Chris Rundfeldt

D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures

The anticonvulsant activity of the novel drug D-23129 (N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl ester) was evaluated in animal models of epileptic seizures. D-23129 was active after oral and intraperitoneal administration in rats and mice in a range of anticonvulsant tests at nontoxic doses. The compound was active against electrically induced seizures (MES, ED50 rat p.o. = 2.87 mg/kg), against seizures induced chemically by pentylenetetrazole (s.c. PTZ, ED50 mouse p.o. = 13.5 mg/kg), picrotoxin and N-methyl-D-aspartate (NMDA) and in a genetic animal model, the DBA/2 mouse. It was not active against seizures induced by bicuculline and strychnine. Motor impairment, evaluated with the rotarod test and by observation in the open field, was minimal at doses showing anticonvulsant activity. D-23129 was very effective in elevating the threshold for electrically and chemically induced seizures. Considering the dose increasing the MES threshold by 50% (TID50 mouse i.p. = 1.6 mg/kg; TID50 rat i.p. = 0.72 mg/kg) and the TD50 obtained in the rotarod test, the protective index of D-23129 is better than that of valproate and phenytoin. During 14 days chronic oral treatment with 15 mg/kg, no development of tolerance was observed. D-23129 thus presents an orally active, safe, broad spectrum anticonvulsant agent, which is structurally unrelated to anticonvulsants currently used. We expect that D-23129 will improve the treatment of refractory seizures in humans.

The new anticonvulsant retigabine (D-23129) acts as an opener of K+ channels in neuronal cells

The patch-clamp technique was used to measure currents passing through K+ channels in neuronal cell preparations. Retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) activated a K+ conductance in slightly depolarized NG108-15 neuronal cells in a dose-dependent manner (0.1-10 microM). At the K+ reversal potential, no current could be elicited and in hyperpolarized cells the current was reversed. A similar current was elicited in primary cultures of mouse cortical neurones and in differentiated hNT cells, a cell line derived from human neuronal cells. At higher concentrations, retigabine also partially blocked voltage activated K+ currents. None of the tested anticonvulsants, phenytoin, carbamazepine and valproate and none of the K+ channel openers cromakalim, diazoxide and pinacidil exerted a similar effect. The current was not affected by the K+ channel blocker glibenclamide (10 microM) but was fully blocked by application of Ba2+ (10.8 mM). Exchange of K+ with cesium in the intracellular space also fully abolished the current. It can be expected that the K+ channel opening effect contributes to the anticonvulsant activity of retigabine.

D-23129: a potent anticonvulsant in the amygdala kindling model of complex partial seizures

The novel anticonvulsant drug D-23129 (N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) was evaluated in the amygdala kindling model of complex partial seizures in rats. D-23129 exerts potent anticonvulsant activity against both focal and generalized seizures in animal models of epilepsy. After intraperitoneal and oral administration in kindled rats, the substance dose dependently increased the threshold for induction of afterdischarges, exerting significant effects already after 0.01 mg/kg. In higher doses (2.5-5 mg/kg i.p., 10-15 mg/kg p.o.) D-23129 also exerted anticonvulsant effects on other seizure parameters of amygdala-kindled rats, i.e. seizure severity, seizure duration, total duration of behavioural changes and afterdischarge duration. The adverse effects of D-23129 were quantitated in the open field and in the rotarod test, a standard test for motor impairment. D-23129 exerted no adverse effects on behaviour in doses up to 5 mg/kg i.p. and 15 mg/kg p.o. Comparing the adverse effects between kindled and non-kindled rats, no differences were found. The data demonstrate that D-23129 is more potent in the amygdala kindling model of complex partial seizures than in other seizure models. D-23129 is orally active and is devoid of neurotoxic effects in anticonvulsant doses, thus indicating that this compound has potential for antiepileptic therapy.

Dose-dependent anticonvulsant and proconvulsant effects of nitric oxide synthase inhibitors on seizure threshold in a cortical stimulation model in rats.

In the central nervous system, nitric oxide (NO) is increasingly being considered as a trans-synaptic retrograde messenger, being involved for instance in cellular responses to stimulation of glutamate receptors of the NMDA subtype. Thus, compounds that modify NO production, such as NO synthase inhibitors, may provide a means of altering NMDA receptor function. The functional consequences of NO synthase inhibition are, however, complicated by the fact that NO not only serves as a messenger to activate guanylyl cyclase and so to raise cGMP in target cells in response to NMDA receptor stimulation but also to induce feedback inhibition of the NMDA receptor via a redox modulatory site on the receptor complex. This may explain the contrasting results obtained previously with NO synthase inhibitors in animal models of ischaemia and seizures. In the present study, we tried to resolve the reported discrepancies about the effects of NO synthase inhibitors in seizure models by studying such drugs at various doses in a novel model of cortical seizure threshold. In this model, the threshold for seizures in rats is determined at short time intervals by applying ramp-shaped electrical pulse-trains directly to the cerebral cortex, allowing one to determine the time course of anti- or proconvulsant drug effects in individual rats. Two NO synthase inhibitors, NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester, were compared with a clinically effective antiepileptic drug, i.e. valproate.(ABSTRACT TRUNCATED AT 250 WORDS)

Low Doses of NMDA Receptor Antagonists Synergistically Increase the Anticonvulsant Effect of the AMPA Receptor Antagonist NBQX in the Kindling Model of Epilepsy

Excitatory amino acid transmitters are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using N‐methyl‐d‐aspartate (NMDA) receptors, although more recent evidence indicates potential roles for the a‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors as well. In the present experiments in amygdala‐kindled rats, i.e. a model of partial epilepsy, competitive and uncompetitive NMDA antagonists exerted only weak anticonvulsant effects, whereas the AMPA antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(F)quinoxaline (NBQX) potently increased focal seizure threshold and inhibited seizure spread from the focus. These effects of NBQX were dramatically increased by pretreatment with low doses of NMDA antagonists, whereas adverse effects of NBQX were not potentiated. These data suggest that both non‐NMDA and NMDA receptors are critically involved in the kindled state, and that combinations of AMPA and NMDA receptor antagonists provide a new strategy for treatment of epileptic seizures.

Phenytoin potently increases the threshold for focal seizures in amygdala-kindled rats.

Previous studies on the effects of phenytoin in the kindling model have yielded equivocal results, in that some groups reported anticonvulsant effects, while others found the drug to be only weakly active, inactive or even proconvulsant. Although part of these discrepancies might relate to factors such as the time of testing, route of administration, doses and administration vehicles, variable results were also obtained by studies in which these factors were comparable. For further investigation of the reasons of the reported discrepancies in the effectiveness of phenytoin in kindled rats, the influence of current intensity on the effects of phenytoin were examined in this model. In fully amygdala-kindled rats, phenytoin was only weakly active against seizures evoked by a fixed suprathreshold current of 500 microA. However, when the current was decreased to 250 microA, phenytoin exerted potent anticonvulsant effects. Determination of the threshold for focal afterdischarges showed that phenytoin, 12.5-75 mg/kg dose-dependently increased the threshold up to about 600% over controls at 75 mg/kg. Average levels in plasma, determined at this dose were about 30 micrograms/ml. In contrast to the potent effect on the seizure threshold, the severity or duration of seizures, evoked in phenytoin-pretreated rats by increasing the current, was not reduced. These data indicate that the primary effect of phenytoin in kindled rats in an increase in focal seizure threshold, while the ability of phenytoin to reduce the spread of seizures appears to be small. The present data might explain the discrepancy in reports on phenytoin in kindled rats, because most studies which found the drug to be ineffective used current intensities far exceeding the seizure threshold.

Anticonvulsant effects of the glycine/NMDA receptor ligands D-cycloserine and D-serine but not R-(+)-HA-966 in amygdala-kindled rats

1 The effects of the glycine/NMDA receptor partial agonists, d‐cycloserine and (+)‐HA‐966 and the full agonist, d‐serine, on focal seizure threshold and behaviour have been determined in amygdala‐kindled rats, i.e. a model of focal (partial) epilepsy. The uncompetitive NMDA receptor antagonist, MK‐801, was used for comparison. 2 The high efficacy glycine partial agonist, d‐cycloserine, did not alter the threshold for induction of amygdaloid afterdischarges (ADT) at doses of 20–80 mg kg−1 i.p., but significant ADT increases were determined after application of higher doses (160 and 320 mg kg−1). The ADT increases after these high doses were long‐lasting; significant elevations were still observed 2 days after drug injection. Determination of d‐cycloserine in plasma and brain tissue showed that it was rapidly eliminated from plasma. Compared to peak levels in plasma, only relatively low concentrations of d‐cycloserine were measured in brain tissue. 3 The low efficacy glycine partial agonist, (+)‐HA‐966, 10–40 mg kg−1 i.p., did not alter the ADT or seizure recordings (seizure severity, seizure duration, afterdischarge duration) at ADT currents. However, the drug dose‐dependently increased the duration of postictal behavioural and electroencephalographic depression in kindled rats. At the higher dose tested, postictal immobilization was dramatically increased from 3 min to about 120 min. This might indicate that glutamatergic activity is decreased postictally, which is potentiated or prolonged by (+)‐HA‐966. 4 Like d‐cycloserine, the glycine receptor full agonist, d‐serine, injected bilaterally into the lateral ventricles at a dose of 5 μmol, significantly increased the ADT, while no effect was seen at a lower dose (2.5 μmol). 5 The anticonvulsant effects observed with d‐cycloserine were completely antagonized by combined treatment with (+)‐HA‐966, indicating that the effects of d‐cycloserine were mediated by the glycine/NMDA receptor complex. 6 MK‐801, 0.1 mg kg−1, did not alter the focal seizure threshold or seizure recordings at ADT current, but induced marked phencyclidine(PCP)‐like behavioural alterations, such as hyperlocomotion, stereotypies and motor impairment. No PCP‐like behaviours were observed after d‐cycloserine, d‐serine or (+)‐HA‐966. High doses of (+)‐HA‐966 induced moderate motor impairment in kindled rats. 7 The long lasting increases in seizure threshold observed after the high efficacy glycine partial agonist, d‐cycloserine but not the low efficacy partial agonist, (+)‐HA‐966, may suggest that the effects of d‐cycloserine are mediated by adaptive changes in the NMDA receptor complex in response to glycine receptor stimulation. 8 Pharmacological intervention at the strychnine‐insensitive glycine receptor by high‐efficacy partial agonists with systemic bioavailability may be an effective means of increasing seizure threshold without concomitantly inducing PCP‐like adverse effects.

Does prolonged implantation of depth electrodes predispose the brain to kindling

Chronically implanted depth electrodes are widely used for the study of electrical signals generated in deep cerebral locations and for electrical stimulation of such locations. Although the effects of lesions resulting from electrode implantation are generally considered minimal, some reports have shown lasting neurochemical, histological, and behavioral alterations in response to such implantation. Furthermore, there is some evidence that prolonged electrode implantation may decrease the seizure threshold of the implanted region and increases the rate of kindling from this region. This prompted us to undertake a study on different periods of post-surgical delay to onset of electrical stimulation and subsequent characteristics of kindling development. Rats were implanted with a bipolar electrode in the basolateral amygdala, and the threshold for induction of focal paroxysmal activity (afterdischarge threshold, ADT) was determined after post-surgical recovery periods of either 1, 2, 4, or 8 weeks. The animals were then kindled by daily administration of an electrical stimulus until all rats exhibited fully kindled seizures. In fully kindled rats, the ADT was redetermined. Compared to animals with 1 week of electrode implantation, the pre-kindling ADT was significantly lower in rats with 2 and 4 weeks of electrode implantation, but returned towards the 1 week values at 8 weeks. An enhanced kindling rate was seen when kindling stimulations were started after 4 and 8 weeks of electrode implantation. Despite the marked differences in pre-kindling ADT, the post-kindling ADT was similar in the groups with 1, 2, or 4 weeks but significantly lower in the group with 8 weeks post-surgical delay to onset of testing. The data suggest that prolonged implantation of a bipolar electrode into a sensitive region of the limbic system predisposes the brain to kindling. Based on previous observation of iron deposits induced by electrode implantation and the epileptogenic effect of iron in cortical and limbic regions, we propose that the present observations are due to deposition of iron from hemoglobin destruction in local microhemorrhages caused by the implantation.

Transmitter amino acid levels in rat brain regions after amygdala-kindling or chronic electrode implantation without kindling: Evidence for a pro-kindling effect of prolonged electrode implantation

Kindling is a chronic model of epilepsy characterized by a progressive increase in response to the same regularly applied stimulus. The biological basis of the kindling phenomenon requires to be determined, but several studies indicate that alterations in amino acidergic neurotransmission may be involved. In the present experiments, levels of glutamate, aspartate, GABA, glycine, and taurine were determined in 12 brain regions by HPLC in 3 groups of animals: (a) a group which was kindled via electrical stimulation of intraamygdala electrodes and was sacrificed 36 days after the last fully kindled seizure for neurochemical determinations; (b) a group of implanted but nonstimulated rats (surgical control group) in which neurochemical measurements were done at the same time after electrode implantation as the kindled group, and (c) a group of non-implanted, naive control rats. Compared to surgical controls, kindling induced a significant reduction of glutamate, GABA, and taurine in the brain stem (pons/medulla), whereas no differences between both groups were found in any of the other regions. However, both electrode-implanted groups differed significantly from non-implanted naive rats in several regions, indicating that electrode-implantation per se induced long-lasting alterations in transmitter amino acids. The most striking difference to naive controls was an increase of glycine levels in several regions in which this amino acid is known to potentiate glutamatergic transmission. In order to examine the functional consequences of prolonged electrode implantation, seizure thresholds were determined in groups of rats with short and prolonged electrode implantation. Data from these experiments indicated that prolonged electrode implantation per se induces pro-kindling effects, i.e. a dramatic decrease of seizure threshold. The data of this study thus demonstrate that the choice of adequate controls is critical in neurochemical and functional studies on the kindling phenomenon.

Characterization of the K+ channel opening effect of the anticonvulsant retigabine in PC12 cells

Retigabine (D-23129) is a new anticonvulsant compound which acts as a K+ channel opener in neuronal cells. The aim of the present study was to further characterize the retigabine induced K+ current. In nerve growth factor treated PC12 cells and in rat cortical neurones the application of retigabine activated a K+ current. In contrast, however, no K+ current activation was observed in untreated PC12 and in glial cells which were cultivated together with the neuronal cells. To characterise the retigabine activated K+ current, K+ channel blockers were used. The retigabine induced current was not affected by 1 and 10 mM 4-aminopyridine (4AP). Ba2+ 1 mM resulted in a reduction of 88.6+/-3.0% (n = 5); 10 mM abolished the current. Tetraetylamonium (TEA), 1 and 10 mM, reduced the current by 23.6+/-3.1 and 61.6+/-3.7%, respectively. To investigate the current/voltage (I/V) relation of the current initiated by retigabine (10 microM), cells were clamped to a holding potential of -80 mV and a ramp stimulation protocol (-120 to +60 mV in 5 s) was applied prior to and during application of retigabine. Subtraction of the two traces yielded the current induced by retigabine. A nearly linear relationship was determined between - 120 and -40 mV. At potentials positive to - 40 mV, the response was variable. This was due to the additionally observed weak blocking effect of retigabine on delayed rectifier (Kdr) currents. If the ramp was applied in the presence of 10 mM 4AP to block Kdr, a nearly linear I/V-relationship was present from -120 to +60 mV. The comparison of the I/V relation and pharmacology with published K+ channel subtypes gives evidence that an unknown neuronal K+ channel subtype may be involved.