Pharmacology: Ouabain-induced Increase in Dopamine Release from Mouse Striatal Slices is Antagonized by Riluzole

We have examined the effects of riluzole, a neuroprotective drug which stabilizes voltage-dependent sodium channels in their inactivated state and inhibits the release of glutamate in-vivo and in-vitro, on the release of newly taken up [³H]dopamine induced by ouabain, a potent and selective inhibitor of Na⁺/K⁺-ATPase in mouse striatal slices in-vitro. Riluzole potently (IC50 (concentration resulting in 50% inhibition) = 0.9±0.3 μM) and dose-dependently antagonized ouabain-stimulated [³H]dopamine release, the effect being observed at low concentrations. Tetrodotoxin (1 μM) and nomifensine (10 μM) also abolished ouabain-induced [³H]dopamine release. Blockade of glutamate receptors with dizocilpine (1 μM) and 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione (YM-90K; 10 μM), alone or in combination, was without effect. Incubation of striatal slices with 50 μM La³⁺, which blocks voltage-dependent calcium channels, did not inhibit [³H]dopamine release induced by ouabain. The potent effects of riluzole observed in this model are probably related to its ability to block voltage-dependent sodium channels. The consequences of this activity are critically discussed in relation to the protective action of riluzole previously reported in various models of Parkinson's disease and other neurodegenerative disorders.     

YM872: a selective,potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist

This review focuses on the in vitro and in vivo neuropharmacology of YM872, a potential neuroprotective agent currently undergoing clinical trials in the United States (trial name: AMPA Receptor Antagonist Treatment in Ischemic Stroke - ARTIST). Its neuroprotective properties in rats and cats with induced focal cerebral ischemia are described. YM872, [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydroquinoxalin-1-yl]-acetic acid monohydrate, is a selective, potent and highly water-soluble competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. YM872 has a potent inhibitory effect on [(3)H]AMPA binding with a K(i) value of 0.096 microM. In contrast, YM872 has very low affinity for other ionotropic glutamate receptors. The solubility of YM872 is approximately 500 to 1000 times higher than that of the other competitive AMPA antagonists: YM90K, NBQX, or CNQX. The neuroprotective efficacy of YM872 was investigated in rats and cats subjected to permanent occlusion of the left middle cerebral artery. The animals were assessed either histologically or neurologically following ischemia. In rats with occluded middle cerebral artery (MCAO) YM872, by i.v. infusion, significantly reduced infarct volume measured at 24 h and 1 week after ischemia. Significant neuroprotection was maintained even when drug administration was delayed for up to 2 h after ischemia. In addition, YM872 significantly improved neurological deficit measured at 1 week after ischemia. In cats with MCAO YM872, by i.v. infusion, dose-dependently reduced infarct volume at 6 h after ischemia. YM872 produced no behavioral abnormalities and was not nephrotoxic. The evidence for the neuroprotective efficacy of YM872 suggests its therapeutic potential in the treatment of acute stroke in humans.     

Delayed Treatment with YM90K,an AMPA Receptor Antagonist,Protects against Ischaemic Damage after Middle Cerebral Artery Occlusion in Rats

The neuroprotective effect of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride] has been examined in a rat middle cerebral artery occlusion model. Intravenous infusion of YM90K (2·5–20mgkg^?1h^?1 for 4 h) starting immediately after occlusion of the middle cerebral artery significantly reduced the cortical infarct volume 24 h after occlusion compared with the control group. The protection at the highest dose was 39% (P<0·05). Similar protective effects were observed when YM90K (20mgkg^?1h^?1 for 4 h) was delayed up to 2 h after middle cerebral artery occlusion (45% reduction, P<0·05). CNS1102 [N-(1-naphthyl)-N′-(3-ethylphenyl)-N′-methylguanidine hydrochloride], a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the cortical infarct volume when 1·3mgkg^?1 was administered by intravenous bolus injection immediately after middle cerebral artery occlusion, followed by intravenous infusion at 0·785 mgkg^?1h^?1 for 4 h (35% reduction, P<0·05). This neuroprotective effect was not observed when administration was delayed 1 h after middle cerebral artery occlusion. These results suggest that AMPA receptors might play a more important role than NMDA receptors in the late development of neuronal cell damage after focal cerebral ischaemia and that AMPA receptor blockade would be one beneficial strategy in treating acute stroke.     

Effect of YM872, a selective and highly water-soluble AMPA receptor antagonist, in the rat kindling and rekindling model of epilepsy

We examined antiepileptogenic and anticonvulsant effects of [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]-acetic acid monohydrate (YM872), a potent and highly water-soluble alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor antagonist, in the rat amygdala kindling model of epilepsy. Administration of YM872 significantly suppressed fully kindled seizures. Daily pretreatment with YM872 markedly retarded development of kindling during drug sessions. We also used the rekindling method to investigate the antiepileptogenic effect of YM872 in an attempt to differentiate between true and false effects in the conventional method of daily administration. The results using the rekindling method suggested that the effect of YM872 was truly antiepileptogenic, indicating its possible clinical usefulness as an antiepileptogenic drug. We also affirmed the importance of AMPA receptors in the seizure expression mechanism and development of kindling-induced epileptogenesis.     

Synthesis and leishmanicidal evaluation of sulfanyl- and sulfonyl-tethered functionalized benzoate derivatives featuring a nitroimidazole moiety

A series of new nitroimidazole-containing derivatives was synthesized by coupling of 2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]ethanol with diversely substituted benzoic acids. Upon treatment with m-CPBA, 12 of these sulfanyl compounds were further oxidized to their sulfonyl analogs. All the 26 synthetic compounds were examined for in vitro activity against Leishmania (V.) braziliensis and Leishmania (L.) mexicana, and some of them displayed an efficient antileishmanial activity. Among the compounds tested, the catecholic derivative 2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]sulfanyl}ethyl 3,4-dihydroxybenzoate ( 9a , LC₅₀ = 13 and 11 µM) and the pyrogallolic derivative 2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]sulfanyl}ethyl 3,4,5-trihydroxybenzoate ( 9b , LC₅₀ = 4 and 1 µM) were the most active ones against the two Leishmania strains.     

Characterization of the renal tubular transport of zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, by human organic anion transporters.

Zonampanel monohydrate (YM872; [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate) is a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. The major elimination route for zonampanel has been reported to be by urine via the kidneys. The purpose of this study is to elucidate the molecular mechanism of the renal excretion of zonampanel using cells stably expressing human organic anion transporters (hOAT) 1, hOAT2, hOAT3, and hOAT4, as well as human organic cation transporters (hOCT) 1 and hOCT2. Another AMPA receptor antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride], a decarboxymethylated form of zonampanel, was also used for comparing the substrate specificity. Zonampanel inhibited the uptake of prototypical organic anion substrates, [14C]para-aminohippurate in hOAT1 and [3H]estrone sulfate in hOAT3 and hOAT4, in a competitive manner. A time- and concentration-dependent increase in [14C]zonampanel uptake was observed in cells expressing hOAT1, hOAT3, and hOAT4. The Km values of zonampanel uptake by hOAT1, hOAT3, and hOAT4 were 1.4, 7.7, and 215 microM, respectively. Considering the localization of each transporter, results suggest that zonampanel is taken up via hOAT1 and hOAT3 from the blood into proximal tubular cells and then effluxed into the lumen via hOAT4. Probenecid and cimetidine competitively inhibited [14C]zonampanel uptake by the hOATs (hOAT1, hOAT3, and hOAT4 for probenecid; hOAT3 for cimetidine). YM90K inhibited the uptake of the prototypical substrate via hOAT3 competitively, but the uptake via hOAT1 noncompetitively. These findings suggest that the prototypical organic anion substrates (para-aminohippurate and estrone sulfate), cimetidine, probenecid, and zonampanel share binding specificity in each hOAT, whereas YM90K does not in hOAT1, possibly due to it being the decarboxymethylated form.     

Neuroprotective effects of an AMPA receptor antagonist YM872 in a rat transient middle cerebral artery occlusion model

The neuroprotective effects of YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl]acetic acid monohydrate), a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist with high water solubility, were examined in rats with transient middle cerebral artery (MCA) occlusion. The right MCA of male SD rats was occluded for 3 h using the intraluminal suture occlusion method. YM872 significantly reduced the infarct volume 24 hours after occlusion, at dosages of 20 and 40 mg/kg/h (iv infusion) when given for 4 h immediately after occlusion. Furthermore, delayed administration of YM872 (20 mg/kg/h iv infusion for 4 h, starting 2 or 3 h after the occlusion) also reduced the infarct volume and the neurological deficits measured at 24 h. Additionally, the therapeutic efficacy of YM872 persisted for at least seven days after MCA occlusion in animals treated with YM872 for 4 h starting 2 h after MCA occlusion. These data demonstrate that AMPA receptors contribute to the development of neuronal damage after reperfusion as well as during ischemia in the focal ischemia models and that the acute effect of the blockade of AMPA receptors persists over a long time period. YM872 shows promise as an effective treatment for patients suffering from acute stroke.     

Effect of AMPA receptor antagonist YM872 on cerebral hematoma size and neurological recovery in the intracerebral hemorrhage rat model

[2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]-acetic acid monohydrate (YM872 or zonampanel), an AMPA receptor antagonist, is in clinical development for acute ischemic cerebral infarction. Stroke patients are prone to have subsequent intracerebral hemorrhages. In order to predict potential adverse effects, YM872 was tested in a rat model with collagenase-induced intracerebral hemorrhage. The morphologically determined hematoma volumes after 24 h were compared between animal groups intravenously infused with 3600 U/kg/h heparin for 30 min, or with 20 or 40 mg/kg/h of YM872, or placebo for 4 h. Heparin enlarged hematoma volume, but neither dose of YM872 affected hematoma size. In a separate study, neurological deficits were scored at various days after intracerebral hemorrhage induction in animals with intravenous infusion for 24 h of 10 or 20 mg/kg/h YM872, or saline. The YM872 groups scored significantly better than the saline group at 14 days. These data suggest that YM872 does not exacerbate intracerebral hemorrhage and might accelerate recovery.     

Glutamate AMPA receptor antagonist treatment for ischaemic stroke

During cerebral ischaemia, glutamate is released in supraphysiological amounts and is toxic to brain tissue. This excitotoxicity is mediated by several glutamate receptor subtypes, including the ionotropic N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Clinical trials of drugs that block the NMDA receptor in acute ischaemic stroke have been disappointing. No improvement in clinical outcome of stroke has been seen with competitive NMDA antagonists (selfotel) and non-competitive NMDA antagonists (dextrorphan, GV150526, aptiganel and eliprodil). The AMPA receptor differs in important ways from the NMDA receptor. It is the principal mediator of fast excitatory neurotransmission. This ligand-gated cation channel is primarily permeable to sodium rather than calcium. It is found in grey and white matter. It is expressed by oligodendrocytes. This distribution may provide neuroprotection for both grey and white matter. In a variety of animal models, reduction in infarct volume with AMPA blockade has been demonstrated. AMPA antagonists also show benefit in spinal cord ischaemia and trauma. The clinical development of safe and effective AMPA blockers has been hampered by poor water solubility and associated renal toxicity. A novel, highly water-soluble, competitive AMPA receptor antagonist, YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydroquinoxalin-1-yl]acetic acid monohydrate; Yamanouchi), has been identified. Phase I clinical trial data indicate that this agent can be safely administered in young and elderly subjects. Sedation and other CNS associated adverse events determine the ceiling dose and become more problematic with infusion times exceeding 24 h. Phase II studies of YM872 in acute ischaemic stroke are ongoing.     

AMPA- and kainate-receptors differentially mediate excitatory amino acid-induced dopamine and acetylcholine release from rat striatal slices

Rat striatal slices, preincubated with [³H]dopamine (DA) and [¹⁴C]choline, were superfused continuously. Detection of radioactivity was used to monitor the release of the neurotransmitters DA and acetylcholine (ACh). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f) quinoxaline (NBQX) caused a concentration-dependent decrease in 100 μM α-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA)-, 100 μM kainate- or 100 mM glutamate-induced release of DA and ACh. The ₅₀ of NBQX is 3-fold higher (for ACh release) and is 2-fold lower (for DA release) than that of CNQX. This is in agreement with the ₅₀ ratio of NBQX and CNQX on kainate- and AMPA-receptor binding. These two antagonists, at doses that produce an equivalent blockade of kainate-receptor binding (5 μM for NBQX and 1.56 μM for CNQX), caused an approximately equal decrease in ACh- but not DA-release induced by 100 μM kainate or AMPA. At doses that produce an equivalent blockade of AMPA-receptor binding (5 μM for NBQX and 10 μM for CNQX), they caused an approximately equal decrease in DA but not ACh release induced by 100 μM AMPA or kainate. Moreover, concanavalin A (0.3 and 0.5 mg/ml), which selectively potentiates kainate-receptor responses, markedly enhanced 100 μM kainate-induced release of ACh but not DA. Cyclothiazide (10 μM), which selectively potentiates AMPA-receptor responses, significantly increased 100 μM AMPA- or kainate-induced release of DA but not ACh. In summary, these results indicate that AMPA- and kainate-receptor activation, respectively, are predominantly involved in excitatory amino acid (EAA)-induced DA and ACh release in the striatum.     

Effects of YM90K, a selective AMPA receptor antagonist, on amygdala-kindling and long-term hippocampal potentiation in the rat.

To investigate the role of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) type glutamate receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxalinedione hydrochloride], a potent and selective new AMPA receptor antagonist, in the rat amygdala-kindling model of epilepsy. Pretreatment with YM90K (7.5-30 mg/kg i.p.) markedly retarded the evolution of kindling. Once kindling was established, administration of YM90K (7.5-30 mg/kg i.p.) significantly and dose-dependently suppressed fully kindled seizures. The maximal effects were observed 15-30 min after injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold, the anticonvulsant effects of YM90K were reversed, suggesting that they were due to elevation of the generalized seizure-triggering threshold. Furthermore, an anticonvulsant dose (15 mg/kg) of YM90K affected neither field potentials nor long-term potentiation in the hippocampus in vivo. These results indicate that AMPA receptors play an important role in the seizure expression mechanism and the development of kindling-induced epileptogenesis, and suggest the possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs.     

Aniracetam, 1-BCP and cyclothiazide differentially modulate the function of NMDA and AMPA receptors mediating enhancement of noradrenaline release in rat hippocampal slices

Aniracetam, 1-(1,3-benzodioxol-5-yl-carbo-nyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the ‘kynurenate test’, a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-d-aspartate (NMDA)-evoked [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 μM) antagonism of the [³H]NA release elicited by 100 μM NMDA with high potency (EC₅₀≤0.1 μM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) added at 5 μM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of cyclothiazide. The AMPA-evoked release of [³H]NA from hippocampal slices or synaptosomes was enhanced by cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [³H]NA release in slices; this antagonism was attenuated by 1 μM cyclo-thiazide and reversed to an enhancement of AMPA-evoked [³H]NA release by 10 μM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP. Received: 14 September 1998 / Accepted: 19 January 1999     

Pharmacology: α-Adrenoceptor Interaction of Tetrandrine and Isotetrandrine in the Rat: Functional and Binding Assays

The action of 1S,1′S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on α₁-adrenoceptors has been compared with that of its isomer 1R,1′S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [³H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to α-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the α₁-adrenoceptors displacing [³H]prazosin from the specific binding site. The K_i values (inhibition constants) were 0.69±0.12 and 1.6±0.4 μM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca²⁺-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 μM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 μM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca²⁺ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 μM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with α₁-adrenoceptors by displacing the [³H]prazosin binding site and that both compounds inhibit mainly the Ca²⁺-dependent process and have less action on α₁-adrenoceptors. Tetrandrine is more potent than isotetrandrine.     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by 4-aminopyridine in rat entorhinal cortex hippocampal slices

The purpose of this study was to evaluate the effects of the new anticonvulsant drug N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid ethyl ester (retigabine, D-23129, ASTA Medica, Dresden, Germany) on different patterns of epileptiform activity induced by 4-aminopyridine (4AP) in rat entorhinal cortex hippocampal slices. Application of 4AP (100 μM) induced in entorhinal cortex two different types of epileptiform activities; seizure-like events (SLE) and interictal epileptiform discharges (IED). Bicuculline (10 μM) changed 4AP-induced SLE and IED to recurrent epileptiform discharges (RED). IED were isolated after blockade of the SLE by glutamate receptor antagonists for α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors, i.e. 1,2,3,4 tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX, 10 μM) and 2-amino-5-phosphonovaleric acid (APV, 30 μM). Anticonvulsant properties of retigabine were evaluated as effect on the frequency and amplitude of SLE, IED and RED. Retigabine suppressed all types of epileptiform events in a dose dependent and reversible manner. SLE were suppressed in 71.4 and 100% of slices by 5 and 10 μM, respectively. The frequency of IED was significantly reduced by 20 μM retigabine (40.9±24.5%) and IED were blocked completely by 50 μM retigabine. When IED were isolated by application of glutamate antagonists 20 μM retigabine was sufficient to block this activity completely. RED induced by combined application of bicuculline and 4AP were blocked in 71.4% of the tested slices with 100 μM retigabine. The frequency of the RED in the remaining slices was reduced by 96.1±6.1%. We conclude that retigabine acts on a large variety of different epileptiform activities in temporal lobe structures that are known to develop readily pharmacoresistant seizures. Received: 15 May 1998 / Accepted: 20 October 1998     

3−Nitropropionic Acid Exacerbates [3H]GABA Release Evoked by Glucose Deprivation in Rat Striatal Slices

3?Nitropropionic acid (3?NPA) is a metabolic poison that produces lesions of striatal intrinsic neurones such as γ-aminobutyric acid (GABA) neurones. This study was carried out to determine whether 3?NPA would impair the ability of striatal GABAergic neurones to withstand hypoglycaemic stress. 3?NPA (500 μM) did not affect [³H]GABA release from striatal slices under normal (11 mM) glucose concentrations. When the glucose concentration was lowered to 0m?3 mM, however, 3?NPA greatly potentiated the leakage of [³H]GABA from the slices. Blockade of N-methyl-d-aspartate (NMDA) or α-amino?3?hydroxy?5?methyl?4?isoxazolepropionic acid (AMPA) receptors with 1 μM 5?methyl?10,11?dihydro?5H-dibenzo[a,d]cyclohepten?5,10?imine (MK?801) or 10 μM 2,3?dihydroxy?6?nitro?7?sulpha-moylbenzo[F]quinoxaline (NBQX), respectively, or a combination of both, had no effect. However, blockade of voltage-dependent sodium channels with tetrodotoxin totally antagonized the [³H]GABA overflow induced by the combination of 3?NPA and hypoglycaemia. Riluzole (10 to 100 μM), a neuroprotective agent that stabilizes the inactivated state of the voltage-dependent sodium channel, also dose-dependently antagonized the increase in [³H]GABA release induced by the combination of the two stresses.     

Transport properties of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxyinosine in the rat choroid plexus

Transport properties of 3′-azido-3′-deoxythymidine (AZT) and 2′, 3′-dideoxyinosine (DDI) were characterized in the isolated rat choroid plexus. AZT and DDI competitively inhibited the active transport of [³H]benzylpenicillin, a prototypic organic anion, with K_i values of 85·4±13·1 and 155±22 μM, respectively. Accumulation of [³H]DDI was against an electrochemical potential via a saturable process (K_m=29·7±4·9 μM, V_max=13·5±2·4 pmol min⁻¹/μL tissue) that was inhibited by metabolic inhibitors (carbonylcyanide p -trifluoromethoxyphenylhydrazone, 10 μM, and rotenone, 30 μM) and sulphydryl reagents (p -chloromercuribenzoic acid, 100 μM, and p -chloromercuribenzenesulphonic acid, 100 μM), but did not require an inwardly directed Na⁺ gradient. Accumulation of [³H]DDI was inhibited by benzylpenicillin and AZT in a dose-dependent manner, with IC₅₀ values of 91·6±28·9 and 294±84 μM, respectively. In contrast, no significant accumulation of [³H]AZT was observed. These results suggest that DDI is transported, at least in part, by the transport system for organic anions located on the rat choroid plexus, whereas AZT is recognized, but not transported by this system. © 1997 John Wiley & Sons, Ltd.     

Kainate receptors are involved in the glutamate-induced indirect,purinergic inhibition of [3H]-noradrenaline release in rabbit brain cortex

Activation of ionotropic but not of metabotropic glutamate receptors causes an indirect inhibition of the release of noradrenaline in slices of rabbit brain cortex. The inhibition is mediated by adenosine which activates presynaptic adenosine A₁-receptors. The present study characterizes the ionotropic receptor types through which glutamate itself produces this indirect inhibition. Rabbit brain cortex slices were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1 M) and stimulated electrically by trains of 6 pulses at 100 Hz.Glutamate (100–3000 M) reduced the electrically evoked overflow of tritium by up to 58 %. The effect did not differ 20 min and 60 min after addition of glutamate. Adenosine deaminase (1 U ml^-1) as well as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 M) and d--glutamylamino-methanesulfonate (GAMS; 30 M), both of which block kainate receptors, attenuated the glutamate-induced inhibition. The NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5; 100 M) and the AMPA receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX; 30 M) did not change the effect of glutamate. Given alone, CNQX and GAMS, but not AP5 and NBQX, slightly increased the evoked overflow of tritium; the increases were abolished in the presence of adenosine deaminase.The results indicate that activation of kainate but not NMDA and AMPA receptors is involved in the indirect, adenosine-mediated inhibition by exogenous glutamate of the release of noradrenaline in rabbit brain cortex slices. Moreover, as shown by the increase caused by CNQX and GAMS, endogenous excitatory amino acids inhibit the release of noradrenaline through the kainate receptor-adenosine mechanism and thus contribute to the purinergic inhibitory control of noradrenaline release in the brain.     

Pharmacokinetics and tissue distribution of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide; a novel ALK5 inhibitor and a potential anti-fibrosis drug

The authors investigated the pharmacokinetics and metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a novel ALK5 inhibitor, which suppresses renal and hepatic fibrosis, and also exerts anti-metastatic effects on breast cancer-bearing MMTV-cNeu mice model. Plasma half-lives of orally administered IN-1130 were 62.6 min in mice, 76.6?±?10.6 min in dogs, 156.1?±?19.3 min in rats, and 159.9?±?59.9 min in monkeys. IN-1130 showed a high apparent permeability coefficient (P_app) of (45.0?±?2.3)?×?10^?6 cm s^?1 in in vitro permeability tests in a Caco-2 cell monolayer model. The bioavailability of orally administered IN-1130 was 84.9% in dogs and 34.4% in monkeys (oral dose, 5.5 mg kg^?1), 11.4% in rats and 8.95% in mice (oral dose, 50.3 mg kg^?1), respectively. Orally given IN-1130 was readily distributed into liver, kidneys and lungs. The major metabolite of IN-1130 (M1) was detected in the systemic circulation of rat and mouse and was purified and tentatively identified as 3-((4-(3-hydroxyquinoxaline-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide or 3-((4-(2-hydroxyquinoxalin-6-yl)-5-(6-methylpyridine-2-yl)-1H-imidazol-2-yl)methyl)benzamide. The highest levels of M1 were found in liver. The results of this study suggest that IN-1130 has the potential to serve as an effective oral anti-fibrotic drug.     

Baclofen (β-p-chlorophenyl-γ-aminobutyric acid) enhances [3H]γ-aminobutyric acid (3H-GABA) release from rat globus pallidus in vitro

The rat globus pallidus has been investigated as a possible model in which to study pre-synaptic G AB A mechanisms in vitro. (±)-Baclofen (300 μM-1 mM) significantly enhanced the release of radioactivity from superfused slices of rat globus pallidus prelabelled with ³H-GABA in vitro. This releasing action was specific to the (+)-isomer of baclofen: neither the (—)-isomer nor another neuronal depressant DL-α-∞-diaminopimelic acid had any significant effect. The releasing effect of baclofen appeared unrelated to the phenethylamine moiety of its structure as neither β–phenethylamine nor dopamine evoked release of ³H-GABA from pallidal slices. Baclofen increased the efflux of radioactivity from pallidal slices prelabelled with either [³H]β-alanine or [³H]diaminobutyric acid in vitro. The use of specific glial and neuronal GABA uptake blocking compounds (β-alanine and (±)-cis-1,3-amino-cyclohexanecarboxylic acid) did not permit resolution of the elements from which baclofen was evoking [³H]GABA release. Baclofen also inhibited uptake of [³H]GABA into pallidal slices with an IC50 value of 6 × 10^?4 M. The GABA-like properties of baclofen may be related to the (+)-isomer while non-specific neuronal depressant actions are an effect of the (—)-isomer. The potential of the (+)-isomer as an antipsychotic agent while (—)-baclofen remains the effective antispastic drug free from unwanted side-effects, is discussed.