Animal Models Of Epilepsy Research Articles

Automated analysis of brain activity for seizure detection in zebrafish models of epilepsy

BackgroundEpilepsy is a chronic neurological condition, with over 30% of cases unresponsive to treatment. Zebrafish larvae show great potential to serve as an animal model of epilepsy in drug discovery. Thanks to their high fecundity and relatively low cost, they are amenable to high-throughput screening. However, the assessment of seizure occurrences in zebrafish larvae remains a bottleneck, as visual analysis is subjective and time-consuming. New methodFor the first time, we present an automated algorithm to detect epileptic discharges in single-channel local field potential (LFP) recordings in zebrafish. First, candidate seizure segments are selected based on their energy and length. Afterwards, discriminative features are extracted from each segment. Using a labeled dataset, a support vector machine (SVM) classifier is trained to learn an optimal feature mapping. Finally, this SVM classifier is used to detect seizure segments in new signals. ResultsWe tested the proposed algorithm both in a chemically-induced seizure model and a genetic epilepsy model. In both cases, the algorithm delivered similar results to visual analysis and found a significant difference in number of seizures between the epileptic and control group. Comparison with existing methodsDirect comparison with multichannel techniques or methods developed for different animal models is not feasible. Nevertheless, a literature review shows that our algorithm outperforms state-of-the-art techniques in terms of accuracy, precision and specificity, while maintaining a reasonable sensitivity. ConclusionOur seizure detection system is a generic, time-saving and objective method to analyze zebrafish LPF, which can replace visual analysis and facilitate true high-throughput studies.

Carvacrol promotes neuroprotection in the mouse hemiparkinsonian model

Carvacrol is a monoterpene that has been linked to neuroprotection in several animal models of neurodegeneration, including ischemia, epilepsy and traumatic neuronal injury. In this study, we investigated the effects of carvacrol (i.p.) upon the neurodegeneration induced by 6-hydroxy-dopamine unilateral intrastriatal injections in mice. We have also used the cylinder test to assess the behavioral effects of carvacrol in that model of Parkinson’s disease, and immunoblots to evaluate the levels of caspase-3 and TRPM7, one of major targets of carvacrol. Behavioral testing revealed that carvacrol largely reduced the asymmetrical use of the forelimbs induced by unilateral 6-hydroxy-dopamine. Carvacrol dramatically reduced the loss of tyrosine hydroxylase immunostaining both in the substantia nigra and in the striatum that are typical of the model. Immunoblots for tyrosine hydroxylase confirmed this effect. Caspase-3 levels were very high after toxin injections, but carvacrol appeared to reduce them to control levels. Finally, TRPM7, observed by immunoblots, increased after 6-hydroxy-dopamine, suggesting the involvement of this cation channel in the ensuing neurodegenerative process. The present data suggest that carvacrol promotes a marked neuroprotection in the 6-hydroxy-dopamine model of Parkinson’s disease, possibly by its non-specific blocking effect upon TRPM7 channels.

4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid attenuates spontaneous recurrent seizures and vasogenic edema following lithium-pilocarpine induced status epilepticus

Vasogenic edema induced by blood brain barrier disruption and neuronal loss play an important role in the epileptogenic process. 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) is a commonly used anion channel inhibitor that has been reported to exert an anticonvulsant effect in rat hippocampus in vitro. The present study aimed to investigate whether DIDS could prevent epileptogenic process in rat lithium-pilocarpine model of temporal lobe epilepsy. The tight junction proteins and serum extravasation were examined in the piriform cortex 3days after status epilepticus. The findings showed that status epilepticus induced vasogenic edema. Based on these findings, rats were intracerebroventricularly infused with saline and DIDS 1 week after surgery, DIDS reduced vasogenic edema and prevented neuronal loss following status epilepticus in the piriform cortex. Moreover, spontaneous recurrent seizures were recorded by continuous video monitoring. DIDS significantly reduced the frequency and duration of spontaneous recurrent seizures from day 28 to day 42 post status epilepticus. These findings demonstrated that DIDS attenuated vasogenic edema and neuronal apoptosis and might exert disease-modifying effect in animal model of temporal lobe epilepsy. These results explored a novel therapeutic strategy for treatment of epilepsy.

Anticonvulsant effects of antiaris toxicaria aqueous extract: investigation using animal models of temporal lobe epilepsy

BackgroundAntiaris toxicaria has previously shown anticonvulsant activity in acute animal models of epilepsy. The aqueous extract (AAE) was further investigated for activity in kindling with pentylenetetrazole and administration of pilocarpine and kainic acid which mimic temporal lobe epilepsy in various animal species.ResultsICR mice and Sprague–Dawley rats were pre-treated with AAE (200–800 mg kg−1) and convulsive episodes induced using pentylenetetrazole, pilocarpine and kainic acid. The potential of AAE to prevent or delay onset and alter duration of seizures were measured. In addition, damage to hippocampal cells was assessed in kainic acid-induced status epilepticus test. 800 mg kg−1 of the extract suppressed the kindled seizure significantly (P < 0.05) as did diazepam. AAE also produced significant effect (P < 0.01) on latency to first myoclonic jerks and on total duration of seizures. The latency to onset of wet dog shakes was increased significantly (P < 0.05) by AAE on kainic acid administration. Carbamazepine and Nifedipine (30 mg kg−1) also delayed the onset. Histopathological examination of brain sections showed no protective effect on hippocampal cells by AAE and nifedipine. Carbamazepine offered better preservation of hippocampal cells in the CA1, CA2 and CA3 regions.ConclusionAntiaris toxicaria may be effective in controlling temporal lobe seizures in rodents.

Phase-Dependent Astroglial Alterations in Li–Pilocarpine-Induced Status Epilepticus in Young Rats

Epilepsy prevalence is high in infancy and in the elderly population. Lithium-pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56days after Li-pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders.

Allopregnanolone decreases interictal spiking and fast ripples in an animal model of mesial temporal lobe epilepsy

The objective of this study was to characterize the impact of allopregnanolone, a neurosteroid that acts as a positive allosteric modulator of synaptic and extrasynaptic GABAA receptors, on interictal spikes and high-frequency oscillations (ripples: 80–200 Hz, fast ripples: 250–500 Hz) in the pilocarpine model of mesial temporal lobe epilepsy. Seven out of 25 Sprague-Dawley rats experiencing 1 h of pilocarpine-induced status epilepticus (SE) began treatment with allopregnanolone (9.6–12.8 mg/kg/day) on the following day. On day 4 after SE, video-depth EEG recordings from the hippocampal CA3 subfield and the entorhinal cortex were initiated and continued for 12 consecutive days. We found that 66.7% (12/18) of untreated animals exhibited seizures compared to 28.6% (2/7) of allopregnanolone-treated animals. Interictal spikes occurred less frequently in the CA3 subfield of allopregnanolone-treated rats (n = 4) than in untreated animals presenting (n = 4) or not presenting (n = 4) with spontaneous seizures (p < 0.05), and were less frequent in the entorhinal cortex compared to both untreated groups (p < 0.05). Finally, allopregnanolone-treated rats had significantly lower rates of interictal spikes with fast ripples (250–500 Hz) compared to untreated animals but only in CA3 (p < 0.05). Our findings show that allopregnanolone reduces the frequency of interictal spikes and fast ripples in CA3, a structure that plays an important role in ictogenesis and epileptogenesis. Neurosteroids may therefore influence pathological network activity leading to spontaneous seizures following pilocarpine-induced SE. Recordings after termination of allopregnanolone treatment will be however required to establish whether allopregnanolone exerts disease-modifying properties.

Evaluation of anticonvulsant potential of aqueous methanolic extract and various fractions of Ajuga bracteosa wall

We aim to assess the anticonvulsant potential of Ajuga bracteosa Wall. The aqueous methanol extract (30:70) of Ajuga bracteosa and its n-hexane, chloroform and ethyl acetate fractions were prepared. Anticonvulsant effect was evaluated using different animal models of epilepsy. Crude extract along with its fractions at 500 and 1000 mg/kg doses, administered orally to albino mice were appraised against pentylenetetrazole-induced, strychnine-induced and picrotoxin-induced convulsions. Moreover, effect of test substances on brain GABA level and phenobarbitone induced hypnosis was also studied at dose level of 350 and 400 mg/kg. The plant extract and its fractions produced potent dose dependent anticonvulsant activity in all models of epilepsy. In addition, plant extract and fractions increased brain GABA level and potentiated phenobarbitone-induced sleep duration. From the results it can be deduced that Ajuga bracteosa possess potent anticonvulsant activity, supporting its folkloric use in the treatment of epilepsy.

Synthesis and Anticonvulsant Properties of New 3,3-Diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetamides and 3,3-Diphenyl-propionamides.

The focused library of new amides derived from 3,3-diphenyl-2,5-dioxo-pyrrolidin-1-yl-acetic acid (2a-t) and 3,3-diphenyl-propionic acid (3a-t) as potential anticonvulsant agents was synthesized. The final products were obtained in the amidation reaction of the given carboxylic acid (2, 3) with appropriate secondary amines in the presence of carbonyldiimidazole (CDI) as a coupling reagent. The initial anticonvulsant screening was performed in mice intraperitoneally (i.p.) using the "classical" maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models, whereas the acute neurological toxicity was determined applying the rotarod test. Additionally, several compounds were studied also in the 6-Hz seizures recognized as the animal model of human pharmacoresistant epilepsy. In this series, compound 3q displayed a broad spectrum of activity across the preclinical seizure models (ED50 MES = 31.64 mg/kg; ED50 scPTZ = 75.41 mg/kg, ED50 6-Hz (32 mA) = 38.15 mg/kg). Consequently, compound 3q revealed a wider spectrum of protection, higher activity or/and a better safety profile than the commonly used antiepileptic drugs such as phenytoin, ethosuximide, valproic acid, or/and levetiracetam. Notably, the in vitro studies showed that the most possible mechanism of action of 3q may be connected to the interaction with neuronal voltage-sensitive sodium channels (site 2). Other substances were active predominantly in the chemically induced seizures. The results of the current studies indicate that the presence of the pyrrolidine-2,5-dione ring is important but not indispensable for anticonvulsant activity.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs.

The identification of potential therapeutic agents for the treatment of epilepsy requires the use of seizure models. Except for some early treatments, including bromides and phenobarbital, the antiseizure activity of all clinically used drugs was, for the most part, defined by acute seizure models in rodents using the maximal electroshock and subcutaneous pentylenetetrazole seizure tests and the electrically kindled rat. Unfortunately, the clinical evidence to date would suggest that none of these models, albeit useful, are likely to identify those therapeutics that will effectively manage patients with drug resistant seizures. Over the last 30 years, a number of animal models have been developed that display varying degrees of pharmacoresistance, such as the phenytoin- or lamotrigine-resistant kindled rat, the 6-Hz mouse model of partial seizures, the intrahippocampal kainate model in mice, or rats in which spontaneous recurrent seizures develops after inducing status epilepticus by chemical or electrical stimulation. As such, these models can be used to study mechanisms of drug resistance and may provide a unique opportunity for identifying a truly novel antiseizure drug (ASD), but thus far clinical evidence for this hope is lacking. Although animal models of drug resistant seizures are now included in ASD discovery approaches such as the ETSP (epilepsy therapy screening program), it is important to note that no single model has been validated for use to identify potential compounds for as yet drug resistant seizures, but rather a battery of such models should be employed, thus enhancing the sensitivity to discover novel, highly effective ASDs. The present review describes the previous and current approaches used in the search for new ASDs and offers some insight into future directions incorporating new and emerging animal models of therapy resistance.

Cav 1.3 channels play a crucial role in the formation of paroxysmal depolarization shifts in cultured hippocampal neurons.

An increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). Distinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. Our data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

A Murine Model to Study Epilepsy and SUDEP Induced by Malaria Infection

One of the largest single sources of epilepsy in the world is produced as a neurological sequela in survivors of cerebral malaria. Nevertheless, the pathophysiological mechanisms of such epileptogenesis remain unknown and no adjunctive therapy during cerebral malaria has been shown to reduce the rate of subsequent epilepsy. There is no existing animal model of postmalarial epilepsy. In this technical report we demonstrate the first such animal models. These models were created from multiple mouse and parasite strain combinations, so that the epilepsy observed retained universality with respect to genetic background. We also discovered spontaneous sudden unexpected death in epilepsy (SUDEP) in two of our strain combinations. These models offer a platform to enable new preclinical research into mechanisms and prevention of epilepsy and SUDEP.

P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

Physical Exercise Restores the Generation of Newborn Neurons in an Animal Model of Chronic Epilepsy.

Neurogenesis impairment is associated with the chronic phase of the epilepsy in humans and also observed in animal models. Recent studies with animal models have shown that physical exercise is capable of improving neurogenesis in adult subjects, alleviating cognitive impairment and depression. Here, we show that there is a reduction in the generation of newborn granule cells in the dentate gyrus of adult rats subjected to a chronic model of epilepsy during the postnatal period of brain development. We also show that the physical exercise was capable to restore the number of newborn granule cells in this animals to the level observed in the control group. Notably, a larger number of newborn granule cells exhibiting morphological characteristics indicative of correct targeting into the hippocampal circuitry and the absence of basal dendrite projections was also observed in the epileptic animals subjected to physical exercise compared to the epileptic animals. The results described here could represent a positive interference of the physical exercise on the neurogenesis process in subjects with chronic epilepsy. The results may also help to reinterpret the benefits of the physical exercise in alleviating symptoms of depression and cognitive dysfunction.

Effects of Jasminum multiflorum leaf extract on rodent models of epilepsy, motor coordination and anxiety

Jasmine flowers and leaves are used extensively in folk medicine in different parts of the world to treat a variety of diseases. However, there are very few published reports on the neuropsychiatric effects of Jasmine extracts. Hence, the objectives of the present study were to examine the effects of an alcohol extract of Jasminum multiflorum leaves on topically-applied bicuculline (a model of acute simple partial epilepsy) and maximal electroshock (MES, a model of generalized tonic-clonic seizure) in male Sprague-Dawley rats. The objectives also included an examination of the anxiolytic properties of the extract using an elevated plus maze and the effect of the extract on motor coordination using a rotarod treadmill. Phytochemical analysis of the extract showed the presence of three flavonoids and four additional compounds belonging to the steroid, terpenoid, phenol or sugar classes of compounds. The Jasmine alcohol extract, diluted with water and given orally or intraperitoneally, reduced the number of bicuculline-induced epileptiform discharges in a dose-dependent manner. The extract did not cause a significant increase in the current needed to induce hind limb extension in MES experiments. The extract significantly affected motor coordination when injected at 500mg/kg but not at 200mg/kg. At the latter dose, the extract increased open-arm entries and duration in the elevated plus maze to a level comparable to that of diazepam at 2mg/kg. We conclude that Jasmine leaf extract has a beneficial effect against an animal model of acute partial complex epilepsy, and significant anxiolytic effect at a dose that does not affect motor co-ordination.

Antiepileptic action of c-Jun N-terminal kinase (JNK) inhibition in an animal model of temporal lobe epilepsy

Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target.

Protective effect of spermine against pentylenetetrazole kindling epilepsy induced comorbidities in mice

Nitric oxide (NO), an important intracellular signaling molecule is involved in modulation of neuronal transmission. The NO level increases during epileptic activity in animal models of epilepsy. However, its role in epileptic activity remains controversial. Spermine is an endogenous polyamine; possesses anti-oxidant property and has ability to modulate ion channels and NO synthase activity. Therefore, the present study was designed to investigate the role of NO pathway in the neuroprotective effect of spermine, in Pentylenetetrazol (PTZ) induced kindling epilepsy in mice. PTZ (35mg/kg; intraperitoneal, i.p.) was administered on every alternate day up to 29days and challenge test was performed on 33rd day. From 15th day, spermine (5 and 10mg/kg; i.p.), L-NAME (10mg/kg; i.p), l-Arginine (50mg/kg; i.p) and sodium valproate (400mg/kg; i.p.) were administered up to 33rd day. Animals were sacrificed on 34th day for estimation of biochemical and neurotransmitters. Pretreatment with spermine, considerably, reversed the PTZ induced alterations. Further, pretreatment of L-NAME and l-Arginine with 5 and 10mg/kg; i.p. spermine, respectively, leads to an increase and decrease in its protective effects. The present study suggests the involvement of NO pathway in the protective effect of spermine against PTZ-induced kindling epilepsy in mice.

NS1209/SPD 502, A Novel Selective AMPA Antagonist for Stroke, Neuropathic Pain or Epilepsy? Drug Development Lessons Learned.

Preclinical Research The selective AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist, NS1209 (also known as SPD 502) has been explored in several research and development campaigns since its selection as a lead drug candidate in the early 1990s by the Danish biotechnology company, NeuroSearch. The compound was successively tested in animal models of stroke, neuropathic pain and epilepsy. The preclinical data to support development for the treatment of stroke were incomplete, as the compound was administered after the stroke episode, and did not protect subcortical areas of the brain. Preclinical data for neuropathic pain and epilepsy appeared more promising, but the design of the Phase IIa studies in both indications was suboptimal, and an exploratory study in neuropathic pain, like one in refractory epilepsy gave inconclusive results. Preclinical data in pain models were much less convincing than reported by the authors in abstract and discussion sections. Due to a long preclinical sequential testing phase and insufficiently powered clinical trials, NS 1209 disappeared from the CNS development pipeline, while its patent protection exclusivity was markedly reduced due to the unfortunate slow speed in development-a phenomenon far from unusual in CNS drug discovery. NeuroSearch ceased operations as an R&D entity in 2012 and its R & D portfolio was transferred to Teva Pharmaceuticals and to a spin off, Saniona A/S. Based on an in-depth case analysis of the development of NS 1209, a number of recommendations are given to reduce chances of failure during clinical development of neuropathic pain compounds and, more generally, of CNS compounds. Drug Dev Res 78 : 75-80, 2017. © 2017 Wiley Periodicals, Inc.

Delineation of the Role of Astroglial GABA Transporters in Seizure Control.

Studies of GABA transport in neurons and astrocytes have provided evidence that termination of GABA as neurotransmitter is brought about primarily by active transport into the presynaptic, GABAergic nerve endings. There is, however, a considerable transport capacity in the astrocytes surrounding the synaptic terminals, a transport which may limit the availability of transmitter GABA leading to a higher probability of seizure activity governed by the balance of excitatory and inhibitory neurotransmission. Based on this it was hypothesized that selective inhibition of astrocytic GABA transport might prevent such seizure activity. A series of GABA analogs of restricted conformation were synthesized and in a number of collaborative investigations between Prof. Steve White at the University of Utah and medicinal chemists and pharmacologists at the School of Pharmacy and the University of Copenhagen, Denmark, GABA analogs with exactly this pharmacological property were identified. The most important analogs identified were N-methyl-exo-THPO (N-methyl-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole) and its lipophilic analog EF-1502 ((RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) both of which turned out to be potent anticonvulsants in animal models of epilepsy.

Analgesic, antiallodynic, and anticonvulsant activity of novel hybrid molecules derived from N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2,5-dioxopyrrolidin-1-yl)butanamide in animal models of pain and epilepsy

The purpose of the present study was to examine the analgesic activity of six novel hybrid molecules, which demonstrated in the previous research anticonvulsant activity in the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole seizure (scPTZ) tests in mice. The antinociceptive properties were estimated in three models of pain in mice—the hot plate test, the formalin test, and in the oxaliplatin-induced neuropathy. Moreover, extended anticonvulsant studies were carried out and the antiseizure activity was investigated in the 6-Hz test. Considering drug safety evaluation, the influence of compounds on locomotor activity and contextual memory were checked. Furthermore, chosen molecules were tested in vitro for potential hepatotoxicity. To explain the probable mechanism of action, the radioligand binding assays were performed. In both phases of formalin test, analgesic activity demonstrated compounds 4, 8, and 9. These agents relieved also mechanical allodynia in oxaliplatin-induced model of neuropathic pain. At active doses, they did not influence locomotor activity of mice. Moreover, for compounds 8 and 9, no deleterious effect on memory was observed, but compound 4 might induce memory deficits. All tested compounds (4, 5, 8, 9, 15, and 16) inhibited psychomotor seizures with the ED50 values = 24.66–47.21 mg/kg. The binding studies showed that compound 4 only at the high concentrations revealed the effective binding to the neuronal sodium channels and moderately binding to the L-type calcium (verapamil site) channels and NMDA receptors. The present preclinical results proved that novel hybrid molecules demonstrate very promising anticonvulsant and analgesic activity.