Predictive validity of endpoints used in electrophysiological modelling of migraine in the trigeminovascular system.

The trigeminovascular system has a pivotal role in the pathomechanism of migraine. The aim of the present study was to further develop existing models of migraine making them more suitable for testing the effects of compounds with presumed antimigraine activity in anaesthetised rats. Simultaneous recording of ongoing activity of spontaneously active neurons in the trigeminocervical complex as well as their discharges evoked by electrical stimulation of the dura mater via activation of A- and C-sensory fibres were carried out. Effects of sumatriptan, propranolol and topiramate were evaluated prior to and after application of a mixture containing inflammatory mediators on the dura. Propranolol (10 mg/kg s.c) and topiramate (30 mg/kg s.c.) resulted in a tendency to decrease the level of both spontaneous and evoked activity, while sumatriptan (1 mg/kg s.c.) did not exhibit any effect on recorded parameters. Application of an inflammatory soup to the dura mater boosted up spontaneous activity, which could be significantly attenuated by propranolol and topiramate but not by sumatriptan. In addition, all compounds prevented the delayed increase of spontaneous firing. In contrast to the ongoing activity, evoked responses were not augmented by inflammatory mediators. Nevertheless, inhibitory effect of propranolol and topiramate was evident when considering A- or C-fibre responses. Findings do not support the view that electrically evoked responses are useful for the measurement of trigeminal sensitization. It is proposed however, that inhibition of enhanced firing (immediate and/or delayed) evoked by inflammatory mediators as an endpoint have higher predictive validity regarding the clinical effectiveness of compounds.

Recent advancements in anti-migraine drug research: focus on attempts to decrease neuronal hyperexcitability.

Migraine is a painful, sometimes debilitating disorder, which is frequently associated with various neurological symptoms. Its prevalence in the population is higher than that of any other neurological disorders, thus the burden of this disease on society is considerable. Although the introduction of triptans nearly two decades ago revolutionized the treatment of the disease there is still a huge unmet need regarding drugs with better properties. Formerly, migraine therapy primarily aimed at treating the pathological alterations of meningeal blood vessels that are thought to directly initiate a migraine headache attack. By now, it has been increasingly recognized by drug companies that abnormal neural function may be more important in the development of the disease and also in triggering an attack. Migraine is associated with an increased neuronal excitability and episodes of cortical spreading depression. Understanding the molecular mechanisms underlying the abnormal functioning of over-activated neuronal circuits may help to identify novel anti-migraine drug targets. Besides a general description of the pathophysiology and pharmacotherapy of migraine this review paper aims at discussing the possible drug targets through which migraine-related hyperexcitability and over-excitation can be attenuated. It will be shown how these new ideas appear in the recent patent literature.

Concerted action of antiepileptic and antidepressant agents to depress spinal neurotransmission: Possible use in the therapy of spasticity and chronic pain.

Chronic pain states and epilepsies are common therapeutic targets of voltage-gated sodium channel blockers. Inhibition of sodium channels results in central muscle relaxant activity as well. Selective serotonin reuptake inhibitors are also applied in the treatment of pain syndromes. Here, we investigate the pharmacodynamic interaction between these two types of drugs on spinal neurotransmission in vitro and in vivo. Furthermore, the ability of serotonin reuptake inhibitors to modulate the anticonvulsant and windup inhibitory actions and motor side effect of the sodium channel blocker lamotrigine was investigated. In the hemisected spinal cord model, we found that serotonin reuptake inhibitors increased the reflex inhibitory action of sodium channel blockers. The interaction was clearly more than additive. The potentiation was prevented by blocking 5-HT(2) receptors and PKC, and mimicked by activation of these targets by selective pharmacological tools, suggesting the involvement of 5-HT(2) receptors and PKC in the modulation of sodium channel function. The increase of sodium current blocking potency of lamotrigine by PKC activation was also demonstrated at cellular level, using the whole-cell patch clamp method. Similar synergism was found in vivo, in spinal reflex, windup, and maximal electroshock seizure models, but not in the rotarod test, which indicate enhanced muscle relaxant, anticonvulsant and analgesic activities with improved side effect profile. Our findings are in agreement with clinical observations suggesting that sodium channel blocking drugs, such as lamotrigine, can be advantageously combined with selective serotonin reuptake inhibitors in some therapeutic fields, and may help to understand the molecular mechanisms underlying the interaction.

Blockers of voltage-gated sodium channels for the treatment of central nervous system diseases.

Voltage gated sodium channels play important roles both in vital physiological functions and several pathological processes of the central nervous system. Epilepsy, chronic pain, neurodegenerative diseases, and spasticity are all characterized by an over-excited state of specific groups of central neurons that is accompanied by an abnormally increased activity of sodium channels. An efficient strategy of controlling such diseases is to use blockers that preferentially act on these over-excited cells. State dependently acting agents, such as phenytoin, or lamotrigine, leave normal physiological functions relatively intact, resulting in a favorable therapeutic window. Nine isoforms of the channel forming alpha subunit are known, which show distinct expression patterns in different tissues. Another possible way to decrease the chance of adverse effects is to develop agents selectively inhibiting the channel subtype involved in the pathomechanism of the disease to be treated. Many recent patents claim sodium channel blockers with improved characteristics regarding state dependency or subtype selectivity. Such agents may offer a breakthrough in the treatment of a variety of central nervous system diseases. This review focuses on the current trends in sodium channel research, surveying the traditional and newly emerging therapeutic fields, and the diverse medicinal chemistry of sodium channel blockers.

Functional characterization of sodium channel blockers by membrane potential measurements in cerebellar neurons: prediction of compound preference for the open/inactivated state.

Voltage-gated sodium channel (VGSC) blockers are widely used in the therapy, but most currently available blockers have suboptimal profile. However, discovery of new drug candidates has been hampered by the lack of appropriate in vitro assays. We established a fluorometric, plate reader-based membrane potential assay for testing the inhibitory potency of various VGSC blocking drugs, using primary cultures of cerebellar neurons, and veratridine, as activator of VGSCs. Since inhibition was strongly dependent on the depolarizing effect of veratridine, the EC(80) value of veratridine was determined on each experimental day, and this concentration was used for drug testing. This strict control on agonist effect seems to improve the reliability of the dose-inhibition measurements with antagonists. Veratridine responses could be completely inhibited by tetrodotoxin (TTX, IC(50)=17 nM), consistent with the exclusive expression of TTX-sensitive VGSCs. A variety of compounds known to block sodium channels inhibited veratridine-induced membrane depolarization concentration-dependently. Furthermore, inhibitory potencies of drugs strongly depended on whether their administration preceded or followed veratridine application. Potency of lamotrigine, carbamazepine, phenytoin and lidocaine was approximately 10-fold higher when applied after a steady-state depolarization had been achieved by a supramaximal veratridine dose, compared with those from a different protocol, where cells were preincubated with the antagonists prior to veratridine application. On the contrary, there was only relatively small difference between the IC(50) values of GBR 12909 obtained from the two different protocols (0.51 microM versus 1.23 microM). In contrast with most sodium channel blockers, this compound lacks binding preference to inactivated channels. We suggest that comparison of the results obtained with a particular blocker in the pretreatment and post-treatment schedules may be suitable for drawing conclusions regarding the state-dependency of its action. Thus, relevant information can be obtained about the potential therapeutic utility of different drugs by applying non-electrophysiological methods.

Simple pharmacological test battery to assess efficacy and side effect profile of centrally acting muscle relaxant drugs.

INTRODUCTION: Centrally muscle relaxants (CMRs) are used mainly for treating muscle spasticities of neurological origin, and painful muscle spasms due to rheumatologic conditions. Their use is frequently associated with dose-limiting adverse effects. New drugs with improved side-effect characteristics are badly needed. However, there is no general agreement in the pharmacological literature on what methods are adequate to assess CMR effect and side effects in behaving rodents, which may hinder the development of new drugs. Here we report on the establishment of a simple pharmacological test battery, which was used to compare efficacies and side effect profiles of 11 compounds with central muscle relaxant action, in mice (intraperitoneal application). METHODS: For measuring muscle relaxant activity, (1) a new tremor model (GYKI 20039-induced tremor) and (2) the morphine-induced Straub-tail assay were used. The former, newly developed method has advantages over harmaline- or LON-954-induced tremor. For detecting side effect liability (ataxia, sedation, impairment of voluntary motor functions), (1) the rota-rod test, (2) measurement of spontaneous motility, (3) the weight-lifting test and (4) the thiopental sleep test were used. RESULTS: Among the 11 muscle relaxant compounds tested (tolperisone, eperisone, silperisone, diazepam, baclofen, tizanidine, afloqualon, mephenesin, zoxazolamine, memantine and carisoprodol), the calculated safety ratios (i.e. ID50 for side effect/ID50 for muscle relaxant effect) varied in a wide range. Silperisone seems to have the most advantageous profile (safety ratios range between 1.7 and 3.3 in the different pairs of assays) compared to the other tested drugs with lower (one or more ratios below 1.5, and often far below 1) and more varying ratios. DISCUSSION: Therapeutic indices calculated from the results of these in vivo experiments for the clinically used muscle relaxants are in agreement with their adverse effect profiles in humans. Thus the present test battery seems to be suitable for predicting the possible clinical utility of newly synthesized compounds.

Tolperisone-type drugs inhibit spinal reflexes via blockade of voltage-gated sodium and calcium channels.

The spinal reflex depressant mechanism of tolperisone and some of its structural analogs with central muscle relaxant action was investigated. Tolperisone (50-400 microM), eperisone, lanperisone, inaperisone, and silperisone (25-200 microM) dose dependently depressed the ventral root potential of isolated hemisected spinal cord of 6-day-old rats. The local anesthetic lidocaine (100-800 microM) produced qualitatively similar depression of spinal functions in the hemicord preparation, whereas its blocking effect on afferent nerve conduction was clearly stronger. In vivo, tolperisone and silperisone as well as lidocaine (10 mg/kg intravenously) depressed ventral root reflexes and excitability of motoneurons. However, in contrast with lidocaine, the muscle relaxant drugs seemed to have a more pronounced action on the synaptic responses than on the excitability of motoneurons. Whole-cell measurements in dorsal root ganglion cells revealed that tolperisone and silperisone depressed voltage-gated sodium channel conductance at concentrations that inhibited spinal reflexes. Results obtained with tolperisone and its analogs in the [3H]batrachotoxinin A 20-alpha-benzoate binding in cortical neurons and in a fluorimetric membrane potential assay in cerebellar neurons further supported the view that blockade of sodium channels may be a major component of the action of tolperisone-type centrally acting muscle relaxant drugs. Furthermore, tolperisone, eperisone, and especially silperisone had a marked effect on voltage-gated calcium channels, whereas calcium currents were hardly influenced by lidocaine. These data suggest that tolperisone-type muscle relaxants exert their spinal reflex inhibitory action predominantly via a presynaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels.

NR2B receptors are involved in the mediation of spinal segmental reflex potentials but not in the cumulative motoneuronal depolarization in vitro.

Windup, the frequency dependent build-up of spinal neuronal responses is an electrophysiological model of the development of the central sensitization in the chronic pain states. NR2B subunit containing NMDA-type glutamate receptors are implicated in the windup of dorsal horn neurons, while their role at the motoneuronal level is controversial. The cumulative motoneuronal depolarization in hemisected rat spinal cord preparation is an in vitro model of windup. The role of NR2B receptors in this process, and in the mediation of dorsal root stimulation evoked ventral root reflex potentials was elucidated. Three selective NR2B antagonists; CP-101,606; CI-1041 and Co-101244 (1 microM) were used. They had only weak, but statistically significant inhibitory effect on the early part of ventral root response, and did not influence the cumulative depolarization. On the contrary, non-selective NMDA antagonist APV (40 microM) decreased both responses markedly. We conclude that the pharmacological sensitivities of windup at the sensory and motor levels are different. NR2B containing NMDA receptors have major role in the mediation of the windup of dorsal horn neurons, but their contribution to this phenomenon at the motor level is negligible.

NR2B containing NMDA receptor dependent windup of single spinal neurons.

Windup, the frequency dependent build-up of spinal neuronal responses, is implicated in the development of central sensitization of nociceptive pathways. N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in these processes but the role of various receptor subtypes at the spinal level is not fully understood. In our experiments, we compared the inhibitory effect of MK-801 (a nonselective NMDA receptor antagonist, 0.01-3 mg/kg i.v.) and CI-1041 (an NR2B subunit specific NMDA receptor antagonist, 0.3-10 mg/kg i.v.) on the formation of dorsal horn neuronal windup in spinalized rats, in vivo. Both types of antagonist blocked windup considerably at doses not affecting the normal synaptic transmission. These results are in agreement with the well-documented effectivity of NR2B subtype selective NMDA receptor antagonists in chronic pain models and give the first direct evidence that spinal mechanisms are involved in this effect.

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission, in rat.

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex. These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.

[Mydeton: a centrally acting muscle relaxant drug from Gedeon Richter LTD]. / Mydeton: a Richter centrális támadáspontú izomrelaxánsa.

Since its introduction in 1959 tolperisone hydrochloride (Mydeton) is still one of the leading products of Gedeon Richter Ltd. It has been successfully applied for treating different painful muscle spasms. The compound is successfully marketed also by several foreign, mostly Japanese, pharmaceutical companies, as a central muscle relaxant agent. The present summary overviews the pharmacology of tolperisone, with special emphasize on its still partly understood way of action. Data from the scientific literature as well as our own experimental results strongly support the hypothesis that inhibition of voltage gated sodium channels is a major component of the mechanism of action of tolperisone. The paper also summarizes the clinical results with tolperisone and the perspectives of the therapeutic use of centrally acting muscle relaxants.

Protective effect of the antiepileptic drug candidate talampanel against AMPA-induced striatal neurotoxicity in neonatal rats.

2,3-Benzodiazepines represent a family of specific, noncompetitive AMPA receptor antagonists with anticonvulsant and neuroprotective properties. In this study, the antiexcitotoxic potency of the clinical antiepileptic drug candidate, talampanel (4 x 2 mg/kg), and that of two related 2,3-benzodiazepines, 5-(4-aminophenyl)-8-methyl-9H-1,3-dioxolo[4,5-h][2,3]-benzodiazepine (GYKI 52466) (4 x 10 mg/kg) and GYKI 53784 (4 x 2 mg/kg), was investigated in 7-day-old rats. The AMPA antagonists were applied in four consecutive i.p. injections at 1-h intervals, the first dosage was given shortly after the intrastriatal injection of (S)-alpha-amino-3-hydroxy-5,7-methylisoxazole-4-propionic acid (AMPA) (2.5 nmol). All tested compounds protected animals from brain damage induced by AMPA as assessed 5 days later by using a tissue volume determination method based on computer-aided serial section reconstruction. GYKI 53784 (56.1 +/- 5.0% protection) and talampanel (42.5 +/- 5.3% protection) were more potent neuroprotective agents than GYKI 52466 (21.8 +/- 2.8% protection). Furthermore, the three compounds attenuated the unilateral AMPA injection-induced turning behavior and seizure-like events.Our present findings are in agreement with those of other investigators who found talampanel neuroprotective in various in vivo experimental models. These data indicate that besides being a promising antiepileptic drug candidate talampanel may have a value in the pharmacotherapy of acute and chronic neurodegenerative diseases, including perinatal ischemia/hypoxia-induced brain injuries, as well.

Non-competitive AMPA antagonists of 2,3-benzodiazepine type.

The discovery of the selective AMPA antagonist character of 2,3-benzodiazepine derivative GYKI 52466 (5) in the late eighties and the recognition of the non-competitive nature of its mode of action some years later set off the world-wide search for novel class of drugs. Notably the quest to develop new antiepileptic and neuroprotective medicines, which allosterically inhibit the AMPA sensitive glutamate operated channels. This review summarises our present knowledge about the allosteric site, dubbed "GYKI site" where the 2,3-benzodiazepines are supposed to bind to. The structure-activity relationships among AMPA antagonist 2,3-benzodiazepines and their structural analogues with similar biological profile are reviewed in a possibly comprehensive fashion. The chemical synthesis of 2,3-benzodiazepines is shortly described. The in vitro and in vivo experimental methods used for pharmacological characterisation of the biologically active compounds are briefly explained. Finally the therapeutic potential of 2,3-benzodiazepines i.e. the main fields of their clinical utility are outlined with special regard to talampanel (20) in the light of the ongoing clinical trials with this new drug candidate.