Search

Epilepsy, one of the most common and most disabling neurological disorders, is characterized by spontaneous recurrent seizures, often associated with structural brain alterations and cognitive and psychiatric comorbidities. In about 30% of patients, the seizures are resistant to current treatments; so more effective treatments are urgently needed. Among the ∼30 clinically approved antiseizure drugs, retigabine (ezogabine) is the only drug that acts as a positive allosteric modulator (or opener) of voltage-gated Kv7 potassium channels, which is particularly interesting for some genetic forms of epilepsy. Here we describe a novel dual-mode-of-action compound, GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-carboxylic acid amide) that activates both Kv7 potassium channels and the mitochondrial translocator protein 18 kDa (TSPO), leading to increases synthesis of brain neurosteroids. TSPO activators are known to exert anti-inflammatory, neuroprotective, anxiolytic, and antidepressive effects, which, together with an antiseizure effect (mediated by Kv7 channels), would be highly relevant for the treatment of epilepsy. This prompted us to compare the antiseizure efficacy of retigabine and GRT-X in six mouse and rat models of epileptic seizures, including the 6-Hz model of difficult-to-treat focal seizures. Furthermore, the tolerability of the two compounds was compared in mice and rats. Potency comparisons were based on both doses and peak plasma concentrations. Overall, GRT-X was more effective than retigabine in three of the six seizure models used here, the most important difference being the high efficacy in the 6-Hz (32 mA) seizure model in mice. Based on drug plasma levels, GRT-X was at least 30 times more potent than retigabine in the latter model. These data indicate that GRT-X is a highly interesting novel anti-seizure drug with a unique (first-in-class) dual-mode mechanism of action.

Background IMI2-PainCare-BioPain-RCT3 is one of four similarly designed clinical studies aiming at profiling a set of functional biomarkers of drug effects on the nociceptive system that could serve to accelerate the future development of analgesics, by providing a quantitative understanding between drug exposure and effects of the drug on nociceptive signal processing in human volunteers. IMI2-PainCare-BioPain-RCT3 will focus on biomarkers derived from non-invasive electroencephalographic (EEG) measures of brain activity. Methods This is a multisite single-dose, double-blind, randomized, placebo-controlled, 4-period, 4-way crossover, pharmacodynamic (PD) and pharmacokinetic (PK) study in healthy subjects. Biomarkers derived from scalp EEG measurements (laser-evoked brain potentials [LEPs], pinprick-evoked brain potentials [PEPs], resting EEG) will be obtained before and three times after administration of three medications known to act on the nociceptive system (lacosamide, pregabalin, tapentadol) and placebo, given as a single oral dose in separate study periods. Medication effects will be assessed concurrently in a non-sensitized normal condition and a clinically relevant hyperalgesic condition (high-frequency electrical stimulation of the skin). Patient-reported outcomes will also be collected. A sequentially rejective multiple testing approach will be used with overall alpha error of the primary analysis split between LEP and PEP under tapentadol. Remaining treatment arm effects on LEP or PEP or effects on EEG are key secondary confirmatory analyses. Complex statistical analyses and PK-PD modeling are exploratory. Discussion LEPs and PEPs are brain responses related to the selective activation of thermonociceptors and mechanonociceptors. Their amplitudes are dependent on the responsiveness of these nociceptors and the state of the pathways relaying nociceptive input at the level of the spinal cord and brain. The magnitude of resting EEG oscillations is sensitive to changes in brain network function, and some modulations of oscillation magnitude can relate to perceived pain intensity, variations in vigilance, and attentional states. These oscillations can also be affected by analgesic drugs acting on the central nervous system. For these reasons, IMI2-PainCare-BioPain-RCT3 hypothesizes that EEG-derived measures can serve as biomarkers of target engagement of analgesic drugs for future Phase 1 clinical trials. Phase 2 and 3 clinical trials could also benefit from these tools for patient stratification. Trial registration This trial was registered 25/06/2019 in EudraCT (2019%2D%2D001204-37).

During drug development, seizure threshold tests are widely used to identify potential proconvulsant activity of investigational drugs. The most commonly used tests in this respect are the timed intravenous pentylenetetrazole (PTZ) infusion seizure test and the maximal electroshock seizure threshold (MEST) test in mice or rats. To our knowledge, no study is available in which proconvulsant drug activities in these models are directly compared, which prompted us to perform such experiments in male Wistar rats. Five drugs with reported proconvulsant activity were tested in the two models: d-amphetamine, chlorpromazine, caffeine, theophylline, and tramadol. Furthermore, the anticonvulsant drug phenobarbital was included in the experiments. While phenobarbital exerted anticonvulsant activity in both models, the five proconvulsant drugs markedly differed in their effects. In the dose range tested, d-amphetamine significantly lowered the PTZ seizure threshold but increased the MEST, caffeine and theophylline did not alter the PTZ seizure threshold but decreased the MEST, and tramadol reduced the PTZ threshold but increased the MEST. These marked differences between seizure threshold tests are most likely a consequence of the mechanisms underlying seizure induction in these tests. Our data indicate that using only one seizure threshold model during preclinical drug development may pose the risk that potential proconvulsant activity of an investigational drug is overseen. However, the label "proconvulsant" may be misleading if such activity only occurs at doses high above the therapeutic range, but the drug is not proconvulsant or even exerts anticonvulsant effects at lower, therapeutically relevant doses.

The nociceptin/orphanin FQ (N/OFQ) receptor (e.g. the human ortholog ORL1) has been shown to be pharmacologically distinct from classic opioid receptors. Recently, we have identified buprenorphine as a full ORL1 agonist using a reporter gene assay. For further functional analysis, buprenorphine’s effects on ORL1 receptors were investigated using a K + channel (GIRK1) assay in Xenopus oocytes and GTPγS assay in CHO-K1 membrane preparations. In both assays, buprenorphine behaved as a partial agonist compared to nociceptin itself. The N/OFQ agonism of buprenorphine might contribute to actions of buprenorphine in pain models in vivo beside its μ- or κ-opioid receptor mediated effects.

In epilepsy research, there is a growing interest in the role of the piriform cortex (PC) in the development and maintenance of limbic kindling and other types of limbic epileptogenesis leading to complex partial seizures. Neurophysiological studies on PC or amygdala-PC slice preparations from kindled rats showed that kindling of the amygdala induces long-lasting changes in synaptic efficacy in the ipsilateral PC, including spontaneous discharges and enhanced susceptibility of PC neurons to evoked burst responses. These long-lasting electrophysiological changes in the PC during kindling appear to be due, at least in part, to impaired function of gamma-aminobutyric acid (GABA)ergic interneurons. The aim of the present study was to develop an anesthetic protocol allowing electrophysiological single-unit recordings from inhibitory, presumably GABAergic PC interneurons in vivo. In addition to recording of spontaneously active PC neurons, microiontophoretic application of glutamate was used to activate silent neurons. Anesthesia of rats with ketamine/xylazine was not suited for single-unit recordings in the PC because of marked cardiovascular depression. Anesthesia with chloral hydrate allowed recording of spontaneous or glutamate-driven single-unit activity in ≈40% of all animals. A similar percentage was obtained when recordings were done with the narcotic opioid fentanyl (plus gallamine), after all surgical preparations were performed under anesthesia with repeated administration of the barbiturate methohexital. To avoid brain accumulation of methohexital by repeated applications, we modified the anesthetic protocol in that methohexital was only injected once for initiation of surgical anesthesia, followed by the short-acting anesthetic propofol which does not accumulate upon repeated application. Again, after surgical preparation, electrophysiological recordings were done under fentanyl (plus gallamine). By this procedure, spontaneous or glutamate-driven single-unit activity could be measured in all rats in either layer II or III of the PC. Based on shape and frequency of action potentials, two types of neurons were recorded. The predominant type was similar in its firing characteristics to GABAergic neurons in other brain regions, was mainly located in layer III, and could be suppressed by the serotonin2A receptor antagonist MDL 100,907, suggesting that this type of PC neuron represents inhibitory, putative GABAergic interneurons. This new in vivo preparation may be useful for evaluation of PC neurons in kindled rats. J. Neurosci. Res. 55:608–619, 1999. © 1999 Wiley-Liss, Inc.

Levetiracetam ((S)-α-ethyl -2-oxo-pyrrolidine acetamide, ucb L059) is a novel anticonvulsant drug presently in clinical development. Its mechanism of action is unknown although a recently reported novel specific binding site for [3H]levetiracetam, unique to brain, may be involved. This binding site has not yet been characterized, but some evidence suggested a possibly indirect interaction with the GABA system. We therefore examined levetiracetam's effects on GABA metabolism and turnover in several rat brain regions after systemic administration of anticonvulsant doses. Furthermore, in order to study functional effects of levetiracetam on a well defined system of GABAergic neurons in a brain region that has been critically involved in anticonvulsant drug action, we examined levetiracetam's action on spontaneous firing of substantia nigra pars reticulata (SNR) neurons in anesthetized rats. Although levetiracetam did not alter the activity of the GABA synthesizing and degrading enzymes glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T) in vitro, systemic administration induced significant alterations in these enzymes in several brain regions, indicating that these enzyme alterations were no direct drug effects but a consequence of postsynaptic changes in either GABAergic or other neurotransmitter-related systems. In the striatum, levetiracetam, 170 mg/kg i.p., induced a significant increase in GABA-T activity while GAD activity markedly decreased. When GABA turnover was estimated after inhibition of GABA-T by aminooxyacetic acid (AOAA), treatment with levetiracetam (given 15 min prior to injection of AOAA) significantly reduced GABA turnover in the striatum. Since the substantia nigra pars reticulata (SNR) receives a strong GABAergic input from the striatum, we examined if the alterations in GABA metabolism and turnover in the striatum led to functional alterations in neuronal activity in the SNR by recording single unit activity of SNR neurons after i.p. injection of levetiracetam. While injection of vehicle did not affect SNR neuronal activity, a significant decrease in spontaneous neuronal firing was recorded after levetiracetam. Since a substantial body of evidence suggests that the SNR is a critical site at which decrease of neuronal firing results in protection against various seizure types, the suppressive effect of levetiracetam on SNR activity may contribute to the anticonvulsant action of this drug.

GluR5 receptors modulate spinal nociception, however, their role in nociceptive hypersensitivity remains unclear. Using behavioural and electrophysiological approaches, we have investigated several GluR5 ligands in acute and hyperalgesic states. Furthermore, as the GABAergic system plays a role in GluR5 mediated effects in the brain, we also analysed the interaction between GluR5 agonists and GABA(A) antagonists in the spinal cord. In young rats in vivo, the GluR5 selective agonist ATPA was antinociceptive and antihyperalgesic in a model of inflammatory hyperalgesia (ED(50) approximately 4.6 and approximately 5.2 mg/kg, respectively), whereas the GluR5/GluR6 agonist SYM2081 was only antihyperalgesic. ATPA, but not SYM2081, was also able to inhibit nociceptive motoneurone responses in anaesthetised adult rats after intrathecal administration. In hemisected spinal cords in vitro, SYM2081 was inactive, whereas ATPA and another GluR5 agonist, (S)-5-iodowillardiine, inhibited nociceptive reflexes (EC(50) 1.1+/-0.4 micro M and 0.36+/-0.05 micro M, respectively). Both GluR5 agonists also inhibited motoneurone responses to repetitive dorsal root stimulation and their cumulative depolarisation, a correlate of wind-up. The GABA(A) antagonists bicuculline (10 micro M) and SR95531 (1 micro M) enhanced polysynaptic responses to single stimuli but abolished the cumulative depolarisation. Both bicuculline and SR95531 significantly attenuated the inhibition of nociceptive responses by 1 micro M ATPA (by approximately 50%). We conclude that selective GluR5 kainate receptor activation inhibits spinal nociception and its sensitisation caused by ongoing peripheral nociceptive drive. GABA(A) receptors are involved in tonic inhibition of segmental responses, but contribute to their sensitisation by repetitive primary afferent stimulation. Furthermore, there is a cross-talk between the two systems, presumably due to GluR5-mediated activation of GABAergic inhibitory interneurones in the spinal cord.

Gabapentin is a novel anti-epileptic drug which enhances GABA (γ-aminobutyric acid) turnover in certain brain regions, including substantia nigra. However, the functional consequences of GABA turnover increases in response to gabapentin and their potential involvement in the anticonvulsant action of this drug are not known. In the present study, we examined the effects of gabapentin on the extracellular, single unit activity of nondopaminergic (presumably GABAergic) neurons of the substantia nigra pars reticulata in rats. During the recordings, the animals were infused with the narcotic opioid analgesic fentanyl, associated with a skeletal muscle relaxant and artificial ventilation. The spontaneous firing of substantia nigra pars reticulata neurons was determined up to about 2 h after i.v. or i.p. administration of gabapentin at doses of 15–30 mg/kg. After both routes of administration, gabapentin markedly reduced neuronal firing when administered at a dose of 20–30 mg/kg, while 15 mg/kg were ineffective in this regard. The suppressive effect of gabapentin was rapid in onset (2 min after i.v. and about 20 min after i.p. injection), reached peak values of about 70% below predrug baseline after about 45–60 min, and remained at this level for at least 2 h. Vehicle administration had no effect on substantia nigra pars reticulata neurons. The ability of gabapentin to alter substantia nigra pars reticulata firing does correlate with its known ability to increase nigral GABA turnover. Since a substantial body of evidence suggests that the substantia nigra pars reticulata is a critical site at which decrease of neuronal firing by potentiation of GABAergic influences results in protection against various seizure types, the suppressive effect of gabapentin on substantia nigra pars reticulata activity may contribute to the anticonvulsant action of this drug.