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(Stroke. 1995;26:1683-1690.)
© 1995 American Heart Association, Inc.

Articles

SB 201823-A Antagonizes Calcium Currents in Central Neurons and Reduces the Effects of Focal Ischemia in Rats and Mice

F. C. Barone, PhD; P. G. Lysko, PhD; W. J. Price; G. Feuerstein, MD; K. A. Al-Baracanji, PhD; C. D. Benham, PhD; D. C. Harrison; M. H. Harries, PhD; S. J. Bailey A. J. Hunter, PhD

From SmithKline Beecham Pharmaceuticals, King of Prussia, Pa (F.C.B., P.G.L., W.J.P., G.F.), and New Frontiers Science Park, Harlow, UK (K.A.Al-B., C.D.B., D.C.H., M.H.H., S.J.B., A.J.H.).

Correspondence to Frank C. Barone, PhD, Department of Cardiovascular Pharmacology, UW2521, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406-0939.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Excessive calcium entry into depolarized neurons contributes significantly to cerebral tissue damage after ischemia. We evaluated the ability of a novel neuronal calcium channel blocker, SB 201823-A, to block central neuronal calcium influx in vitro and to reduce ischemic injury in two rodent models of focal stroke.

Methods Patch-clamp electrophysiology and intracellular Ca²⁺ imaging in rat hippocampal and cerebellar neurons were used to determine effects on neuronal calcium channel activity. Middle cerebral artery occlusion was performed in Fisher 344 rats and CD-1 mice to determine the effects on rodent focal ischemic injury and neurological deficits. Cardiovascular monitoring in conscious rats was conducted to determine cardiovascular liabilities of the compound.

Results In cultured rat hippocampal cells, calcium current measured at plateau was reduced by 36±8% and 89±4% after 5 and 20 µmol/L SB 201823-A, respectively. In cerebellar granule cells in culture, pretreatment with 2.5 µmol/L SB 201823-A totally prevented initial calcium influx and reduced later calcium influx by 50±2.5% after N-methyl-D-aspartate/glycine stimulation (P<.01). KCl depolarization–induced calcium influx also was reduced by more than 95%. In rats, a single treatment with 10 mg/kg IV SB 201823-A beginning 30 minutes after focal ischemia decreased (P<.05) hemispheric infarct by 30.4% and infarct volume by 29.3% and reduced (P<.05) forelimb deficits by 47.8% and hindlimb deficits by 36.3%. In mice, treatments with 10 mg/kg IP SB 201823-A beginning 30 minutes after focal ischemia significantly reduced infarct volume by 41.5% (P<.01). No blood pressure effects were observed with the therapeutic dose of the compound.

Conclusions These results indicate that the new neuronal calcium channel blocker SB 201823-A can block stimulated calcium influx into central neurons and can provide neuroprotection in two models of focal cerebral ischemia without affecting blood pressure. Data from several different studies now indicate that the neuronal calcium channel antagonists are a promising therapy for the postischemic treatment of stroke.

Key Words: calcium channel blockers • cerebral ischemia, focal • neuroprotection • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
High intracellular calcium concentrations that occur during brain ischemia contribute to neuronal cell death.^{1 2} In ischemia, the depletion of neuronal ATP results in an inability to maintain ion gradients, leading to membrane depolarization^{1 2} and excessive excitatory amino acid release (eg, glutamate release^{3 4 5} ). The excessive glutamate stimulation and membrane depolarization opens VOCCs and results in toxic neuronal calcium overload.^{1 2} This "excitotoxic hypothesis" of neuronal mortality in cerebral ischemia is now widely accepted.^{6 7 8} Attempts to restrict depolarization and/or resolve the toxic calcium influx have been directed at all stages of the process: excitatory amino acid antagonists, Na⁺ channel modulators, presynaptic modulators of transmitter release (adenosine agonists), and calcium channel antagonists.⁹ The strongest evidence demonstrates that glutamate receptor antagonists can reduce neuronal injury in models of cerebral focal ischemia.^{9 10 11 12 13}

Attempts to develop calcium antagonists as a treatment for cerebrovascular disease originated from the successful clinical use of these agents for cardiovascular diseases such as hypertension and coronary disease.¹⁴ In neuronal tissue, there are at least four types (L, N, P, and T) of functionally identified calcium channels,^{15 16 17} and molecular biology has defined at least five classes of calcium channels that could admit large amounts of calcium into depolarized neurons.^{15 18} The L-type calcium channel is important for excitation-response coupling in cardiac muscle, smooth muscle, and neurons and is blocked by dihydropyridines (eg, nifedipine, nimodipine) and other organic (eg, diltiazem and verapamil) VOCC blockers. In smooth muscle and cardiac muscle, the L-type channel is the major influx route for calcium ions,¹⁹ and brain-penetrating dihydropyridines such as nimodipine and nicardipine have shown variable efficacy in in vivo models of ischemia.^{14 20} The N-type channel is important in neuronal transmission and is blocked by -conotoxin (GVIA).¹⁹ A peptide blocker of the neuronal N channel has been shown to be effective in at least one model of stroke when given up to 6 hours after ischemia.²¹ To date, VOCC antagonist selectivity for neuronal versus vascular tissue has been poor, and the available VOCC antagonists have limited efficacy against all neuronal channels. These factors help explain the variable efficacy of current VOCC antagonists in cerebral ischemia and/or stroke.¹⁴

Since many different VOCCs are present in neuronal tissue, compound(s) with broader neuronal VOCC activity may be required to provide efficacy in cerebral ischemia. Precedent for such a potent and selective compound is available in the spider toxin -Aga 1A,²² which appears to block all high-voltage activated channels in dorsal root ganglion neurons at low nanomolar concentrations while having little activity on sodium channels in the same cells. Recently, nonpeptide calcium channel antagonists have been developed that show broad activity against dorsal root neuronal calcium channels and neuroprotective efficacy when administered after cerebral ischemia.^{23 24}

SB 201823-A (4-[2(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride) is a molecule that reduces calcium currents in dorsal root neurons and has been shown to provide significant neuroprotection in cerebral ischemia (ie, it reduces CA1 cell damage in gerbil transient forebrain ischemia and reduces cerebral tissue infarction in rat photothrombotic stroke).^{24 25} The purpose of the present series of experiments was to examine the efficacy of SB 201823-A in blocking calcium currents of central neurons (both hippocampal and cerebellar) and in reducing the histological and neurological consequences of focal stroke in both rats and mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare, Bethesda, Md). All procedures using lab animals were reviewed by an internal ethics committee and approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Hippocampal Central Neurons
Primary cultures of rat hippocampal neurons were prepared from fetal rats. Pregnant rats were killed by cervical dislocation. The amniotic sac containing the embryos was surgically removed from the mother and placed into a sterile petri dish. Embryos were decapitated, and the heads were placed in cold KRB that contained (in mmol/L) NaCl 120, KCl 4.8, KH₂PO₄ 1.25, NaHCO₃ 25, and glucose 14. Hippocampi were suspended in 10 mL KRB containing 2.5 mg trypsin and incubated at 37°C for 10 minutes, being swirled occasionally. A further 10 mL KRB containing 7.5 mg deoxyribonuclease I was added, mixed by inversion, and then centrifuged at 1500 rpm for 10 seconds in a Beckman TJ6 centrifuge. The mixture was aspirated, and the pellet was resuspended in 3 mL of medium. Tissue was triturated through a 1-mL blue plastic Gilson tip 10 times and then filtered through a cell strainer, washing the container out with 5 mL medium. Cells were counted in a hemocytometer and diluted to 1.5x10⁶ cells per mL, and 2 mL was plated out onto coverslips coated with polyethylenimine (50 mg/mL in Tris buffer, pH 8.3, diluted 1:100 with H₂O, soaked for at least 2 hours and rinsed with H₂O). Cells were incubated at 37°C until required. Glial growth was arrested after 7 days in culture by the addition of cytosine arabinoside (10 µmol/L final concentration).

For electrophysiological measurement of Ca²⁺ current, patch-clamp recordings were made from 7-day-old hippocampal neurons in culture as described previously.²⁴ The charge carrier was 10 mmol/L Ba²⁺. All experiments were performed at room temperature. SB 201823-A was dissolved in DMSO as a 20-mmol/L stock from which 5- and 20-µmol/L dilutions were made with the external solution for recording calcium channel currents. Maximum DMSO concentrations (0.1%) have been shown previously to have no effect on Ca²⁺ currents.

Cerebellar Central Neurons
Primary cultures of rat CGC neurons were prepared from 8-day-old Sprague-Dawley rat pups (Taconic Farms) and were used after 8 to 12 days in culture by washing and incubating in a buffer composed of (in mmol/L) NaCl 154, KCl 5.6, CaCl₂ 2.3, glucose 5.6, and HEPES 8.6, adjusted to pH 7.4 with NaOH. We have previously characterized the pharmacology of the NMDA response in these neurons, which offers a glutamate excitotoxicity model that is dependent on compromised energy levels and relief of the voltage-dependent Mg²⁺ block.^{26 27 28 29 30}

For measurements of [Ca²⁺]i, cells were grown on 14-mm² ACLAR plastic coverslips³¹ (Allied-Signal Engineered Plastics; available from Pro Plastics Inc) in 60-mm dishes (Nunc), loaded with 2 µmol/L fura 2-AM (Calbiochem) in the above buffer for 1 hour, and allowed to equilibrate at 37°C for 5 minutes in fresh buffer. Fluorescence of fura 2 in cell monolayers immersed into 2 mL of buffer in a stirred cuvette was measured as previously described^{30 31 32} with a University of Pennsylvania Biomedical Instruments Group dual-channel fluorometer. Data were captured on-line as voltage recordings with the aid of a personal computer running the LABWINDOWS application (National Instruments) under Microsoft WINDOWS (Microsoft Corp) and were transferred to a Macintosh terminal for analysis by IGOR version 1.2 software (WaveMetrics).

Rat Focal Ischemia
Methods were as described previously.^{23 33} Briefly, Fisher F-344 rats, with free access to food and water and weighing 250 to 350 g, were anesthetized with halothane. Right MCAO by electrocoagulation (Aspen Labs Inc; MF 180 Electrosurgical Unit) was performed at the level of the inferior cerebral vein under stereotaxic control. Right CCAO was performed immediately by double ligation and transection. The right femoral artery was cannulated for drug or vehicle administration. SB 201823-A (10 mg/kg IV, dose expressed as the free base) was dissolved in 3 mL of 20% DMSO/saline and administered over 1 hour beginning 30 minutes after MCAO. The effective dose for this compound was selected on the basis of previous data.²⁴ Control animals received 3 mL of 20% DMSO/saline administered over 1 hour beginning 30 minutes after MCAO. Body temperature was maintained at 37°C throughout surgery and drug administration with a heating pad. Animals were allowed to recover from anesthesia under a heating lamp to maintain normal body temperature and were then returned to their cages.

At 24 and 48 hours after MCAO, a neurological examination was performed. Each animal was classified as one of four neurological grades.³⁴ The grades of 0 (no observable deficit), 1 (any amount of consistent contralateral forelimb flexion), 2 (reduced resistance to lateral push toward the paretic, contralateral side), or 3 (circling behavior toward the paretic side) basically defined the degree of contralateral hemiparalysis that occurs as a consequence of focal ischemia and the associated ipsilateral hemispheric infarct. A hindlimb placement test^{23 33} was performed for each rat. In this test, the rat is held facing away from the edge of a table, and the contralateral hindlimb is pulled over the edge of the table and extended downward. A normal response seen in nonsurgically treated animals or ipsilateral to the cerebral surgery is an immediate placement of the hindlimb back onto the table, thus appropriately coordinating sensory/motor stimuli. An abnormal response is no limb placement and/or movement.

At 48 hours, animals were killed by an overdose of sodium pentobarbital, and their brains were removed; seven coronal forebrain slices (2 mm thick) were made from the level of the olfactory bulbs to the cortical-cerebellar junction. Slices were immersed immediately in a 1% solution of triphenyltetrazolium chloride in phosphate buffer at 37°C for 20 to 30 minutes³⁵ and then fixed by infiltration in 10% phosphate-buffered formalin. Both sides of each triphenyltetrazolium chloride–stained section were photographed in color using a Polaroid camera and analyzed for the quantification of ischemic damage using an image analysis system (Amersham RAS 3000; Loats Associates Inc). Morphological changes after surgery were evaluated in the entire forebrain (total of 14 planar surfaces) for each animal as described previously.^{23 33} Hemispheric swelling, infarct size, and infarct volume were determined for each slide. Hemispheric swelling, which was expressed as the percent increase in size of the ipsilateral hemisphere over the contralateral hemisphere, was calculated as ipsilateral hemispheric area minus contralateral hemispheric area divided by contralateral hemispheric area multiplied by 100. Infarct size was expressed as the percent infarcted tissue in reference to the contralateral hemisphere and was calculated as infarct area divided by contralateral hemispheric area multiplied by 100. The total volume of infarction was also calculated by summation of the infarct area (in square millimeters) from all the brain planar images that were considered 1 mm thick.

Mouse Focal Ischemia
Adult male CD1 mice (Charles River Labs) in the weight range of 25 to 30 g were anesthetized with 250 mg/kg IP tribromoethanol. Focal cerebral ischemia was induced as follows. A temporal approach was adopted to occlude the right MCA. With the aid of an operating stereomicroscope, an incision was made between the outer canthus of the eye and the external auditory meatus. The temporalis muscle was bisected and retracted to expose the temporolateral surface of the skull. The MCA was exposed by means of a burr-hole craniotomy. A thin layer of bone was preserved to protect the dura mater and cortex surface from mechanical damage and thermal injury. Remaining bone was removed with watchmaker's forceps. The dura immediately overlying the MCA was cut with fine-gauge needles and removed, and the MCA was occluded distal to the branch point by microbipolar diathermy (Surgitron, Ellman International UK Ltd) and severed to ensure the occlusion was complete. The temporalis muscle and skin incision were then sutured.

SB 201823-A (10 mg/kg IP) was dissolved in 10% ß-cyclodextrin in isotonic saline and administered 30 minutes after MCAO. Further doses were administered bi-daily for 3 days after MCAO. Control animals received 10% ß-cyclodextrin in saline administered under an identical dosing regimen.

Throughout preparation and during the surgical procedure, body temperature was maintained at 37±1°C with a heating blanket with feedback control. After the surgical procedure, mice were maintained in an environmental temperature of 37°C for 120 minutes, during which time they recovered from the anesthetic, and were then returned to normal housing.

After recovery, body weight was monitored daily. Four days after ischemia, mice were killed with a lethal dose of sodium pentobarbitone, and their brains were removed and stored in neutral buffered formalin. Preliminary studies (data not shown) indicated that lesions were mature by day 4 after occlusion and edema was minimal. Brains were fixed for a minimum of 48 hours before processing for histological examination. Coronal sections (60 µmol/L) were taken and stained with cresyl fast violet. Infarct volume was determined planimetrically using a Quantimet 920 image analysis system (Cambridge Instruments) and computed using Simpson's rule.²⁴

Cardiovascular System
Adult male Hooded-Lister rats (Charles River), 400 to 500 g body weight, were anesthetized with a mixture of fentanyl-medetomidine hydrochloride (10:0.5 vol/vol, 6.3 mL/kg). Medical-grade Tygon catheters were implanted in the abdominal aorta for recording blood pressure and in the vena cava (through the femoral artery) for administration of drug or vehicle. Twelve days after this surgical preparation, animals were placed in a restrainer cage, and mean arterial blood pressure in millimeters of mercury and heart rate in beats per minute were recorded for 1.5 hours, during which steady-state recordings were observed. Blood pressure and heart rate were then continuously recorded from these conscious rats for 30 minutes before SB 201823-A administration, during 30 minutes of SB 201823-A administration (total dose, 10 mg/kg IV), and for an additional 60 minutes after drug administration. Vehicle was administered to rats in a similar manner for comparative purposes.

Statistical Analysis
Data were expressed as mean±SEM. For nonparametric data (ie, the hindlimb placement test), the ² test was used.³⁶ For all the parametric data, comparisons were made using an ANOVA and/or t test.³⁷ Statistical significance was accepted when P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hippocampal Neuronal Calcium Currents
Electrophysiological studies in cultured hippocampal cells indicated peak inward barium currents ranging from 0.4 to 0.7 nA (n=4). SB 201823-A was applied after the current had stabilized. SB 201823-A caused a dose-dependent reduction in current, but current subsequently returned to approximately 40% of control after washout (Fig 1A). A dose of 5 µmol/L caused 36±8% reduction in current measured at the end of a 100-millisecond test depolarization after 3 minutes of contact time. This is a conservative estimate of block, since 3 minutes does not allow equilibrium to be attained but limits the degree of rundown of the current. Current was further reduced by 89±4% relative to control after application of 20 µmol/L SB 201823-A. Fig 1B shows a plot of peak current evoked at 0 mV against time for one cell. The rapid onset of block after application of 20 µmol/L SB 201823-A is clearly evident. Recovery after washout was slow and incomplete after 6 minutes in this cell.

View larger version (11K): [in this window] [in a new window]   Figure 1. Graphs show dose-dependent block of calcium currents in hippocampal neurons by SB 201823-A. A, Leak subtracted traces from a single hippocampal neuron depolarized from -80 mV to 0 mV for 100 milliseconds. Control current (a) and the effects of 5 µmol/L (b) and 20 µmol/L (c) SB 201823-A followed by washout (d) are displayed. Calcium currents were reduced by 36% and 89% of control values after 3-minute applications of 5 and 20 µmol/L SB 201823-A, respectively, and recovered to 40% of control after washout. B, Peak calcium current amplitude plotted against time from the same cell. Application of two doses of SB 201823-A and washout are indicated by vertical bars. Currents were evoked by depolarizing from a holding potential of -80 mV to 0 mV every 15 seconds.

Cerebellar Neuronal Calcium Flux
Measurements of [Ca²⁺]_i in cultured CGC neurons gave resting values of 153.4±12.8 nmol/L (n=7), similar to our and others' recently published values.^{30 31 32} Stimulation with 5 µmol/L glycine and 100 µmol/L NMDA increased [Ca²⁺]_i threefold to 469±45 nmol/L (n=3) over basal values, providing sustained levels of [Ca²⁺]_i (Fig 2A). Depolarization of neurons with 50 mmol/L KCl caused a sharp increase in [Ca²⁺]_i, which stabilized to a net increase of 192±9 nmol/L (n=5). Pretreatment with 2.5 µmol/L SB 201823-A diminished the steady-state NMDA/glycine-stimulated [Ca²⁺]_i by an average of 50±3% (n=3, P<.01) and totally prevented the initial [Ca²⁺]_i spike as indicated by Fig 2B. In addition, subsequent increases in [Ca²⁺]_i elicited by KCl depolarization were diminished to an average of 8.1±3.7% of untreated controls (Fig 2B; P<.01).

View larger version (14K): [in this window] [in a new window]   Figure 2. Graphs show inhibition of SB 201823-A of KCl- and NMDA/glycine-induced increases in [Ca2+]i in cultured rat cerebellar granule cells. A, Normal response of untreated neurons. B, 2.5 µmol/L SB 201823-A was added to neurons just before the addition of 5 µmol/L glycine (GLY) and 100 µmol/L NMDA. The NMDA/glycine-induced increases in [Ca2+]i were inhibited 50%, and the KCl-induced increase in [Ca2+]i was inhibited by 92%.

Rat Focal Ischemic Injury
A dose of SB 201823-A that was predicted to be neuroprotective on the basis of previous data was studied.⁷ Fig 3A and 3B illustrate the neuroprotective effect of SB 201823-A. A dose of 10 mg/kg IV administered beginning 30 minutes after MCAO significantly (P<.05) decreased hemispheric infarct by 30.4% and infarct volume by 29.3%. SB 201823-A did not significantly affect hemispheric swelling. Fig 4A and 4B demonstrate the reduction in infarction throughout the forebrain after SB 201823-A administration. At 24 and 48 hours after surgery, SB 201823-A significantly (P<.05) reduced the neurological grade by 40% and 47.8%, and the hindlimb placement test score was significantly (P<.05) reduced by 36.3% at 48 hours after surgery (Fig 5A and 5B).

View larger version (11K): [in this window] [in a new window]   Figure 3. Bar graphs show hemispheric infarct (A) and infarct volume (B) for rats treated with vehicle (n=23) and SB 201823-A (n=18) after focal ischemia. SB 201823-A significantly reduced the cortical infarctions produced by focal ischemia. *P<.05.

View larger version (17K): [in this window] [in a new window]   Figure 4. Plots show profiles of hemispheric infarcts (A) and infarct areas (B) for the same animals as in Fig 3 presented as a function of different forebrain section surfaces estimated at various distances from the skull landmark bregma. Measurements for animals treated with vehicle () and SB 201823-A () are depicted.

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graphs show neurological grade (A) and hindlimb placement (B) results for vehicle- and SB 201823-A–treated animals (same as in Figs 3 and 4). Results at 24 hours and 48 hours after surgery are indicated by solid and hatched bars, respectively. SB 201823-A significantly reduced neurological deficits produced by focal ischemia. *P<.05.

Mouse Focal Ischemic Injury
The dose regimen of SB 201823-A that was observed to be neuroprotective in other models of stroke⁷ was studied. Fig 6 illustrates the neuroprotective effect of SB 201823-A. This dose regimen of 10 mg/kg beginning 30 minutes after MCAO decreased lesion volume by 41.5% (P=.01).

View larger version (11K): [in this window] [in a new window]   Figure 6. Bar graph shows infarct volume for mice treated with vehicle (n=7) and SB 201823-A (n=10) after focal ischemia. SB 201823-A significantly reduced cerebral infarctions produced by focal ischemia. *P<.01.

Cardiovascular System
Intravenous administration of the effective dose (10 mg/kg) of SB 201823-A did not produce any effects on the blood pressure of conscious rats (Fig 7A). SB 201823-A did produce a relatively small and transient reduction in heart rate (Fig 7B). Neither measure was affected by administration of the vehicle in the same manner.

View larger version (14K): [in this window] [in a new window]   Figure 7. Graphs show blood pressure (A) and heart rate (B) effects for vehicle (n=4) and SB 201823-A (n=5) delivered intravenously from 0 to 30 minutes to conscious rats. SB 201823-A did not affect blood pressure and produced a small, transient reduction in heart rate. *P<.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although calcium antagonists often show neuroprotection when given before ischemia, positive results with postischemic dosing have not been consistently reported.^{14 20} Since the L-type calcium channel is only one of a number of VOCCs that could contribute a toxic calcium influx to a chronically depolarized neuron, this is not surprising. N-type and P-type channels (and possibly others) might also contribute to toxic calcium influx.

In addition to entry through VOCCs, calcium influx can also occur directly through excitatory amino acid gated ion channels, both NMDA gated and non-NMDA gated.³⁸ NMDA receptor antagonists, such as MK-801, are effective after ischemia in focal models of ischemia, but much more variable effects are seen in global ischemia models, with positive results attributed to the hypothermic effects of these compounds.³⁹ Recently, attention has switched to antagonists of non-NMDA receptors with the encouraging results for NBQX, a quisqualate receptor antagonist.¹¹ It seems clear that calcium influx occurs through both NMDA and quisqualate gated channels. However, parallel to this influx will be calcium influx through VOCCs, providing a viable alternative target to the excitatory amino acid receptors.

There is a strong rationale for examining the potential of calcium antagonists with a broader selectivity that can inhibit calcium influx through all high-voltage activated neuronal calcium channels. We have previously used sensory neurons cultured from rat dorsal root ganglia as a convenient assay because they express the same range of calcium channels identified pharmacologically in central neurons.⁴⁰ The dorsal root ganglion high-threshold calcium current can be divided into P (23%), N (43%), L (18%), and remaining (18%) components.⁴¹ Our rat dorsal root ganglion neuronal cultures expressed similar proportions of N and L currents.²⁴ We previously identified SB 201823-A as a compound that was able to block almost all voltage-gated calcium current in these neurons.²⁴ SB 201823-A was also able to reduce calcium influx into cortical synaptosomes, suggesting efficacy in blocking presynaptic central calcium channels.²⁴ These initial studies indicated that SB 201823-A would be able to limit calcium influx through a range of VOCCs in depolarized central neurons. The present studies extend these observations and clearly demonstrate that SB 201823-A blocks VOCCs recorded from cultured hippocampal neurons. In cultured CGC, SB 201823-A blocks [Ca²⁺]_i. We have previously shown in CGC that prior treatment with 5 µmol/L nifedipine, nicardipine, or nimodipine to inhibit voltage-sensitive L-type calcium channels lowered the total NMDA/glycine-stimulated [Ca²⁺]_i responses.³⁰ Dihydropyridines did not significantly change the basal intracellular calcium levels but did significantly lower the NMDA/glycine-stimulated response to 64% of that of untreated controls. Those results suggested that NMDA-mediated depolarizations do indeed open voltage-sensitive calcium channels, which contribute to the overall calcium influx. Here we have noted that the novel piperidine SB 201823-A also significantly lowered the NMDA/glycine-stimulated response in CGC neurons. These results suggest that this class of calcium channel blocker can also prevent in vivo ischemic neuronal damage by indirect interference with glutamate excitotoxicity. The ability of SB 201823-A to almost totally inhibit sustained KCl-induced calcium influx into CGC neurons is consistent with its action as a potent VOCC.

Previously, we demonstrated that in two in vivo models of cerebral ischemia postischemic dosing with SB 201823-A was effective in reducing neuronal death.²⁴ SB 201823-A treatment resulted in a significantly higher survival of pyramidal cells in the CA1 region after transient forebrain ischemia in the gerbil and in 75% to 85% protection against lesion development in the rat photothrombotic model. The present in vivo studies demonstrate that SB 201823-A can also produce a significant reduction in focal ischemic damage in both mice and rats. In addition, the present data in rat focal ischemia demonstrate significant reduction in neurological deficits that parallels the reduced cerebral infarction due to SB 201823-A administration. Similar but less dramatic reduction of neurological deficits in focal ischemia²³ and neuroprotection in global ischemia⁴² have been demonstrated for another neuronal calcium channel blocker. SB 201823-A was effective when administered after ischemia, as would be necessary in the acute-phase treatment of human focal stroke. Since the compound appears to act at more than one neuronal channel, it may provide cerebral protection after MCAO by blocking both the calcium-dependent neurotransmitter release (ie, excitotoxic amino acids such as glutamate) as well as toxic calcium influx produced by the concomitant tissue depolarization during focal ischemia.² Certainly, more work needs to be done in evaluating repetitive dosing to extend the duration of therapy on increasing efficacy of the compound. Also, the maximal delay time before the treatment effect is lost should be evaluated. Finally, although SB 201823-A does exhibit protection in gerbil transient forebrain ischemia, a model of ischemia with reperfusion,²⁴ its effects in MCAO with reperfusion should also be determined in future studies.

The protective effects of SB 201823-A after MCAO in the rat are modest but consistent in both neurological deficits and tissue injury without any noticeable side effects. NMDA-receptor antagonism can produce protection, but it also exhibits dramatic side effects associated with its central nervous system activity.^{10 23} In the mouse MCAO model, glutamate antagonism and peripherally active calcium channel blockers failed to exhibit any efficacy (data not shown), suggesting greater efficacy with SB 201823-A than with more traditional neuroprotective compounds. Also, in the gerbil transient forebrain ischemia model, postischemic SB 201823-A administration is effective,²⁴ but postischemic glutamate antagonist administration provides no protection (data not shown). It is possible that glutamate receptor antagonism combined with the present neuronal calcium channel approach might be very efficacious. For example, glutamate receptor antagonism greatly reduces glycine/NMDA-stimulated calcium fluorescence (influx) in CGC neurons even after dihydropyridine pretreatment.^{30 32}

SB 201823-A is neuroprotective at a dose that is virtually devoid of blood pressure effects in conscious rats. Although heart rate was decreased by the compound, this effect was relatively minor and transient. Clearly, at the therapeutic dose in the rat, this compound has much less effect on blood pressure than previously used calcium antagonists, suggesting much less cardiovascular liability that would limit the dose in humans.

In conclusion, we have extended the initial evaluation of a broad-spectrum neuronal calcium channel antagonist. SB 201823-A blocks calcium currents in sensory and central neurons and in a model of synaptic activity. It also displays significant neuroprotective activity in several different experimental models and species when administered after cerebral ischemia without affecting blood pressure. SB 201823-A also significantly reduces the neurological dysfunction that follows experimental focal stroke but does not exhibit any obvious neurological and/or behavioral side effects. These data suggest that this therapeutic approach, exemplified by SB 201823-A, may be useful in stroke patients.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular calcium concentration CCAO = common carotid artery occlusion CGC = cerebellar granule cell DMSO = dimethyl sulfoxide KRB = Krebs-Ringer bicarbonate buffer MCA = middle cerebral artery MCAO = middle cerebral artery occlusion NMDA = N-methyl-D-aspartate VOCC = voltage-operated calcium channel

	Acknowledgments

 
The authors thank Shirley Wilson and Linda Meoli for careful preparation of this manuscript.

Received June 30, 1994; revision received March 13, 1995; accepted May 5, 1995.

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