Ervin Horvath

Inhibition of evoked glutamate release by the neuroprotective 5-HT1A receptor agonist BAY x 3702 in vitro and in vivo

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT(1A) receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC(50)=1 microM). This effect was blocked by the selective 5-HT(1A) receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT(1A) receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 microg/kg or 10 microg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Neuroprotective and behavioral effects of the selective metabotropic glutamate mGlu1 receptor antagonist BAY 36-7620

This study characterized the neuroprotective and behavioral effects of (3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), a novel, selective and systemically active metabotropic glutamate (mGlu)(1) receptor antagonist. In the rat, neuroprotective effects were obtained in the acute subdural hematoma model (efficacy of 40-50% at 0.01 and 0.03 mg/kg/h, i.v. infusion during the 4 h following surgery); whereas in the middle cerebral artery occlusion model, a trend for a neuroprotective effect was obtained after triple i.v. bolus application of 0.03-3 mg/kg, given immediately, 2 and 4 h after occlusion. Hypothermic effects were mild and only obtained at doses which were considerably higher than those at which maximal neuroprotective efficacy was obtained, indicating that the neuroprotective effects are not a consequence of hypothermia. BAY 36-7620 protected against pentylenetetrazole-induced convulsions in the mouse (MED: 10 mg/kg, i.v.). As assessed in rats, BAY 36-7620 was devoid of the typical side-effects of the ionotropic glutamate (iGlu) receptor antagonists phencyclidine and (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801). Thus, BAY 36-7620 did not disrupt sensorimotor gating, induce phencyclidine-like discriminative effects or stereotypical behavior, or facilitate intracranial self-stimulation behavior. Although behavioral stereotypies and disruption of sensorimotor gating induced by amphetamine or apomorphine were not affected by BAY 36-7620, the compound attenuated some behavioral effects of iGlu receptor antagonists, such as excessive grooming or licking, and their facilitation of intracranial self-stimulation behavior. It is concluded that mGlu(1) receptor antagonism results in neuroprotective and anticonvulsive effects in the absence of the typical side-effects resulting from antagonism of iGlu receptors.

Selective intermediate-/small-conductance calcium-activated potassium channel (KCNN4) blockers are potent and effective therapeutics in experimental brain oedema and traumatic brain injury caused by acute subdural haematoma

Early deterioration and death after brain injury is often the result of oedema in the injured and peri‐lesional tissue. So far, no pharmacotherapy is available that exhibits significant brain oedema‐reducing efficacy in patients. We selected two low molecular weight compounds from different chemical classes, a triazole (1‐[(2‐chlorophenyl)diphenylmethyl]‐1,2,3‐triazole) and a cyclohexadiene (methyl 4‐[4‐chloro‐3‐(trifluoromethyl)phenyl]‐6‐methyl‐3‐oxo‐1,4,7‐tetrahydroisobenzofuran‐5‐carboxylate) to characterize their pharmacological properties on KCNN4 channels (intermediate/small conductance calcium‐activated potassium channel, subfamily N, member 4) in vitro as well as in vivo. In vitro we replaced potassium by rubidium (Rb+) and determined Rb+ fluxes evoked by 10 µm of the calcium ionophore A23187 on C6BU1 rat glioma cells. Compared with known KCNN4 blockers, such as clotrimazole (IC50 = 360 ± 12 nm) and charybdotoxin (IC50 = 3.3 ± 1.9 nm), the triazole and cyclohexadiene were considerably more potent than clotrimazole and displayed similar potencies (IC50 = 12.1 ± 8.8 and 13.3 ± 4.7 nm, respectively). In the rat acute subdural haematoma model, both the triazole and cyclohexadiene displayed reduction of brain water content (−26% at 0.3 mg/kg and −24% at 0.01 mg/kg) and reduction of the intracranial pressure (−46% at 0.1 mg/kg and −60% at 0.003 mg/kg) after 24 h when administered as a 4‐h infusion immediately after brain injury. When infarct volumes were determined after 7 days, the triazole as well as the cyclohexadiene displayed strong neuroprotective efficacy (−52% infarct volume reduction at 1.2 mg/kg and −43% at 0.04 mg/kg, respectively). It is concluded that blockade of KCNN4 channels is a new pharmacological approach to attenuate acute brain damage caused by traumatic brain injury.

Neuroprotective and brain edema-reducing efficacy of the novel cannabinoid receptor agonist BAY 38-7271

BAY 38-7271 is a new high-affinity cannabinoid receptor agonist with strong neuroprotective efficacy in a rat model of traumatic brain injury (acute subdural hematoma, SDH). In the present study we investigated CB1 receptor signal transduction by [35S]GTPgammaS binding in situ and in vitro to assess changes in receptor functionality after SDH. Further, we continued to investigate the neuroprotective properties of BAY 38-7271 in the rat SDH and transient middle cerebral artery occlusion (tMCA-O) model as well as the efficacy with respect to SDH-induced brain edema. [35S]GTPgammaS binding revealed minor attenuation of CB1 receptor functionality on brain membranes from injured hemispheres when compared to non-injured hemispheres or controls. In the rat SDH model, BAY 38-7271 displayed strong neuroprotective efficacy when administered immediately after SDH either as a 1 h (65% infarct volume reduction at 0.1 microg/kg) or short-duration (15 min) infusion (53% at 10 microg/kg). When administered as a 4 h infusion with a 5 h delay after injury, significant neuroprotection was observed (49% at 1.0 microg/kg/h). This was also observed when BAY 38-7271 was administered as a 5 h delayed 15 min short-duration infusion (64% at 3 microg/kg). In addition, the neuroprotective potential of BAY 38-7271 was demonstrated in the rat tMCA-O model, displaying pronounced neuroprotective efficacy in the cerebral cortex (91% at 1 ng/kg/h) and striatum (53% at 10 ng/kg/h). BAY 38-7271 also reduced intracranial pressure (28% at 250 ng/kg/h) and brain water content (20% at 250 ng/kg/h) when determined 24 h post-SDH. Based on these data it is concluded that the neuroprotective efficacy of BAY 38-7271 is mediated by multiple mechanisms triggered by cannabinoid receptors.

4-Phenyl-4H-pyrans as IKCa channel blockers

4-Phenyl-4H-pyrans have been identified as potent and specific IK(Ca) channel blockers. Their synthesis and structure-activity relationships are described. A selected derivative, rac-11, reduces the infarct volume in a rat subdural hematoma model of traumatic brain injury after iv administration.

Neuroprotective efficacy of repinotan HCl, a 5-HT1A receptor agonist, in animal models of stroke and traumatic brain injury

Repinotan is a highly potent 5-HT1A receptor agonist with strong neuroprotective efficacy in animal models of middle cerebral artery occlusion and traumatic brain injury. In this study, we characterized the time window for neuroprotective effects of repinotan in animal models. In the permanent middle cerebral artery occlusion model, repinotan showed neuroprotective efficacy when administered as a triple bolus injection (0.3–100 μg/kg) or an intravenous infusion (0.3–100 μg/kg per hour). A 73% reduction in infarct volume was observed with a 3 μg/kg intravenous bolus, and a 65% reduction was observed with a 3 and 10 μg/kg per hour intravenous infusion. When delayed until 5 hours after occlusion, repinotan (10 μg/kg per hour) reduced infarct volume by 43%. In the transient middle cerebral artery occlusion model, repinotan (10 μg/kg per hour) administered immediately after occlusion reduced infarct volume by 97%, and a delay to 5 hours reduced infarct volume by 81%. In the acute subdural hematoma model, repinotan (3 and 10 μg/kg per hour) reduced infarct volume by 65%. In this model, repinotan (3 μg/kg per hour) administered 5 hours after occlusion reduced infarct volume by 54%. The favorable neuroprotective efficacy, broad dose–response curve, and prolonged therapeutic window observed in all models strongly suggest that repinotan is a promising candidate for treating acute ischemic stroke in humans.

Sensorimotor impairments in rats with cerebral infarction, induced by unilateral occlusion of the left middle cerebral artery : strain differences and effects of the occlusion site

Enormous differences exist between rat strains with respect to the infarct volume induced by unilateral middle cerebral artery (MCA) occlusion. We performed three experiments to address the following questions. Firstly, whether the pattern of MCA-occlusion (MCA-O) induced sensorimotor impairments in rats are strain dependent; secondly, whether proximal (i.e., close to its origin) and distal occlusions (above the lenticulostriate branch) of the MCA affect infarct volume and the behavioral impairments to a different extent; and thirdly, whether there is a relationship between the infarct volume and behavioral deficits. We found that the pattern of sensorimotor malfunctions induced by proximal unilateral MCA-O were highly strain dependent. Of the eight strains tested, Winkelmann-Wistar rats, Spontaneously Hypertensive Stroke-Prone rats, and Wistar-Kyoto rats were most severely affected. By contrast, Brown-Norway rats showed only mild behavioral deficits after the MCA-O. The second experiment confirmed that proximal occlusions induced slightly more behavioral malfunctions than distal occlusions did. Histological evaluation of the brain damage caused by proximal and distal MCA-O, confirmed that distal MCA-O damaged nearly exclusively cortical areas, and spared the caudate/putamen. An exploratory analysis of the relationship between infarct volume and behavioral deficits did not indicate that the severity of sensorimotor malfunctions can be predicted from the size of the infarct.

Effect of the 5-HT1A receptor agonist ipsapirone on the local cerebral glucose utilization of the rat hippocampus

Local cerebral glucose utilization (LCGU) was measured in the hippocampus of the rat brain following i.p. injection of the anxiolytic drug and 5-HT1A receptor agonist ipsapirone (TVX Q 7821). Administration of ipsapirone (5 mg/kg) reduced glucose utilization in the various hippocampal areas to variable extent. The most subtle reduction took place in the dorsal subiculum, while the most pronounced decrease was found in sector CA4 of Ammons horn. The degree of LCGU reduction can be related to the 5-HT1A receptor density in the respective areas.

Sensorimotor impairments in wistar kyoto rats with cerebral infarction, induced by unilateral occlusion of the middle cerebral artery: recovery of function

Wistar Kyoto (WKY) rats with cerebral infarction induced by permanent unilateral occlusion of the middle cerebral artery (MCA) and sham-operated rats were tested in a series of simple behavioral test 2, 16 and 37 days after surgery. In addition, the motility of the animals was measured over a period of 62 h, after the third test series. A subset of the tests appeared to be suitable to assess the effects of cerebral infarction, namely, grasping reflex of contralateral hindpaw, circling behavior, forelimb flexion, hindlimb flexion, and latency to fall off a square bridge. Except for the impaired grasping reflex of the contralateral hindpaw, there was spontaneous complete recovery of function by the third test session, 37 days after surgery. Some of the other tests might not have been sensitive enough to detect the effects of the unilateral MCA-occlusion (MCA-O) on behavior. Moreover, the WKY rats were very inactive in some of the tests, so that reliable scoring of the effects was not always possible. A rat strain other than the WKY strain might be more suitable to study the behavioral consequences of MCA-O.

Semiquantitative expression analysis of ephrine-receptor tyrosine kinase mRNA's in a rat model of traumatic brain injury.

The molecular mechanisms involved in recovery of function of the central nervous system (CNS) after injury to the brain are incompletely understood. Here the expression of ephrine (Eph) kinases following traumatic brain injury (subdural haematoma) was analysed in order to find out whether these developmentally regulated genes may be involved in tissue remodelling after brain damage. mRNA was isolated from ipsilateral cortices 7, 18, and 28 days after surgery and semiquantitative reverse transcription-polymerase chain reaction was performed. Most Eph kinases did not show significant regulation at gene expression level during the time course of recovery from acute brain injury but there is some evidence that mRNA of EphB1 might be slightly upregulated.