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Vol. 289, Issue 3, 1343-1349, June 1999

BIIR 561 CL: A Novel Combined Antagonist of alpha -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors and Voltage-Dependent Sodium Channels with Anticonvulsive and Neuroprotective Properties

T. Weiser, M. Brenner, R. Palluk, W. D. Bechtel, A. Ceci, A. Brambilla, H. A. Ensinger, A. Sagrada and M. Wienrich

Boehringer Ingelheim Pharma KG, Department of CNS Research, Ingelheim, Germany (T.W., W.D.B., H.A.E.); Boehringer Ingelheim Pharma KG, Department of Medicinal Chemistry, Ingelheim, Germany (M.B.); Boehringer Ingelheim GmbH, Ingelheim, Germany (R.P., M.W.); and Boehringer Ingelheim Italy, Milan, Italy (A.C., A.B., A.S.)


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Antagonists of glutamate receptors of the alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, as well as of voltage-gated sodium channels, exhibit anticonvulsive and neuroprotective properties in vivo. One can postulate that a compound that combines both principles might be useful for the treatment of disorders of the central nervous system, like focal or global ischemia. Here, we present data on the effects of dimethyl-{2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxy]ethyl}-amine hydrochloride (BIIR 561 CL) on neuronal AMPA receptors and voltage-dependent sodium channels. BIIR 561 CL inhibited AMPA receptor-mediated membrane currents in cultured cortical neurons with an IC50 value of 8.5 µM. The inhibition was noncompetitive. In a cortical wedge preparation, BIIR 561 CL reduced AMPA-induced depolarizations with an IC50 value of 10.8 µM. In addition to the effects on the glutamatergic system, BIIR 561 CL inhibited binding of radiolabeled batrachotoxin to rat brain synaptosomal membranes with a Ki value of 1.2 µM. The compound reduced sodium currents in voltage-clamped cortical neurons with an IC50 value of 5.2 µM and inhibited the veratridine-induced release of glutamate from rat brain slices with an IC50 value of 2.3 µM. Thus, BIIR 561 CL inhibited AMPA receptors and voltage-gated sodium channels in a variety of preparations. BIIR 561 CL suppressed tonic seizures in a maximum electroshock model in mice with an ED50 value of 2.8 mg/kg after s.c. administration. In a model of focal ischemia in mice, i.p. administration of 6 or 60 mg/kg BIIR 561 CL reduced the area of the infarcted cortical surface. These data show that BIIR 561 CL is a combined antagonist of AMPA receptors and voltage-gated sodium channels with promising anticonvulsive and neuroprotective properties.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Glutamate receptors of the alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype provide excitatory synaptic transmission in the vertebrate central nervous system (CNS). However, overstimulation of these receptor-gated ion channels can induce severe neuronal damage. A deleterious role of AMPA receptors has been demonstrated for brain ischemia and is discussed for, for example, epilepsy (Meldrum, 1992; Gill, 1994; Lees, 1996). Consequently, antagonists acting on this glutamate receptor subtype exhibit anticonvulsive and neuroprotective activity. This has been shown for competitive, as well as noncompetitive, antagonists (Le Peillet et al., 1992; Smith and Meldrum, 1992). Such compounds are effective in a variety of in vivo models for neuroprotection and anticonvulsive activity.

Similarily, voltage-gated sodium channels are essential for neuronal signal transduction under physiological conditions, but their overstimulation during excitotoxic events induces neuronal degeneration. Thus, another useful principle for anticonvulsive or neuroprotective therapy is the inhibition of voltage-gated sodium channels in the CNS. Compounds like phenytoin, carbamazepine, or lamotrigine are blockers of this type of ion channels and have found widespread use as medications against epileptic seizures. Moreover, inhibitors of voltage-gated sodium channels have neuroprotective properties in models of focal brain ischemia (Lang et al., 1993; Rataud et al., 1994; for a review, see Urenjak and Obrenovitch, 1996; Stys, 1998).

One can therefore postulate that a drug that combines these two therapeutic principles should have profound anticonvulsive properties and might be protective in disorders like ischemic stroke of the brain.

Here we describe the effects of the novel AMPA antagonist and sodium channel blocker BIIR 561 CL. In addition, we compare the data with those for the reference compounds 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, a noncompetitive blocker of AMPA receptors) and mexiletine (a sodium channel inhibitor), respectively. We provide evidence that BIIR 561 CL is an inhibitor of glutamate receptors of the AMPA subtype, as well as of voltage-gated sodium channels, with promising anticonvulsive and neuroprotective properties.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Patch-Clamp Experiments. Experiments were performed in the "whole-cell" configuration of the patch-clamp technique using primary cell cultures from rat embryonic cortex as described previously (Weiser and Wienrich, 1996). Recording pipettes were pulled from borosilicate glass (Hilgenberg, Malsfeld, Germany) and had resistances of 3 to 5 MOmega . Membrane currents were measured using an EPC 7 or EPC 9 amplifier (HEKA, Lambrecht/Pfalz, Germany); data were collected, stored, and analyzed using the TIDA data acquisition system (HEKA). All solutions were applied using a gravity-driven perfusion system, which was also controlled by the TIDA software package and allowed the medium change at the cell under study within 50 ms. The intracellular medium consisted of 15 mM NaCl, 20 mM tetraethylammonium-Cl, 110 mM CsF, 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 1 mM ATP, pH 7.2.

The extracellular medium consisted of 140 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 21.5 mM glucose, and 10 mM HEPES, pH 7.4. For investigation of transmitter-activated ion channels, the extracellular medium in addition contained 300 nM tetrodotoxin. Stock solutions (100-1000-fold the final concentration) of BIIR 561 CL, kainate, mexiletine (as hydrochloride), and tetrodotoxin were prepared in water; AMPA and GYKI 52644 were dissolved in dimethyl sulfoxide.

CoCl2 (2 mM) was added to the extracellular solution for the investigation of sodium currents to suppress voltage-gated calcium channels.

All experiments were performed at room temperature (20-23°C).

Cortical Wedges. Male Sprague-Dawley rats (200-300 g) were sacrificed, the brains were rapidly removed, and 500-µm-thick coronal slices were cut at 4°C in artificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 10 mM glucose, gassed with 95% O2/5% CO2, pH 7.4. From two to four slices were retained starting slightly anterior to bregma and proceeding caudally. The wedges were incubated for 30 to 60 min at room temperature (20-24°C) in aCSF and then placed in a two-compartment tissue bath. Each compartment was perfused independently at 2.5 ml/min via a peristaltic pump. The DC potential between the two compartments was continuously monitored using Ag/AgCl electrodes embedded in 2% agar and displayed on a chart recorder. No consistent changes in this DC potential were measured after insertion of the slices; drug-induced deviations from this baseline DC potential were measured at the peak amplitude.

AMPA was applied for 50 s, and the test compounds were administered cumulatively 15 min before the addition of AMPA. AMPA was dissolved in aCSF. BIIR 561 CL and GYKI 52466 were dissolved in aCSF with 1% of dimethyl sulfoxide.

[3H]Batrachotoxin Binding. Membrane preparation and binding assay followed the references (Gray and Whittaker, 1962; Creveling and Daly, 1992) with minor modifications. Briefly, fresh synaptosomal membrane suspensions from the total brain (without cerebellum) were prepared using male rats, strain Chbb:Thom (bred at Boehringer Ingelheim Pharma KG, Biberach, Germany) (weight, 200-350 g). The assays containing the membrane suspension, 1 nM [3H]batrachotoxin (KD = 20.3 nM), and the test compound in a total volume of 1.0 ml were incubated, and the incubation was terminated by rapid filtration through Whatman GF/B filters under vacuum and washing with ice-cold buffer. The radioactivity on the filter disks was measured by the usual liquid scintillation counting. Each assay was performed in duplicate, and experiments were repeated as indicated in the tables. Specific binding was defined as total binding minus that determined in the presence of 100 µM aconitine.

[3H]AMPA Binding. Rats (Chbb:Thom, 200-250 g, male) were decapitated, and the cerebral cortex was immediately removed and after washing with solution B (50 mM tris[hydroxymethyl]aminomethane-acetate, pH 7.4) was transferred into ice-cold solution A (solution B supplemented with 320 mM sucrose). The tissue was weighed and homogenized in a 10-fold volume of ice-cold solution A with a Teflon piston (800 rpm,1 min, 12 strokes), followed by a centrifugation step of 10 min at 1000g at 0-5°C. The supernatant then was centrifuged for 20 min at 20,000g, and the pellet was resuspended in 10 ml of buffer A, homogenized, and then again centrifuged at 20,000g. The pellet was resuspended in 10 ml of buffer B, followed by an incubation for 20 min in the ice bath, then centrifuged for 20 min at 48,000g, and the supernatant was discarded. The last step was repeated. Then, the pellet was resuspended in buffer B, and the suspension was stored frozen at -20°C for 24 h. A freeze-thaw cycle was added. After thawing at room temperature, the suspension was diluted with buffer B to about 500 mg tissue/1 ml (initial wet weight about 1000 mg tissue) and then stored frozen in liquid nitrogen and before used diluted to 5 mg tissue/100 µl buffer C (50 mM tris[hydroxymethyl]aminomethane-acetate, 100 mM KSCN, 2.5 mM CaCl2, pH 7.4). The binding assay was performed as described in the literature (Murphy et al., 1987).

Veratridine-Induced Glutamate Release. The assay followed procedures reported (Leach et al., 1985; Meldrum et al., 1992) with some modifications. Cross-chopped slices, 0.2 mm thick, from rat brain cortex (strain Chbb:Thom) were prepared and preincubated at 37°C for 45 min in Krebs-Henseleit buffer (120 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, pH 7.4). In addition, the buffer contained 2 g of glucose, 100 mg of EDTA, and 200 mg of ascorbic acid per liter. The medium was continuously saturated with 95% O2/5% CO2 and changed completely three times during this preincubation period. Slices randomly distributed in microcentrifuge tubes (equivalent to about 430 µg protein) were then incubated for 15 min with (stimulated) or without (basal) 3 µM veratridine using an Eppendorf model 5437 Thermomixer (Eppendorf, Hamburg, Germany). The test drug was present throughout the incubation period or absent in the controls. Incubation was terminated by centrifugation with 16,000g for 5 min in an Eppendorf model 5415C centrifuge. The supernatant was used for glutamate determination by means of usual HPLC and fluorimetric detection. Protein was determined according to the Coomassie blue procedure (Bradford, 1976).

Traction and Maximum Electroshock Test. Male mice (OF1, IFFA Credo, France) weighing approximately 21 to 32 g were used in the experiments. The animals were kept in groups of 10, without individual identification, in Makrolon cages type III, bedded with soft wood granulate. They had free access to a standard pellet diet and tap water in an air-conditioned animal room (approximately 25°C).

Test compounds were administered via s.c. injections in the lower dorsal region 15 min before the experiments. Stock solutions of the test compounds were prepared in distilled water. All doses are calculated as free base.

The traction test (Courvoisier, 1956; Boissier and Simon, 1960, modified) was performed as follows. Each animal was individually placed for accommodation in a 1-liter beaker containing soft wood granulate bedding for approximately 15 min. Then, the mice were trained to hang to a horizontal steel rod of 3 mm diameter for a period of 15 s. Thereafter, the test compound was administered. After the respective pretreatment time, the mouse was again held in such a position that it touched the bar with its forepaws, and it was tested for its ability to hang to it for at least 15 s. If a mouse fell from the rod within 15 s, this was considered as motor impairment.

The maximum electroshock test (Toman et al., 1946, modified) was performed as follows. Immediately after the traction test, an electroshock (20 mA/50 Hz/200 ms) was applied to the mouse via saline-moistened eye electrodes (rodent shocker Type 221; HSE Electronics, March-Hugstetten, Germany). This was determined in previous control experiments to be a supramaximal stimulus, resulting in a fully developed tonic convulsion in 100% of the mice; therefore, no concurrent control group was considered necessary. If the application of the electroshock after administration of the test compound resulted only in clonic convulsions, this was considered to be anticonvulsive activity (protection from maximal, tonic convulsion) of the compound.

Focal Ischemia. Male CD-1 mice (24-26 g; Charles River, Calco, Italy) were used for the experiments. Animals were anesthetized with tribromoethanol (400 mg/kg i.p.), and the left middle cerebral artery was occluded by cauterization as described in the literature (Welsh et al., 1987, Backaubeta et al., 1992). Body temperature was maintained at 37.4 ± 0.1°C during surgery by means of a heating lamp. Ten minutes after occlusion, animals received the first injection of placebo or BIIR 561 CL and were then placed in an environment of 34°C for 2 h; then, they were returned to the normal environmental conditions and given the second injection.

Forty-eight hours after surgery, animals were administered tribromoethanol (400 mg/kg i. p.), the chest was opened, the right atrium was cut, and the mice were transcardially perfused with 1 ml of a 2,3,5-triphenyltetrazolium chloride solution (2% in physiological saline). The animals were kept at 37°C for 1 h, and the brains were removed and photographed at 6× linear magnification. The ischemic surface of the left hemisphere was measured by means of an electronic planimeter (Planix 7; Tamaya Technics Inc., Japan).

Compounds. BIIR 561 CL, GYKI 52466, mexiletine, and AMPA were synthesized at the Department of Medicinal Chemistry of Boehringer Ingelheim Pharma KG. With the exception of AMPA, hydrochlorides of the compounds were used. Radiolabeled batrachotoxin and AMPA were obtained from NEN (Dreieich, Germany). All other chemicals were at least of reagent grade and were purchased from reputable suppliers.

Data Analysis. If not otherwise stated, concentrations or doses for half-maximum effects were obtained by linear interpolation. Data from the maximal electroshock and traction test were analyzed using probit analysis. For the middle cerebral artery occlusion experiments, one-way ANOVA, followed by the two-tailed Dunnett's test for unequal-size groups, was applied. Data are given as mean ± S.E.M.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The structure of BIIR 561 CL is depicted in Fig. 1. The compound was identified in a screening campaign targeted to the identification of new structures active at AMPA receptors and voltage-gated sodium channels. The compound has a molecular weight of 345.83 g/mol (hydrochloride). The solubility of BIIR 561 CL in physiological buffer is about 9%.


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  		Fig. 1.   Chemical structure of BIIR 561 CL.

Effects on AMPA Receptors. In the first set of our experiments, we investigated the effects of BIIR 561 CL on AMPA receptors in different preparations from rat cortex.

In voltage-clamped primary cultured rat cortical neurons, the application of the selective agonist kainate induced nondesensitizing membrane currents. These currents were concentration-dependently and reversibly reduced by concomitant applications of BIIR 561 CL. With 100 µM kainate, half-maximum inhibition was reached with 8.5 ± 0.3 µM BIIR 561 CL (Fig. 2, A and B). The inhibition was not voltage dependent over the range of -120 to -30 mV (data not shown). GYKI 52644, a noncompetitive inhibitor of AMPA receptors, inhibited kainate-induced inward currents in this model with an IC50 value of 12.8 ± 1.4 µM (Fig. 2B).


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  		Fig. 2.   Effects of BIIR 561 CL on AMPA receptors in voltage-clamped cultured rat cortical neurons. A, inhibition of the currents induced by 100 µM kainate by the application of 100 µM BIIR 561 CL. The compound potently and reversibly inhibited the response. The "hook" (arrowhead) is typical for this compound; further investigations of this phenomenon will be performed in the future. Holding potential was -30 mV. B, concentration-response curve for the inhibition of the response to 100 µM kainate by various concentrations of BIIR 561 CL (closed symbols), and GYKI 52466 (open symbols). Linear interpolation of the data gave IC50 values of 8.5 µM for BIIR 561 CL and 12.8 µM for GYKI 52466 (n = 5 for each compound). C, concentration-response curve for kainate alone (, n = 10) and in the presence of either 33 µM BIIR 561 CL (open circle , n = 5) or 50 µM GYKI 52466 (black-triangle, n = 6). Data were normalized to the response achieved with 3000 µM kainate. The dotted line represents the simulated curve of kainate plus 33 µM BIIR 561 CL under the assumption of a competitive antagonism. Obviously, this curve does not match the experimentally achieved data for this compound.

We next investigated the mechanism of action of BIIR 561 CL on cortical AMPA receptors in greater detail. Concentration-response curves for kainate with and without 33 µM BIIR 561 CL were recorded. The EC50 value for kainate responses was 100.8 ± 2.0 µM in this experimental setting. BIIR 561 CL did not shift the agonist concentration-response curve toward higher concentrations. In contrast, the curve was depressed (Fig. 2C). We mathematically simulated the effect of a competitive antagonist with an assumed IC50 value of 8.5 µM on the kainate concentration-response curve under the assumption of a 1:1 competition between agonist and antagonist. The curve that we obtained was clearly different from the experimental data (Fig. 2C).

GYKI 52466 (50 µM) also suppressed the kainate concentration-response without shifting it to higher concentrations. These findings favor the hypothesis that BIIR 561 CL inhibits AMPA receptors noncompetitively.

The effects of BIIR 561 CL on AMPA receptors were further characterized using glutamate or AMPA as agonists. These experiments were targeted to the questions of 1) whether BIIR 561 CL also suppressed the membrane currents induced by these agonists and 2) whether the compound affected the kinetics of the current responses.

Pulse applications of 1 mM glutamate or 100 µM AMPA to voltage-clamped cortical neurons induced biphasic current responses, consisting of a transient peak and a persisting steady-state component (Fig. 3, A-C and D-F, respectively). BIIR 561 CL suppressed both components with similar potencies. With glutamate as agonist, peak and steady-state responses were inhibited with IC50 values of 7.6 ± 0.7 and 8.8 ± 1.1 µM, respectively (Fig. 3G). Similar values were obtained with 100 µM AMPA as agonist (9.3 ± 0.5 and 13.3 ± 2.2 µM for peak and steady-state, respectively; data not shown), demonstrating that BIIR 561 CL had only negligible effects on receptor kinetics.


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  		Fig. 3.   Inhibition of responses induced by glutamate or AMPA in voltage-clamped cultured rat cortical neurons. A and B, response of a cortical neuron to the pulse application of 1 mM glutamate in the presence (B) or absence (A) of 10 µM BIIR 561 CL. C, traces were normalized to the peak of the control response. BIIR 561 CL did not markedly alter the kinetics of the glutamate response. Agonist application is indicated by the bar; holding potential was -80 mV. D-F, similar experiment as shown in A-C, with 100 µM AMPA as agonist. Also under these conditions, BIIR 561 CL reduced the AMPA-induced membrane current (E) compared with the control (D) without markedly altering the current kinetics. F, traces normalized to the peak of the control. G, concentration-response curve for the effects of BIIR 561 CL on the peak (closed symbols) and steady-state responses (open symbols) of glutamate-induced membrane currents. Both IC50 values were comparable (7.6 and 8.8 µM, respectively; n = 3-6 for each data point); moreover, they are in good agreement with those obtained with kainate as agonist (see Fig. 2B).

In a binding study, up to 10 µM BIIR 561 CL did not inhibit the binding of tritiated AMPA to rat cortical cell membranes (n = 3, data not shown).

To test whether BIIR 561 CL also inhibited AMPA receptors in an ex vivo preparation, we characterized the effects in cortical wedges. The application of AMPA to one compartment of the test chamber induced depolarizations in a concentration-dependent and reversible manner, with a concentration for half-maximum depolarization of 5.4 ± 0.6 µM (Fig. 4A). BIIR 561 CL reduced the depolarizations in response to 5 µM AMPA with an IC50 value of 10.8 ± 1.53 µM. GYKI 52466 inhibited the responses with comparable potency (IC50 = 7.8 ± 2.03 µM, Fig. 4B).


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  		Fig. 4.   Inhibition of AMPA responses in the rat cortical wedge preparation. A, concentration-response curve of AMPA in the cortical wedge preparation. AMPA concentration-dependently depolarized the wedges with an EC50 value of 5.3 µM (n = 10). B, effects of BIIR 561 CL on the depolarizations induced by 5 µM AMPA. The compound reduced the effects of AMPA with an IC50 of 10.8 µM (closed symbols, n = 3). The concentration-response curve for GYKI 52466 is given for comparison. Here, the IC50 value was 7.8 µM (open symbols, n = 3).

Effects on Voltage-Gated Sodium Channels. In addition to the effects on AMPA receptors, BIIR 561 CL turned out to be a potent blocker of voltage-gated sodium channels. We investigated this effect electrophysiologically in voltage-clamped primary cultures of rat cortical neurons. Here, trains of step polarizations from a holding potential of -80 to 0 mV were applied with 5-Hz frequency (100 pulses) to the cells under study. In the presence of BIIR 561 CL, the current responses were concentration-dependently and reversibly suppressed (Fig. 5A). Plotting the responses at the last pulse in the trains against the drug concentration gave an IC50 value of 5.2 ± 1.1 µM in this experimental setting (Fig. 5B). Under these conditions, the sodium channel blocker mexiletine had an IC50 value of 84.9 ± 25.4 µM.


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  		Fig. 5.   Effects of BIIR 561 CL on voltage-dependent sodium channels in primary cultured cortical neurons. A, inhibition of voltage-dependent sodium currents by BIIR 561 CL. A train of depolarizing stimuli was applied to the cells under study (100 pulses, 5 Hz frequency, holding potential: -80 mV, test potential: 0 mV) in the absence or presence of increasing concentrations of BIIR 561 CL. The responses to the 100th pulse in each train are shown. B, concentration-dependence of the BIIR 561 CL effect. Under the experimental conditions described in A, the IC50 value for the inhibition of sodium channels was 5.2 µM (closed symbols, n = 5). Mexiletine inhibited sodium currents in this experimental setting with an IC50 value of 84.9 µM (open symbols, n = 4).

Investigations of the effect of BIIR 561 CL on the binding of radiolabeled batrachotoxin to rat brain synaptosomes showed that the compound inhibited the toxin binding with a Ki value of 1.2 ± 0.2 µM (n = 3; nonlinear regression analysis). The Ki value of mexiletine was 21.0 ± 1.9 µM.

In a third set of experiments, we studied the effect of BIIR 561 CL on the veratridine-induced release of glutamate from rat brain slices. BIIR 561 CL inhibited the glutamate release induced by 3 µM veratridine with an IC50 value of 2.3 ± 0.64 µM, whereas 21.8 ± 0.8 µM mexiletine was necessary for the same amount of inhibition (Fig. 6). Thus, the compound exhibits robust effects on voltage-dependent sodium channels in a variety of models using different preparations from the rat CNS.


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  		Fig. 6.   Inhibition of veratridine-induced glutamate release in rat brain slices by BIIR 561 CL. Sodium channels in rat brain slices were stimulated by the application of 3 µM veratridine, and the release of glutamate was measured. BIIR 561 CL reduced the release of glutamate concentration-dependently with an IC50 value of 2.3 µM (closed symbols, n = 8). The reference compound mexiletine affected the release of glutamate with an IC50 value of 21.8 µM (open symbols, n = 4).

Anticonvulsive and Neuroprotective Properties In Vivo. The maximum electroshock test in mice was used to test for anticonvulsive properties of BIIR 561 CL in vivo. The compound was administered s.c. 15 min before the experiments. BIIR 561 CL prevented tonic seizures after the electrical stimulation with an ID50 value of 3.0 mg/kg (calculated as free base; confidence interval, 2.5-3.8 mg/kg; Fig. 7A). GYKI 52466 and mexiletine had ID50 values of 6.9 mg/kg (confidence interval, 5.0-10.0 mg/kg; Fig. 7B) and 2 mg/kg (confidence interval not defined; Fig. 7C), respectively.


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  		Fig. 7.   Anticonvulsive and neuroprotective effects of BIIR 561 CL in vivo. A, the potency of various doses of BIIR 561 CL on the suppression of tonic seizures after maximum electrical shock in mice was tested (). The compound was administered s.c. 15 min before the shock. The ED50 value under these conditions was 3.0 mg/kg (free base). BIIR 561 CL dose-dependently impaired motor function in the mice: the ED50 value in the traction test 15 min after s.c. administration was 34.4 mg/kg (free base, ). Data were obtained from four independent experiments. B, effects of the AMPA antagonist GYKI 52466 in an experimental setting as described in A. Tonic seizures were reduced with an ED50 value of 6.9 mg/kg (). This compound affected motor function with an ED50 value of 14.1 mg/kg (). C, mexiletine was protective against tonic convulsions with an ED50 value of 2.5 mg/kg (). The compound reduced motor function without a clear dose dependence (). D, neuroprotective effect of BIIR 561 CL in a mouse model of focal ischemia. The area of the cortical infarct was reduced from 32.5 mm2 in the controls to 27.2 mm2 (with two doses of 6 mg/kg i.p.) and 21.5 mm2 (with two doses of 60 mg/kg i.p.), respectively. Doses are given as free base. The reduction by the higher dose was statistically significant (P < .01). n = 12-14 for each group.

Possible disturbances of motor coordination induced by BIIR 561 CL were investigated using the traction test in mice. Here, BIIR 561 CL reduced the performance with an ID50 value of 34.4 mg/kg (free base; confidence interval, 26.3-46.7 mg/kg) after s.c. administration (Fig. 7A). GYKI 52466 disturbed motor coordination with an ID50 value of 14.1 mg/kg (confidence interval, 9.9-24.1 mg/kg). The mexiletine-induced effects could not be fitted appropriately (Fig. 7C).

We now investigated whether BIIR 561 CL can reduce the neuronal damage after an ischemic challenge. In a model of focal cerebral ischemia in mice, BIIR 561 CL significantly reduced the cortical infarct area at 60 mg/kg (free base, two injections i.p.) by 44%. With 6.0 mg/kg, there was a trend toward neuroprotection (reduction by 16% of the infarcted area; Fig. 7D).

Thus, BIIR 561 CL demonstrated anticonvulsive and neuroprotective properties in vivo.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The results of the current study demonstrate that BIIR 561 CL inhibits glutamate receptors of the AMPA subtype, as well as voltage-dependent sodium channels, in a variety of preparations. The effects on these ion channels were compared with those of two reference compounds: the noncompetitive AMPA antagonist GYKI 52466, a well-characterized representative of the 2,3-benzodiazepines with anticonvulsant properties (Donevan and Rogawski, 1993; Zorumski et al., 1993, Donevan et al., 1994), and the antiarrhythmic and sodium channel blocker mexiletine, which also has been shown to have anticonvulsive properties (Alexander et al., 1986).

In voltage-clamped cultured neurons from embryonic rat cortex, kainate induced typical membrane currents, which were inhibited by BIIR 561 CL (Fig. 2, A and B). With a 100 µM concentration of the agonist, which is approximately the EC50 value in this system (Weiser and Wienrich, 1996), current responses were inhibited with an IC50 value of 8.5 µM. BIIR 561 CL did not shift the agonist concentration-response curve to higher concentrations (Fig. 2C), which argues against a competitive mode of action.

The noncompetitive AMPA antagonist GYKI 52466 induced comparable effects: The potency was similar to the potency of BIIR 561 CL (IC50 = 12.8 µM); moreover, this compound did not shift the kainate concentration-response curve to the right (Fig. 2, B and C). The ratios between the IC50 values and the applied concentrations of BIIR 561 CL and GYKI 52466 for the experiments shown in Fig. 2C are comparable for both compounds (8.5/22 µM for BIIR 561 CL and 12.8/50 µM for GYKI 52466, respectively), and the two compounds had similar effects on the kainate concentration-response curve. This suggests that BIIR 561 CL, as well as GYKI 52466, inhibits AMPA receptors in a noncompetitive manner. Moreover, the experimental data are clearly different from the mathematically simulated effects of a competitive antagonist on the kainate concentration-response curve (Fig. 2C).

Kainate is a useful tool for the investigation of AMPA receptors. Nevertheless, the kinetics of the responses differ considerably from those with physiological agonists (e.g., glutamate) or agonists that induce quasiphysiological responses (e.g., AMPA). BIIR 561 CL also inhibited current responses induced by these agonists. These experiments showed that the receptor kinetics were not altered by BIIR 561 CL (Fig. 3, A-F) and that the IC50 values for the inhibition of peak and steady-state responses were comparable (Fig. 3G). This finding is in accordance with the effects of other noncompetitive antagonists (e.g., GYKI 52466; Donevan and Rogawski, 1993). In contrast, competitive antagonists have pronounced effects on the current kinetics (Parsons et al., 1994), which is obviously not the case for BIIR 561 CL.

Thus, BIIR 561 CL is representative of a new class of AMPA antagonists. The inhibition is clearly noncompetitive because 1) the inhibition cannot be overcome by high agonist concentrations (Fig. 2C); 2) IC50 values for the inhibition of peak, as well as steady-state, components with glutamate or AMPA as agonists are comparable, which would not be the case for a competitive antagonist (Fig. 3G); and 3) the compound does not inhibit the binding of radiolabeled AMPA rat brain membranes (up to 10 µM).

Moreover, the data obtained from experiments using the cortical wedge preparation show that BIIR 561 CL also inhibits AMPA receptors in an ex vivo preparation from adult rat brain. The application of AMPA concentration-dependently depolarized the wedges, and this effect was suppressed by BIIR 561 CL, as well as GYKI 52466, with comparable potencies (IC50 = 10.8 and 7.8 µM, respectively; Fig. 4B).

Taken together, these data show that BIIR 561 CL inhibits AMPA receptors under various experimental conditions in a noncompetitive manner. The effects are comparable to those of the noncompetitive antagonist GYKI 52466.

In addition to these effects on AMPA receptors, BIIR 561 CL potently affected voltage-dependent sodium channels in a variety of preparations. In voltage-clamped cultured neurons from embryonic rat brain, sodium currents induced by trains of 100 voltage pulses were inhibited with an IC50 value of 5.2 µM (Fig. 5). Under these conditions, mexiletine had an IC50 value of 84.9 µM, showing that the potency was about a factor of 16 higher for BIIR 561 CL. Various compounds, such as local anesthetic agents, have been shown to allosterically interact with the binding of batrachotoxin to the sodium channel pore (Postma and Catterall, 1984). BIIR 561 CL inhibited the binding of radiolabeled batrachotoxin to rat brain membranes (IC50 = 1.2 µM), and the same was true for mexiletine (IC50 = 21.0 µM). One might hypothesize that both compounds, like local anesthetic agents, allosterically interact with the binding site for batrachotoxin. The ratio between the IC50 values for the two compounds was about 17 in this model and thus correlates well with the findings from the voltage-clamp experiments.

The data obtained so far were confirmed in another functional model using an ex vivo preparation from adult rat brain: veratridine stimulates the release of glutamate from brain slices via the activation of presynaptic sodium channels. This release was suppressed by BIIR 561 CL (IC50 = 2.3 µM), as well as mexiletine (IC50 = 21.8 µM; Fig. 6). The ratio between the IC50 values was approximately 10 in this experimental setting. Thus, BIIR 561 CL inhibited voltage-dependent sodium channels in a variety of preparations with 10- to 20-fold higher potency compared with mexiletine. The affinity is in the range that has been reported for other putative neuroprotective or anticonvulsive sodium channel blockers like phenytoin (Lang et al., 1993, Rataud et al., 1994, Brown et al., 1995), BW619 (Xie and Garthwaite, 1996), or lamotrigine (Xie et al., 1995, Kuo and Lu, 1997).

Bearing the promising in vitro properties of BIIR 561 CL in mind, one should postulate that the compound should be effective in models for anticonvulsive drug action in vivo. This was, indeed, the case. After s.c. administration, BIIR 561 CL suppressed tonic seizures in the maximum electroshock test with an ED50 value of 3.0 mg/kg, providing evidence that the compound crosses the blood-brain barrier and shows the expected in vivo effect. At higher doses, the compound also induced impairment of motor function (Fig. 7). The ratio of the ED50 values for the maximum electroshock and the traction test, however, was about 11 (34.4 versus 3.0 mg/kg). For the noncompetitive AMPA antagonist GYKI 52466, this ratio was only about 2 (6.9 versus 14.1 mg/kg), which is in good agreement with data from the literature (Donevan et al., 1994). It was not possible to make this comparison for the sodium channel blocker mexiletine because the impairment of motor function was not clearly dose dependent. This might be explained by the effects of this compound on peripheral sodium channels (e.g., in skeletal muscle). It can therefore be hypothesized that the dual mechanism of action of BIIR 561 CL might provide a larger "safety margin" for anticonvulsant or neuroprotective therapy compared with side effects at higher doses (like disturbances of motor coordination).

In addition to this anticonvulsive effect, BIIR 561 CL reduced the cortical infarct area in a mice model of focal ischemia (Fig. 7D). Thus, BIIR 561 CL might be a member of a promising new compound class for anticonvulsive and neuroprotective therapy.

    Acknowledgments

We are grateful to the skillful technical assistance of B. Reich, H. Wölfel, K. Kappertz, M. Vogt, G. Mengeling, S. Kurtze, E. Weghofer, P. Heubeta lein-Hoffmann, M. Schiavone, F. Berton, and A. Baschirotto. The mathematical support by G. Weckesser (Department of Research and Development Coordination, Boehringer Ingelheim) is highly acknowledged. We also thank G. D. Bartoszyk for critical reading of the manuscript.

    Footnotes

Accepted for publication February 8, 1999.

Received for publication September 28, 1998.

Send reprint requests to: Dr. T. Weiser, Department of CNS Research, Boehringer Ingelheim Pharma KG, D-55218 Ingelheim, Germany. E-mail: weiser{at}ing.boehringer-ingelheim.com

    Abbreviations

aCSF, artificial cerebrospinal fluid; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BIIR 561 CL, dimethyl-{2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxy]ethyl}-amine hydrochloride; CNS, central nervous system; GYKI 52466, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine.

    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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