methods: Hind limb tonic extension (HLTE) in Maximal electroshock (MES) seizure and clonic seizure in Pentylenetetrazol (PTZ) seizure models were assessed. Group I (control) mice received 1% gum acacia in distilled water (1 ml/100 g). Topiramate (50 mg/kg) was administered in group II (standard) animals. Group III and IV mice were treated with 200 and 400 mg/kg of MESE, respectively. Mice in group V and VI were given MEAC at the dose of 200 and 400 mg/kg, respectively. Drugs were given orally suspended in 1% gum acacia suspension (1 ml/100 g) for 7 d. Next day after 1 h of drug administration, the seizure was induced for evaluation.
 Results: Anticonvulsant property of both extracts was confirmed by reduction (p<0.001) in HLTE phase in MES model; delayed onset of the clonic seizure (p<0.001) and its shortened phase (p<0.001) in PTZ model when compared with the control. MESE-200 mg/kg produced significantly longer (p<0.001) HLTE phase with lower protection (40.34%) among the different doses of the extracts. Clonic seizure onsets and durations in PTZ model were comparable among the different extract-treated groups; however, mortality was higher (66.6%) with MESE-200 mg/kg.
 Conclusion: Anticonvulsant activity of MESE and MEAC was evident; however, MESE at the dose of 200 mg/kg was less effective.

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using Drerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in Celegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

Intracerebroventricular administration of N-type calcium channel blocker ziconotide displays anticonvulsant, anxiolytic, and sedative effects in rats: A preclinical and pilot study

ObjectiveZiconotide (ω-conotoxin MVIIA peptide) is a novel analgesic agent acting on voltage-gated calcium channels and is administered intrathecally for neuropathic pain. While antiepileptic activities of other types of calcium channel blockers (T- or L-type) are well established, there is no information regarding the effect of ziconotide as an N-type calcium channel antagonist in pentylenetetrazol-induced seizures or its anxiolytic and sedative activities. The present study is the first to report on these effects. MethodsTo evaluate the anticonvulsant activity of ziconotide in the pentylenetetrazol (60 mg/kg) seizure model, ziconotide was administered intracerebroventricular (i.c.v.) as a single dose (1 μg/rat) or repeatedly (chronic administration: 0.1, 0.3, or 1 μg/rat once a day for seven days). The anxiolytic and sedative actions of ziconotide were evaluated with the elevated plus maze, light/dark (LD) box, and pentobarbital-induced sleep tests. Immediately after behavioral testing, the amygdala was completely removed bilaterally to determine corticosterone levels by immunoassay. ResultsIn all dosing regimens, ziconotide significantly decreased the seizure frequency and also delayed the latency period compared with control. Chronic administration affected the percentage of mortality protection, while a single dose of ziconotide did not. In behavioral tests, ziconotide significantly increased both the number of entries and the percentage of time spent in the open arms of the elevated plus maze. Furthermore, ziconotide significantly increased the latency period and the number of entries into the light compartment during the LD box examination. Chronic administration of ziconotide significantly reduced the latency to sleep and increased sleeping time, whereas these parameters were not affected by a single dose. Additionally, amygdala corticosterone levels were significantly decreased in rats treated with ziconotide compared with control. ConclusionZiconotide displays beneficial neurobehavioral effects in a model of epilepsy with anxiety as its comorbid event. It seems that at least one of the mechanisms involved in these effects is associated with a decrease in brain corticosterone levels. The main advantage of ziconotide over benzodiazepines (routine anxiolytic and sedative drugs) is that it does not cause tolerance, dependency, and addiction. Therefore, more than ever, it is necessary to improve the convenience of drug delivery protocols and attenuate the adverse effects associated with ziconotide-based therapies.

Experimental Models for the Discovery of Novel Anticonvulsant Drugs: Focus on Pentylenetetrazole-Induced Seizures and Associated Memory Deficits.

Epilepsy is a chronic neurological disorder characterized by irregular, excessive neuronal excitability, and recurrent seizures that affect millions of patients worldwide. Currently, accessible antiepileptic drugs (AEDs) do not adequately support all epilepsy patients, with around 30% patients not responding to the existing therapies. As lifelong epilepsy treatment is essential, the search for new and more effective AEDs with an enhanced safety profile is a significant therapeutic goal. Seizures are a combination of electrical and behavioral events that can induce biochemical, molecular, and anatomic changes. Therefore, appropriate animal models are required to evaluate novel potential AEDs. Among the large number of available animal models of seizures, the acute pentylenetetrazole (PTZ)-induced myoclonic seizure model is the most widely used model assessing the anticonvulsant effect of prospective AEDs, whereas chronic PTZ-kindled seizure models represent chronic models in which the repeated administration of PTZ at subconvulsive doses leads to the intensification of seizure activity or enhanced seizure susceptibility similar to that in human epilepsy. In this review, we summarized the memory deficits accompanying acute or chronic PTZ seizure models and how these deficits were evaluated applying several behavioral animal models. Furthermore, major advantages and limitations of the PTZ seizure models in the discovery of new AEDs were highlighted. With a focus on PTZ seizures, the major biochemicals, as well as morphological alterations and the modulated brain neurotransmitter levels associated with memory deficits have been illustrated. Moreover, numerous medicinal compounds with concurrent anticonvulsant, procognitive, antioxidant effects, modulating effects on several brain neurotransmitters in rodents, and several newly developed classes of compounds applying computer-aided drug design (CADD) have been under development as potential AEDs. The article details the in-silico approach following CADD, which can be utilized for generating libraries of novel compounds for AED discovery. Additionally, in vivo studies could be useful in demonstrating efficacy, safety, and novel mode of action of AEDs for further clinical development.

Anticonvulsant Effect of Alcea aucheri on Pentylenetetrazole and Maximal Electroshock Seizures in Mice.

Introduction:This study was designed to investigate the possible anticonvulsant effect of acute administration of an aqueous extract of flowers of Alcea aucheri (EFA) in two in vivo seizure models.Methods:Seizures were induced in male adult Swiss mice by administration of Pentylenetetrazol (PTZ) or Maximal Electroshock (MES). Mice were randomly subjected to receive saline, EFA (8.75–175 mg.kg−1), or diazepam intraperitoneally (i.p.) 15 or 30 min before PTZ injection. In another experiment, mice were treated (i.p.) with saline, EFA (8.75–350 mg.kg−1), or phenytoin 15 or 30 min before the MES test. Diazepam and phenytoin were used as reference drugs.Results:EFA (175 mg.kg−1) significantly increased the PTZ-induced seizure threshold compared with the saline control group 15 min after its administration. In the MES test, the extract (35 mg.kg−1) increased the latency to onset of tonic Hind Limb Extension (HLE) (seizure activity) compared with the saline group 15 min after treatment. Also, 30 min after treatment, EFA (35, 70, and 175 mg.kg−1) increased the latency to onset of the seizure, decreased the duration of the seizure (70 mg.kg−1), and decreased seizure occurrence (350 mg.kg−1) compared with those of the saline group. At both time points, the extract at all doses significantly reduced the mortality rate compared with the saline group.Conclusion:These findings provide evidence of a possible anticonvulsant effect of A. aucheri in PTZ and MES seizure models in mice.

Zebrafish-Based Discovery of Antiseizure Compounds from the North Sea: Isoquinoline Alkaloids TMC-120A and TMC-120B.

There is a high need for the development of new and improved antiseizure drugs (ASDs) to treat epilepsy. Despite the potential of marine natural products (MNPs), the EU marine biodiscovery consortium PharmaSea has made the only effort to date to perform ASD discovery based on large-scale screening of MNPs. To this end, the embryonic zebrafish photomotor response assay and the larval zebrafish pentylenetetrazole (PTZ) model were used to screen MNP extracts for neuroactivity and antiseizure activity, respectively. Here we report the identification of the two known isoquinoline alkaloids TMC-120A and TMC-120B as novel antiseizure compounds, which were isolated by bioactivity-guided purification from the marine-derived fungus Aspergillus insuetus. TMC-120A and TMC-120B were observed to significantly lower PTZ-induced seizures and epileptiform brain activity in the larval zebrafish PTZ seizure model. In addition, their structural analogues TMC-120C, penicisochroman G, and ustusorane B were isolated and also significantly lowered PTZ-induced seizures. Finally, TMC-120A and TMC-120B were investigated in a mouse model of drug-resistant focal seizures. Compound treatment significantly shortened the seizure duration, thereby confirming their antiseizure activity. These data underscore the possibility to translate findings in zebrafish to mice in the field of epilepsy and the potential of the marine environment for ASD discovery.

Synthesis, in-vitro cholinesterase inhibition, in-vivo anticonvulsant activity and in-silico exploration of N-(4-methylpyridin-2-yl)thiophene-2-carboxamide analogs

In our current research, a diverse effect of acetylcholinesterase inhibitors was studied on BALB-C mice by using pentylenetetrazole (PTZ) seizure model. A series of carboxamide analogs (4a–4i) have been synthesized via Suzuki coupling reaction in moderate to good yields (35–84%). To study the efficacy of the synthesized compounds against AD, in-vitro inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was performed. A number of compounds showed inhibition in low micromolar range. Subsequently, these compounds were evaluated for anticonvulsive effects in BALB-C mice by using pentylenetetrazole (PTZ) seizure model. The compound 4e displayed potential anticonvulsive effect and displayed 50% and 80% protection from mortality at the dose of 10 mg/kg, and 30 mg/kg respectively. The compound 4h showed some protection (33%) from mortality at 10 mg/kg and was not further explored based on non-significant delay in onset of myoclonic seizures. While, other compounds from the series did not show any anticonvulsive activity. To rationalize the observed biological activity, we performed docking studies against AChE and BChE targets. To explore the rationale of the mechanism of in-vivo anticonvulsant activity, docking studies were performed on GABAergic receptors. Moreover, in order to establish a relationship between physiochemical data of the synthesized compounds and their in-vivo performance, we employed in-silico pharmacokinetic predictions. Our in-silico predictions suggest that the plasma protein binding, low to moderate blood brain barrier penetration and low solubility are the main attributes of low in-vivo performance.

New anticonvulsant candidates prevent P-glycoprotein (P-gp) overexpression in a pharmacoresistant seizure model in mice

Despite the approval of a considerable number of last generation antiepileptic drugs (AEDs) (only in the last decade, six drugs have gained Food and Drug Administration approval), the global figures of seizure control have seemingly not improved, and available AED can still be regarded as symptomatic treatments. Fresh thinking in AEDs drug discovery, including the development of drugs with novel mechanisms of action, is required to achieve truly innovative antiepileptic medications.The transporter hypothesis proposes that inadequate penetration of AEDs across the blood–brain barrier, caused by increased expression of efflux transporters such as P-glycoprotein (P-gp), contributes to drug-resistant epilepsy. Neuroinflammation due to high levels of glutamate has been identified as one of the causes of P-gp upregulation, and several studies in animal models of epilepsy suggest that antiinflammatory drugs might prevent P-gp overexpression and, thus, avoid the development of refractory epilepsy.We have applied ligand-based in silico screening to select compounds that exert dual anticonvulsant and antiinflammatory effects. Five of the hits were tested in animal models of seizure, with protective effects. Later, two of them (sebacic acid (SA) and gamma-decanolactone) were submitted to the recently described MP23 model of drug-resistant seizures. All in all, SA displayed the best profile, showing activity in the maximal electroshock seizure (MES) and pentylenetetrazol (PTZ) seizure models, and reversing resistance to phenytoin (PHT) and decreasing the P-gp upregulation in the MP23 model. Furthermore, pretreatment with SA in the pilocarpine status epilepticus (SE) model resulted in decreased histamine release in comparison with nontreated animals.This is the first report of the use of the MP23 model to screen for novel anticonvulsant compounds that may avoid the development of P-gp-related drug resistance.

Design, synthesis, and evaluation of anticonvulsant activities of benzoxazole derivatives containing the 1,2,4-triazolone moiety.

A novel series of benzoxazole derivatives containing 1,2,4-triazolone (5a-m) was designed. These compounds were synthesized in order to screen their anticonvulsant activities by the maximal electroshock seizure (MES) model and the subcutaneous pentylenetetrazole (sc-PTZ) seizure model in mice. The rotarod test was used to evaluate their neurotoxicities. Most of the compounds showed anti-MES activities at 100 and 300 mg/kg. Compound 5f, which showed potential anticonvulsant activity in the MES model with ED50 values of 22.0 mg/kg, was considered as the most promising one in this study. It exhibited greater safety than that of carbamazepine and valproate regarding neurotoxicity. The efficacy of compound 5f in inhibiting the tonic seizures and death induced by the convulsants 3-mercaptopropionic acid and BIC was also verified. In an enzyme-linked immunosorbent assay, compound 5f and the positive drug phenytoin significantly increased the γ-aminobutyric acid (GABA) level in the mouse brain. Further, pretreatment with an inhibitor of the GABA synthesizing enzyme dramatically raised the ED50 value of 5f in the MES model. These results confirmed that the compound 5f plays its anticonvulsive action via regulating the GABA function in the brain. Also, a docking study of the compound 5f in the benzodiazepine (BZD) binding site of the GABAA receptor confirmed possible binding of the compound 5f with BZD receptors.

Evaluation of Anticonvulsant and Antioxidant Activity of Senna occidentalis Seeds Extracts

Aim: The aim of present work was to determine the anticonvulsant and antioxidant activity of Senna occidentalis L. ethanolic seed extract by different mod­els. Methods: For evaluation of anticonvulsant activity, Pentylenetetrazole (PTZ) seizure model and Maximal electroshock (MES) seizure model were used. For antioxidant activity, (1, 1-diphenyl - 2-picryl hydrazine (DPPH) and hydrogen peroxide (H2O2) method were used. Results: The finding suggested that the ethanolic extract (EAE) of Senna occidentalis in the dose 400 mg/kg body weight posses potent anticonvulsant activity. The EAE showed anticonvulsant action in dose dependent fashion. It was observed that upon increasing the concentration of extract, it showed reduced absorbance and increased free radical inhibition, and when comparison was made with Ascorbic acid, it showed marked antioxidant property in DPPH as well as H2O2 method. The IC50 of Ascorbic acid and EAE by DPPH method were found to be 14.56 and 14.8 respectively whereas the IC50 of Ascorbic acid and EAE by H2O2 method were found that 14.3and 14.8 respectively. Conclusion: The results of the present study concluded hat the EAE of Senna occidentalis L. possesses significant antioxidant and anticonvulsant activity. The activity was in dose dependent fashion. This study will assist in future research associated with formulation development of seeds of Senna occidentalis L.
 Keyword: Senna occidentalis L., Anticonvulsant, Antioxidant, DPPH model

Bioassay-guided isolation of anti-seizure principles from Semen Pharbitidis using a zebrafish pentylenetetrazol seizure model

Ethnopharmacological relevanceSemen Pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae) is a well-known traditional Chinese medicinal plant used for treating helminthiasis and epilepsy in China. Aim of the studyThis study aims to identify the anti-seizure components from Semen Pharbitidis. MethodsA bioassay-guided isolation of anti-seizure compounds from Semen Pharbitidis was performed using a zebrafish pentylenetetrazol seizure model. The structures of active compounds were elucidated by high resolution mass spectrometry. The fragments of active compounds were tested for anti-seizure activity as well. ResultsThe bioassay-guided isolation of ethanol extract of Semen Pharbitidis led to a group of resin glucosides, namely pharbitin. One of the fragments of pharbitin, 2-methylbutyric acid, also showed anti-seizure activity. ConclusionsWe provided further experimental scientific evidence to support the traditional use of Semen Pharbitidis for the treatment of epilepsy. Pharbitin was identified to be the main anti-seizure component in Semen Pharbitidis.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A2, pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

Synthesis and Evaluation of the Anticonvulsant Activities of 4-(2-(Alkylthio)benzo[d]oxazol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-ones.

In this study, a novel series of 4-(2-(alkylthio)benzo[d]oxazol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-ones (4a–m) was designed and synthesized. The anticonvulsant activities of these compounds were evaluated by using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. The neurotoxicity of these compounds was evaluated using the rotarod neurotoxicity test. The majority of compounds showed anti-MES activities at 100 or 300 mg/kg. Compound 4g was considered to be the most promising, based on its potency against MES- and PTZ-induced seizures with ED50 values of 23.7 and 18.9 mg/kg, respectively. The TD50 value of 4g was 284.0 mg/kg, which resulted in a higher protective index (PI = TD50/ED50) value than that of carbamazepine and valproate. In an ELISA test, compound 4g significantly increased the γ-aminobutyric acid (GABA) content in mouse brain. In addition, pretreatment with thiosemicarbazide (an inhibitor of the GABA synthesizing enzyme) significantly decreased the activity of 4g in the MES model, which suggests that the mechanism through which compound 4g elicits its anticonvulsive action is at least in part through increasing the GABA level in the brain.