Introduction

Abstract Introduction Methods Procedures Results Discussion Conclusion References

The discovery that a major underlying mechanism for neuronal damage after anoxia/ischemia is related to the massive release and postsynaptic action of excitatory amino acid transmitters (Olney, 1978) has led to a host of pharmacological investigations that have demonstrated a therapeutic role for the modulation of the NMDA subtype of the glutamate receptor (Pulsinelli and Buchan, 1990; Gee, 1994; Harris, 1995). In focal models of cerebral ischemia, NMDA receptor antagonists are effective in reducing cortical infarct volume, but findings regarding beneficial actions in animal models of global ischemia remain controversial (Buchan, 1990). Moreover, MK801 and the other high affinity uncompetitive NMDA receptor antagonists cause numerous side effects including motor hyperactivity and ataxia, deficits in learning and the potential for phencyclidine-like abuse. As a result, low therapeutic indices are characteristic for this drug group (Rogawski, 1992). With the discovery of low affinity uncompetitive NMDA receptor antagonists such as remacemide hydrochloride (Palmer et al., 1995a), ARL 13950AA (Palmer et al., 1995b), ADCI (Rogawski et al., 1991) and HU-211 (Biegon and Joseph, 1995) many of these unfavorable attributes of the high affinity antagonists are surmounted (Rogawski, 1992).

In our laboratories a rational chemical synthesis and screening approach to discover useful, safe, single enantiomeric forms of low affinity uncompetitive NMDA receptor antagonists identified ARL 15896AR [(S)-alpha-phenyl-2-pyridine-ethanamine dihydrochloride, formerly designated FPL 15896AR] (fig. 1). In addition to its neuroprotective activity in rodent models of global and focal ischemia, ARL 15896AR is nonsedative. It also lacks phencyclidine-like stereotyped behavior (open field observations) nor, unlike phencyclidine, will rats self administration ARL 15896AR. Furthermore, ARL 15896AR does not influence either motor activity or learning (Hudzik and Palmer, 1995; Hudzik et al., 1996; Palmer et al., 1996). In addition, ARL 15896AR enters the brain rapidly and exhibits a favorable pharmacokinetic profile (Mahmood et al., 1994). With the successful completion of 30-day toxicology evaluation and phase I of clinical trials, phase IIa of clinical testing in stroke patients is in progress. Our investigation using rodent models of anoxia/ischemia details the preclinical work to date which provided the rational for ARL 15896AR to be tested in patients.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structure of ARL 15896AR

	Methods

Abstract Introduction Methods Procedures Results Discussion Conclusion References

Experimental Subjects

Experimental protocols as well as housing and care of animals were in accordance with stringent requirements established by the Institutional Animal Care and Use Committees from the centers involved in the various studies. All drug doses are calculated to the free base form of ARL 15896AR.

Mice. For hypoxia studies male CF1 mice weighing 18-30 g were purchased from Harlan Laboratories (total used = 170).

Rats. Young adult male Sprague Dawley rats weighing approximately 140 to 200 g used for the hypoxia investigation were obtained from Harlan Laboratories. The weight range of the male Sprague Dawley rats (Harlan Laboratories) used for the 4-vessel occlusion studies varied from 275 to 350 g. For the 2-vessel occlusion models, adult male Sprague Dawley rats weighing from 250 to 350 g, were obtained from either Harlan Labs (Astra Arcus USA study) or Charles River, Montreal (University of Manitoba studies). For the anoxia/global ischemia investigations rats were maintained on low vitamin E content Rodent Lab Chow (Teklad no. 5001, Harlan) (total Sprague Dawley rats used = 133). SHR weighing from 230 to 280 g (Charles River, Montreal, Canada) were used for the focal ischemia experiments (total used = 90). Rats used in the various ischemia investigations were fasted overnight before surgery.

Gerbils. Female Mongolian gerbils weighing 50 to 70 g were obtained from High Oak Ranch, Goodwood, Ontario, Canada (total used = 46).

Global Ischemia Models

Hypoxia studies: mice. Testing for efficacy of compounds to extend survival time of mice exposed to an hypoxic environment has been described (Palmer et al., 1995b). Separate groups of mice received different doses of ARL 15896AR (10 per dose, 4-6 doses). Five min after i.v. dosing (5, 10, 20 and 25 mg/kg) and 20 min after oral dosing (5, 15, 25, 50, 62.5 and 75 mg/kg) the animals were placed in a plexiglass chamber consisting of five interconnecting compartments. This apparatus was in turn placed into a QUEUE environmental chamber maintained at 35 ± 0.2°C. The mice remained in the QUEUE chamber for 10 min before induction and throughout the period of hypoxia. Hypoxia consisted of allowing a gas mixture (96% N₂/4% O₂) to flow into the individual compartments at a pressure of 1.79 kg/cm² (10 pounds per square inch) and at a flow rate of 0.01 m³/min. The time to last gasp was monitored with an electric timer controlled by an observer viewing the animal through a glass door. The environmental chamber eliminates drug-induced hypothermia which produces a "false positive" extension in survival time (Milde, 1988). Care was also taken to reduce AM/PM bias by running the experiments between 09:00-11:00 A.M. Groups of control mice were run concurrently with each dose of ARL 15896AR. The doses at which survival times increased by 50 and 100% were determined from linear regression analysis using least squares (Regrs Program, ISS).

Hypoxia studies: rats. Efficacy for ARL 15896AR in extending the times to loss of the righting reflex and mortality was evaluated in rats maintained at ambient temperature for two reasons; 1) to determine proper dose ranges for the more labor intensive global ischemia studies and 2) to ascertain any effects of drug doses on body temperature. Starting oral doses were determined from previous information using ED₅₀ values obtained from antiseizure tests using maximal electroshock (Knowles et al., 1995, Palmer et al., 1996). Rats were dosed (p.o., n = 9-15/dose) with ARL 15896AR, placed in turn into individual clear plastic sealed chambers and 30 min later exposed to the gas mixture (97%:3% N₂/O₂). An observer with a stop watch recorded the times to loss of the righting reflex and death (Palmer et al., 1995b).

Rat 4-VO model of global ischemia. An oral dose of 24 mg/kg of ARL 15896AR (active in the rat hypoxia studies) was selected for further evaluation in the rat 4-VO model of global ischemia.

2-VO/hypotension model of global ischemia. The protocols for the surgical procedures between the two laboratories (Astra Arcus USA and the University of Manitoba) were standardized. The procedure is adapted from the original by Smith et al., (1984). On the day of the experiment, anesthesia was induced with either: 1) atropine (0.5 mg/kg i.p.) to decrease esophageal secretions followed by pentobarbital (65 mg/kg, i.p., dose volume 0.1 ml/100 g) or 2) nitrous oxide/oxygen (70%/30%) and 2% halothane administered via a cup fitted over the nose and mouth. For the pentobarbital experiments the rats were intubated and ventilated on a respirator. Two incisions were made (ventral neck and tail). Throughout the procedure and recovery, brain temperature was monitored by a thermocouple placed through the tympanic membrane. A temperature of 37.5°C was maintained by means of thermostatically controlled heat lamps and water blankets. Blood pressure was monitored via a 24-gauge catheter inserted into the tail artery. Blood gas samples were taken before ischemia and the ventilation adjusted to maintain normal measurements of PO₂ (80-200 mm Hg), PCO₂ (35-45 mm Hg) and pH. Both common carotid arteries were exposed and isolated using loose ligatures. Normal blood pressure was recorded for 5 min followed by exsanguination of blood from the arterial line to lower pressure to 45 to 50 mm Hg. Once the <50 mm Hg pressure stabilized, ischemia was induced for 12 min by occlusion of the common carotids using temporary aneurysm clips. During the ischemic period, blood pressure was maintained from 45 to 50 mm Hg by withdrawal or infusion of blood. Ischemia was terminated by removal of the arterial clips, the exsanguinated blood reinfused into the animal over a 1-min period and the arterial line flushed with 1 ml heparinized saline. Monitoring of blood pressure and blood gases were continued over 5 additional min at which time the incisions were secured. Anesthesia was discontinued for the halothane anesthetized rats whereas the pentobarbital anesthetized animals were taken off the ventilation system and placed on a heating pad for recovery.

Gerbil model of global forebrain ischemia. Great care was taken with gerbils to maintain brain and body temperature because of the extreme susceptibility of this species to the ameliorative actions of hypothermia (Corbett et al., 1990). One week before ischemia gerbils were implanted with a guide cannula to allow a brain temperature probe to be inserted into the striatum. Baseline brain temperatures were recorded 2 days before ischemia. Rectal and brain temperatures were maintained near 37°C during carotid occlusion and for several hours after reflow. For the ischemia procedure, animals were anesthetized under 1.5 to 2.0% halothane anesthesia, the common carotid arteries rapidly isolated and clamped for 3 min. Treatment with ARL 15896AR (24 mg/kg s.c., b.i.d./4 days) or vehicle (phosphate buffered saline, pH 7.4) commenced at the time of reflow.

Focal Ischemia ModelsMCAO Surgical Procedure

The procedure for focal ischemia consisted of a unilateral carotid artery ligation in tandem with 120 or 90 min of transient MCAO in the SHR as described in detail by Buchan et al., (1992). Anesthesia for the SHR was with 3% halothane/70% N₂ /30% O₂ with subsequent maintenance on 1 to 2% halothane. The anesthesia was adjusted to sustain mean arterial blood pressure above 90 mm Hg during the surgical procedure. The tail artery was cannulated for physiological monitoring. Mean arterial pressure, blood gases (pH, PCO₂ and PO₂), CBF, hematocrit and serum glucose were measured before the onset of ischemia, during ischemia and at the time of reperfusion. Body temperature was rigorously maintained at 37.5 ± 0.5°C during and after surgery with a rectal thermistor coupled to a heating lamp. For surgery the ipsilateral common carotid artery was isolated. A 1-cm incision perpendicular to and bisecting a line between the lateral canthus of the right eye and the external auditory canal was made, the underlying temporalis muscle excised and under direct visualization, the right MCA was exposed through a 3-mm burr hole drilled 2 to 3 mm rostral to the fusion of the zygomatic arch with the squamosal bone. The MCA was visualized where it crosses the inferior vein, which lies within the rhinal fissure. This site is proximal to the MCA bifurcation, but distal to the origin of the lenticulostriate arteries. After obtaining basal physiological and CBF measurements, the ipsilateral (right) common carotid was occluded, the dura overlying the MCA, cut and retracted, a no. 1 microclip (Codman, Boston, MA), placed on the MCA and flow interruption observed. The incisions were secured, anesthesia discontinued and the animals returned to their home cages.

In selected studies, to establish baseline information, regional CBF was monitored in the "core" ischemic region using laser Doppler flow measurements (TSI, St. Paul, MN) at a single location, which corresponds to the region that histologically becomes the center of the infarction. A 1- to 2-mm diameter burr hole was drilled 3 mm dorsal to the site of MCAO and 3 mm caudal to the bregma. The dura at the laser Doppler flow probe site was left intact and the probe (0.8 mm diameter) advanced with a micromanipulator (Narishige Instruments, Tokyo, Japan) under microscopic guidance to a site free of large pial vessels. The probe was positioned to rest on the surface of the dura without indenting the cortex. Cerebral blood flow was measured prior to the MCAO (determine baseline flow) and immediately after occlusion. Blood flow was recorded as the mean of the maximum and minimum recorded flows over a period of 5 min and expressed as a percentage of preischemic baseline values.

MCAO therapeutic window: SHR. The study was carried out at the Neuroscience Unit of the Ottawa Civic Hospital, Ottawa, Canada. The period of focal MCAO was 2 hr followed by 22 hr reflow. Rats were given i.p. injections of saline (1 ml × 3 doses) or ARL 15896AR (12 mg/kg × 3 doses). Initial doses were administered 30 min preischemia, at the onset of ischemia, or at 30, 60 or 120 min post-MCAO. The two subsequent doses were injected at 4 and 12 hr from the time of the initial doses.

Magnetic Resonance Imaging

Magnetic resonance imaging was performed using a Bruker MSL-X Biospec 7/21 Spectrometer. For each imaging study taken on day 2 and day 6 post-MCAO, the rat was anesthetized with pentobarbital (50 mg/kg, i.p.) and placed in a holder with the head positioned in a 3-cm diameter saddle coil using an incisor bar. Spin-echo scout images were acquired first in the coronal and then in the sagittal planes to select reproducible coronal slice positions. Sixteen contiguous coronal slices, centered 1.5 mm posterior to bregma, were imaged in two interleaved sets to minimize interslice excitation. For all coronal images, the matrix size was 256 × 256. Four averages were accumulated using a field of view of 3.5 × 3.5 cm² and a slice thickness of 1 mm. A field of view extension factor of 2 was used in the read direction, and 256 phase-encoding steps were acquired. Multislice multi-echo T₂-weighted spin-echo images were acquired with echo times of 20, 40 and 60 msec and TR of 1500 msec. Total acquisition time was 52 min.

	Histological Procedures

Abstract Introduction Methods Procedures Results Discussion Conclusion References

For all procedures involving processing of the brain the animals were anesthetized with sodium pentobarbital (64 mg/kg, i.p.) and perfused transcardially first with heparinized saline and then with 10% formalin fixative solution. The head was removed, wrapped in plastic or left in fixative and placed overnight in a refrigerator to allow postfixation in situ. The next day the brain was removed and placed in perfusion solution for another 24 to 48 hr.

4-VO and 2-VO experiments conducted at Astra Arcus USA. One coronal brain block from the fixed rat brain that includes the entire hippocampus was prepared using a brain dissection guide (Harvard Instruments Large Rat Brain MatrixCoronal, no. 52-4512c). The block is defined by the blade-guide grooves in the dissection guide that are the 9th and 15th from the rostral pole of the brain. Individual blocks were imbedded in paraffin and processed to obtain 6-µ thick coronal sections. Staining was by H & E, selected because it shows overall damage (including microglia infiltration, neovascularization) to the brain, as well as assessment of neuronal loss. One section was collected every 100 µ through the entire depth of the block. For histological ratings, six consecutive sections from each animal were evaluated beginning with the section that most-closely matches a coronal section approximately 3.2 mm caudal to the bregma according to the Paxinos and Watson (1982) atlas. The extent of ischemic damage was evaluated bilaterally septal to midsep- to temporal hippocampus in six separate hippocampal subregions. Two raters blinded to the treatment condition classified the slides. Gross ischemic damage (evidenced by loss of the CA1 pyramidal cells, the presence of acidophilic cell "ghosts," extent of neovascularization and microglial infiltration) was scored using a 4-point scale: 0 = no damage, 1 = less than 33% cell loss, 2 = 34 to 66% damage and 3 = 67 to 100% damage. Individual neuronal counts of left and right sides of the entire CA1 region were made in alternate sections (2, 4 and 6) using 20 × magnification. The beginning of the CA1 zone was designated from the CA2/CA1 transition zone. The end of CA1 was determined by an imaginary line drawn from the apex of the dentate gyrus, perpendicular to the CA1 cell layer.

2-VO conducted at Winnipeg. Perfusion-fixed rat brains were coded and processed for histology (8-µm sections stained with H&E). Striatal damage was quantified by examining two 630 µm diameter microscopic fields in the dorsolateral caudate at the coronal level of septal nuclei (+0.2 mm from Bregma)and involved direct counting of acidophilic neurons. The hippocampus was examined at 6 levels along its septotemporal axis. Damage at each level was expressed as a percentage of the total CA1 sector. If only a few necrotic neurons were present, these were counted and divided by the total number of known neurons. If the majority of neurons were necrotic, then the remaining viable neurons were counted and necrosis obtained by subtracting this value from the total neuronal counts. Statistical analyses were performed using non-parametric tests.

Forebrain ischemia gerbils. On day 10 of the experiment the brains were perfusion-fixed as described above followed by sectioning (10 µm) and Cresyl violet staining. The degree of hippocampal CA1 injury was quantified according to the following rating (Hewitt and Corbett, 1992): 4 = 90-100% normal appearing neurons; 3 = 60 to 89%, 2 = 30 to 59%, 1 = 6 to 29% and 0 = less than 5% appearing normal. The rating scale was applied to each of three sectors of the dorsal CA1 region (medial, middle and lateral) from both hemispheres in the rostral hippocampus (1.7 mm posterior to Bregma). In addition the ventral hippocampus from the middle and lateral sectors was rated separately. Thus an animal with little or no CA1 loss would be rated as a 23 or 24 whereas an animal with severe loss would be rated close to 0. Previous work demonstrated the rating method correlated well (r = 0.97, P = .0001, n = 56) with actual cell counting techniques. All rating techniques were performed blind with respect to the treatment condition.

MCAO rats. Perfusion-fixed brains from SHR were processed for histology at 22 hr post- MCAO in the acute "therapeutic window" investigation. In the collaborative study the histology was performed at 42 days after MCAO. Brains were cut into 2-mm thick coronal slices, dehydrated in graded ethanols, followed by clearing in xylol and embedding in paraffin. Coronal sections were taken every 500 µm, yielding 25 coronal planes encompassing the infarct. Under a light microscope the necrotic areas of the cerebral cortex and the striatum were traced on images of the slices, controlling for the infarct borderline. Areas of the ipsilateral and contralateral hemispheres were also measured. This allowed for calculation of damage as percent of the hemisphere, thus controlling for variations in atrophy or brain size from one rat to another. Data were compared using one- way ANOVA (Zhu and Auer, 1995).

Pharmacokinetics and Plasma Analysis

Plasma levels. For sample analysis, 50 µl of internal standard containing 500 ng of ARL 15759XX (1-methyl-1-phenyl-2-pyridine ethanamine free base) were added to each tube containing 250 µl of plasma followed by the addition of 40 µl of 1 M NaOH. Mixing was followed by addition of 2 ml of 10% n-butanol/hexane, mixing for 10 min with low speed centrifugation (822 × g for 10 min). The organic phase was removed and added to tubes containing 125 µl of 0.1 M HCl followed by mixing, centrifugation (822 × g) and removal of the organic layer. Fifteen µl of 1.0 M NaOH were added to the tubes, followed by brief mixing and transfer to an HPLC autosampler. Standards were prepared in plasma from naive rats at concentrations between 5 and 10,000 ng/ml. The method is specific and the peak height ratios versus concentration are linear over the measured range of standards. Samples were analyzed by HPLC using reverse phase chromatography with a mobile phase of 13.8% acetonitrile, 86% 0.05 M KH₂PO₄, pH 3.75, and 0.2% triethylamine. Using a variable wavelength detector at 260 nm with a 250 × 4 mm LiChrospher 60 RP with 5 µM particle size, the detection limit for ARL 15896AR was 5.0 ng/ml in plasma. Levels of ARL 15896AR were reported as the free base.

Pharmacokinetics: rat. Determination of pharmacokinetic parameters of ARL 15896AR was made in Sprague-Dawley rats after an acute dose of 100 mg/kg, s.c. Blood samples were taken at 0.083, 0.5, 0.833, 1.5, 2, 4, 8 and 24 hr from six rats with jugular vein cannulas. The data were fit to a one-compartment model using the computer program PCNONLIN V4 (SCI Software, Statistical Consultants, Inc., Lexington, KY). For comparison with profiles from other studies, the plasma concentration versus time profile from our study was normalized to doses used in other studies (Mahmood et al., 1994; Palmer et al., 1996), as well with data from the present investigations namely, 4-VO, 2-VO, gerbil ischemia study and the collaborative investigation with the MCAO.

Pharmacokinetics: gerbils. Elimination kinetics of ARL 15896AR were determined in 30 female Mongolian gerbils after a single s.c. dose of 24 mg/kg, a dose previously shown to be protective in the rat 4-VO study. The study compared parameters of ARL 15896AR between gerbils and rats. Blood was taken from gerbils (CO₂-induced mortality) at 0.083, 0.5, 0.833, 1.5, 2, 4, 6, 8, 12 and 24 hr after dosing (three animals per time point). Vena cava samples were transferred to heparinized Vacutainer tubes on ice, centrifuged and the plasma removed and stored at 70°C until analyzed

Testing for Motor Behavior

Crude motor behavior. The tests conducted on SHR included the following.

Skilled forepaw use. The "Staircase Testing Paradigm" for evaluation of complex motor behavior was conducted on the SHR which were food deprived for 24 hr before testing. The apparatus consists of an elevated platform on which the rat rests with staircases on either side of the animal. A staircase contains seven steps with a food well in each step. Each day the rat was placed in the apparatus with two Noyes pellets on the platform and each food well baited with 3 Noyes pellets. The animals were left in the apparatus for 20 min and the number of pellets consumed noted. After the first test session each rat was given 6 Noyes pellets and returned to the home cage which was devoid of food. The next day the rats were returned to the staircase apparatus, again with 2 Noyes pellets present on the platform and the food wells baited. At the end of the second test session and after subsequent test sessions all rats were given 10 g of rat food in their home cages. This amount of food plus whatever food was obtained in the staircase test maintained the animals at approximately 85% of their free-feeding weight. A total of nine test sessions were conducted per animal in the staircase apparatus. The data obtained were based on the average number of pellets consumed over the last three test sessions. The animals were unable to retrieve any spilled pellets. Thus the amount actually consumed was determined from the number of pellets present at the start of the test session minus the number left in food wells or spilled to the chamber floor. Because the rats could reach the top two steps of the staircase and obtain pellets by using their tongues or the "good limb" (i.e., limb ipsilateral to the occlusion) the total number of pellets consumed per test session did not include pellets eaten from the top two steps nor the two "free" pellets placed on the platform. In other words, data were based on food eaten/spilled/uneaten from the bottom five steps of each staircase (Montoya et al., 1991; Grabowski et al., 1993).

Experimental Plan for MCAO Collaborative Study: SHR

Surgery for the 90 min MCAO, dosing, sampling of plasma, T₂ weighted MRI and crude motor assessments were carried out at the University of Manitoba. Analyses of plasma samples were conducted at Astra Arcus USA, staircase testing was performed at Memorial University and resultant histopathological examination of the brains at Foothills Hospital. All findings were coded until final completion of the study.

Four groups of animals were prepared: group Isham-operated control (n = 6), group IIpost MCAO treatment (30 min) with phosphate buffered saline vehicle (pH = 7.4, n = 6), group IIIpost MCAO treatment (30 min) with ARL 15896AR (n = 6) and group IVpre-MCAO treatment (15 min) with ARL 15896AR (n = 4). Dosing with ARL 15896AR was 10 mg/kg, s.c. The second and third doses were administered at 4.5 and 12 hr post-MCAO. A plasma sample for determination of ARL 15896AR levels was removed 5.5 hr after MCAO from groups II and III. Physiological measurements were made before and after ischemia from six rats selected at random from the different experimental MCAO groups. Now that the reproducibility of the model has been established these served as spot checks only. Multi-slice T₂ weighted MRI were taken at 2 and 6 days post-MCAO. After MCAO the rats were tested for crude motor performance at 4 hr and 1, 2, 4, 6, 8 and 10 days post-MCAO. At 12 days after surgery the rats were flown to Memorial University and tested from days 30 to 42 for complex motor behavioral assessments involving the staircase apparatus. Histopathological evaluation included comparisons of both the volumes of infarction (mm³), as well as percent change from the contralateral undamaged hemisphere. Cortical infarction contains dead or dying tissue, although cortical atrophy represents loss of tissue. Atrophy is determined as the difference in volume between the control and damaged hemispheres.

	Results

Abstract Introduction Methods Procedures Results Discussion Conclusion References

Global Ischemia Models

Hypoxia: mice. Pretreatment with ARL 15896AR extended the time to mortality after exposure of mice to an hypoxic environment. The i.v. dose to extend survival time by 50% was 21.0 mg/kg and by 100% it was 40.4 mg/kg (slope ± S.E. for the i.v. dose response curve = 2.6 ± 0.70). The mean ± S.E. time to mortality in the 30 control animals run with this experiment was 2.4 ± 0.1 min.

Hypoxia: rats. Because hypothermia may influence therapeutic outcome in hypoxia/ischemia investigations (Corbett et al., 1990; Hewitt and Corbett, 1992), we first looked for possible influence of oral doses (12 and 24 mg/kg) of ARL 15896AR on rectal temperature over a 2-hr time course using naive control rats. The stress of measuring rectal temperature in both control and ARL 15896AR-treated rats (n = 10/treatment group) indicated a tendency (nonsignificant) for a slight elevation when measured 30 min later. However, neither dose of ARL 15896AR affected body temperature when assessed at 30-min intervals for up to 2 hr after administration of the compound. The 3 × 5 ANOVA indicated the following: TREATMENT: P = .1081; INTERVAL: P = .0273; TREATMENT × INTERVAL: P = .2271.

4-VO: rats. Monitoring of rectal temperature at 5-min intervals during and after 20 min of 4-VO global ischemia did not result in any significant change in body temperature among the sham-operated controls, the ischemic controls or the ischemic rats receiving 24 mg/kg, p.o. of ARL 15896AR (overall ANOVA: TREATMENT - P = .15; TIME - P = .14; TREATMENT x TIME - P = .99). Moreover, the mean weights of each group as monitored throughout the 7-day treatment period were unremarkable (ANOVA: TREATMENT: P = .98; DAYS - P = .001; TREATMENT × DAYS - P = .10).

View larger version (65K): [in this window] [in a new window]   Fig. 2.   Mean ± S.E. Gross histological scores from six sections taken from the left and right hippocampal CA1 neuronal regions after 20 min 4-VO global ischemia in rats. The values are the mean gross scores (0 = no damage; 1 = up to 33% damage, 2 = 34% up to 66% damage and 3 = >67% damage). Hippocampal sections are taken from the septal to the middle pole. ARL 15896AR is administered p.o. at 24 mg/kg (b.i.d.)/day for 7 days. The initial dose was administered at the time of reperfusion (20 min after 4-VO).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Mean ± S.E. Counts of viable CA1 pyramidal neurons from three sections taken from the left and right hippocampal regions after 20 min 4-VO global ischemia in rats. Hippocampal sections are taken from the septal to the middle pole. ARL 15896AR is administered p.o. at 24 mg/kg (b.i.d.)/day for 7 days. The initial dose was administered at the time of reperfusion (20 min).

2-VO: rats. Regardless of the mode of anesthesia, no 7-day treatment regimen with ARL 15896AR resulted in any significant degree of neuronal protection to the CA1 hippocampal region or the striatum (see table 1).

Forebrain ischemia: gerbils. The ARL 15896AR dosing schedule did not significantly influence brain or body temperature in either sham-operated or ischemic gerbils. Moreover, this regimen did not afford protection to the CA1 neurons from the dorsal hippocampus. The mean ± S.E. histological scores for the experimental groups were: 1) sham-operated, ARL 15896AR-treated controls, score = 24 ± 0 (n = 5); 2) ischemia, ARL 15896AR treatment, score = 2.5 ± 3.9 (n = 7); 3) ischemia, vehicle treatment, score = 5.3 ± 5.4 (n = 7).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Plasma concentration vs. time profiles of ARL 15896 (free base form) in female gerbils after s.c. dosing at 24 mg/kg (n = 3/time point) overlayed with values obtained from rats. For comparison the rat data at 24 mg/kg are estimated using measurements from a 100 mg/kg dosing experiment (n = 6/time point) with the assumption that plasma levels are proportional to the dose administered (see Mahmood et al., 1994; Palmer et al., 1996).

Focal Ischemia Studies

MCAO therapeutic window: SHR. After 2 hr of MCAO, the one-way ANOVA analyses of the data indicated highly significant (P < .001) reductions in the volume of cortical infarction when the initial dosing occurred at 30 min preischemia, at the time of MCAO or 30 min post-MCAO. Protection remained significant, albeit less (P < .05), when the initial dosing regimen began at 60 min post-MCAO. No protection was observed in the SHR if the initial dosing with ARL 15896AR was delayed to 2 hr post-MCAO (fig. 5).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Mean volumes (mm3) ± S.D. of cortical infarction in SHR after 2 hr MCAO and 22 hr reflowtherapeutic treatment window for ARL 15896AR. ARL 15896AR is administered in three doses (12 mg/kg, i.p.) the second and third doses given 4 and 12 hr after the initial time of drug administration. The darkened circles represent individual data points.

MCAO Collaborative Study: SHR

For the physiological measurements (plasma glucose, hematocrit, PO₂, PCO₂, plasma pH) made from the six selected rats, the mean pre-ischemia values did not differ significantly from the mean post-ischemia values (see table 3).

Magnetic resonance imaging. A representative multi-slice T₂ weighted MRI taken at 2 days post-MCAO from an SHR is depicted in figure 6. In all animals the area of cortical infarction on day 2 was considerably greater (P < .001, overall ANOVA followed by post hoc Newman Keuls analysis) than on day 6. The data shown in figures 7 and 8 make these comparisons. Figures 7 (day 2) and 8 (day 6) represent apparent lesion areas on consecutive T₂ weighted MR images for the 3 MCAO groups. The group treated with ARL 15896AR 30 min after MCAO (group III) had a significantly smaller lesion volume on both days (P < .05) when compared to the control MCAO group (group II). Possibly due to the smaller n value, the values for the group pretreated with ARL 15896AR (group IV) were not significantly different than the controls (group II). The composite mean volumes (mm³) ± S.D. of all the serial T₂ weighted images taken at both 2 or 6 days from the three ischemic groups were: 1) group II - 2 days = 248 ± 34, 6 days = 123 ± 18; 2) group III - 2 days = 185 ± 39, 6 days = 99 ± 20; 3) group IV - 2 days = 204 ± 42, 6 days = 109 ± 25. 

View larger version (136K): [in this window] [in a new window]   Fig. 6.   Representative contiguous T2 weighted magnetic resonance images taken from a control SHR at 2 days post 90 min MCAO. The hyperintense regions represent the area of apparent infarction.

View larger version (16K): [in this window] [in a new window]   Fig. 7.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Apparent mean infarct volumes (mm3) 2 days (fig. 7) or 6 days (fig. 8) post MCAO measured from contiguous coronal T2 weighted MR images (slice thickness = 1 mm, see fig. 6) centered at the indicated distance (mm) from bregma. Groups of SHR subjected to 90 min MCAO received either saline (control), or treatment with ARL 15896AR initiated before (PreTX or after (PostTx) onset of cerebral ischemia.

Plasma levels. Blood samples were taken from post-treatment group III and the control group II at 5.5 hr post-MCAO. The mean ± S.D. levels of free base ARL 15896 were 3527 ± 930 ng/ml plasma. With the exception of one outlier, the plasma levels were within the predicted range (present experiments and Mahmood et al., 1994; Palmer et al., 1996) expected after a second s.c. dose of 10 mg/kg (fig. 9).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Individual plasma concentrations vs. time modeled for a s.c. dose of 10 mg/kg of ARL 15896AR free base at time 0.5 and 4.5 hr (see "Methods"). The measured plasma levels at 5.5 hr from individual rats are shown as numbered symbols.

Crude motor behavior. After MCAO, SHR were tested for crude motor performance. The findings for the neurological deficit scores of all rats receiving MCAO or sham operations are given in figure 10. Out of a total possible deficit score of 16 from the 4 motor/neurological impairment tests the maximal scores attained ranged from 6.5 to 8, an observation occurring immediately after the MCAO (4 hr). However, at this time the animals had not fully recovered from the effects of the anesthesia/stroke operation. By 24 to 48 hr after MCAO, significant impairment was again noted in all groups, however, the scores dropped from 5 to 6 at 24 hr to ~4 by 48 hr. From day 4 through day 10 after MCAO the crude motor impairment scores were in the range of the sham-operated control group. Neither treatment regimen with ARL 15896AR was associated with any significant improvement when compared to the control SHR subjected to MCAO.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Crude neurological deficit scores after MCAO in SHR with or without treatment with ARL 15896AR. The mean ± S.D. scores represent combined scores from four separate tests, each graded as 0 (no motor deficit) to 4 (exhibiting the most severe deficit). Testing involves an evaluation of ipsilateral circling, contralateral hindlimb retraction, beam walking and bilateral grasp. A maximal attained deficit score would thus be a 16.

Complex motor behavioral assessments. In the staircase test there were definite, significant (ANOVA with post hoc Newman Keuls analyses, P < .05) motor deficits with the contralateral forepaw regarding the number of "steps reached." This observation was evident in all ischemic groups (fig. 11, top panel). As the staircase test also measures the number of food pellets actually picked up from the lower steps and eaten, it was readily apparent in the ischemic control SHR that there were severe deficits with both the contralateral (ANOVA, P = .0004) and ipsilateral forepaws (ANOVA, P = .03). Pre- and post-MCAO treatment of SHR with ARL 15896AR did not avert the subsequent deficit on the contralateral side, but did indeed significantly (P < .05, post hoc Newman Keuls analyses) prevent the motor deficit on the side ipsilateral to the lesion (fig. 11, bottom panel).

View larger version (40K): [in this window] [in a new window]   Fig. 11.   Staircase testcomplex motor analysis of SHR from 30 to 42 days post MCAO. The values are the mean ± S.D. Top panel, steps reached. The contralateral paw values for the ischemic groups are significantly different from the respective sham-operated control group (P < .05, ANOVA with post hoc Newman Keuls test; ANOVA values are for steps reached with contralateral paw F = 6.43, P = .003; for steps reached with ipsilateral paw F = 2.32, NS). Bottom panel, staircase testpellets retrieved and eaten. Data were analyzed using ANOVA with Post Hoc Newman Keuls test. For the contralateral paw the control and ARL 15896AR treatment groups subjected to MCAO are significantly different from the respective sham-operated group (P < .05). For the ipsilateral paw the pre- and post-ARL 15896AR treatment groups including the respective sham-operated group are significantly different (P < .05) from the ischemic control group. The ANOVA values arepellets eaten with contralateral limb F = 9.634, P = .0004; pellets eaten with ipsilateral limb F = 3.66, P = .03.

Histopathology. Histopathological evaluation of the serial brain slices indicated no significant difference between treatment groups (MCAO control and MCAO ARL 15896AR) regarding the extent of total brain damage, brain atrophy, as well as cortical or subcortical infarction volumes. Data analysis included comparisons of both the volumes of infarction (mm³), as well as percent change from the contralateral undamaged hemisphere. With regard to measurements of volume of infarction, the cerebral cortex experienced, as expected, the greatest degree of damage (all treatments = ~ 20 mm³). Subcortical damage was minimal (all treatments ranged from 0.75-1.25 mm³). The volume of cortical atrophy ranged from ~50 to 67 mm³ and the total brain damage ranged from ~63 to 90 mm³ for all treatments. The data in figure 12 are presented as the percent damage on the right (MCAO) side with respect to the contralateral left control side.

View larger version (30K): [in this window] [in a new window]   Fig. 12.   Comparisons of histopathological assessment of cerebral damage in SHR receiving 90 min MCAO. The data are expressed as mean ± S.D. percent damage of the lesioned hemisphere compared to the contralateral control side of the brain. The negative values represent areas with no brain damage, where the ipsilateral hemisphere happened to be slightly larger than the contralateral hemisphere (edema is not possible any longer at 42 days).

	Discussion

Abstract Introduction Methods Procedures Results Discussion Conclusion References

General

ARL 15896AR is currently under development primarily as a neuroprotective compound with treatment of acute focal cerebral ischemia as the ultimate clinical target. In addition, ARL 15896AR possesses good antiepileptic potency in rodents. Prevention of seizures elicited by the excitatory amino acids, NMDA and kainate, as well as, tonic hind limb extension as a consequence of maximal electroshock, 4-aminopyridine or bicuculline have been observed (Knowles et al., 1995; Palmer et al., 1996). Unlike the more potent uncompetitive NMDA receptor antagonists, ARL 15896AR does not produce abuse liability (Hudzik et al., 1996), affect learning (Hudzik and Palmer, 1995) nor elicit motor deficits (Palmer et al., 1996).

In vitro experiments indicate that ARL 15896AR possesses a moderate degree of receptor affinity as an uncompetitive NMDA receptor antagonist. The IC₅₀ for displacement of MK801 binding in rat brain synaptosomal preparations in vitro is 1.6 µM. When added to neuronal cultures from rat cerebral cortex, ARL 15896AR prevents excitatory amino acid-elicited cell death, translocation of protein kinase C, plus the associated Ca⁺⁺ surge into the cells (Black et al., 1995). NMDA-induced depolarization in rat hippocampal slices is also prevented by ARL 15896AR (Cregan et al., 1995).

Role in Ischemia

Rationale. The ability of ARL 15896AR to influence both NMDA and possibly sigma-1 receptors (Palmer et al., 1996) indicates potential to lessen the degree of neuronal damage as a result of ischemic/anoxic insult to the brain. Such compounds have been shown repeatedly in the past to afford neuroprotection in animals subjected to various experimental paradigms of global and focal ischemia (Chapman et al., 1990; Meldrum, 1992; McCulloch, 1992; Jensen and Auer, 1991; Buchan, 1990). Notably, eliprodil, a mixed NMDA/sigma receptor antagonist, was effective in a mouse focal ischemia model (Poignet et al., 1992).

Anoxia. ARL 15896AR lengthened the time of survival in mice and rats plus increased the duration to the loss of the righting reflex in rats exposed to an anoxic environment. Anoxic conditions do indeed evoke a massive release of excitatory amino acid transmitters in the central nervous system and such conditions are amenable in part to treatment by both NMDA and sigma receptor antagonists (Chapman et al., 1990; Meldrum, 1992; Jensen and Auer, 1991; Poignet et al., 1992). If a chemical series is related in structure and individual compounds are active regarding lengthening survival time, the method is then useful for general rapid drug screening and for selection of initial doses for the more labor intensive investigations involving ischemia (Palmer et al., 1995a, b). Unless body temperature was controlled (e.g., mice in our study) anoxic conditions may, however, lead to identification of false positives (Milde, 1988). Nevertheless, the model has been shown to have applicability, even to the clinical situation (Collins et al., 1989).

Global ischemia. In the 4-VO model of global ischemia, oral ARL 15896AR treatment beginning at reflow and continuing for 7 days resulted in protection of the highly vulnerable hippocampal CA1 neurons. We did, however, observe greater damage to the CA1 neurons on the left side. A plausible explanation for this phenomenon was the possibility of incomplete cauterization of the right vertebral artery in some of the rats. Two structurally related compounds, namely remacemide hydrochloride (Palmer et al., 1995a; Ordy et al., 1992) and ARL 13950AA (Palmer et al., 1995b), were also highly effective in the 4-VO model and we did not observe the laterality effect in these studies.

Focal ischemia. NMDA receptor antagonists are especially effective as neuroprotective agents in focal models of ischemia (for reviews see McCulloch, 1992; Meldrum, 1992; Chapman, 1990; Buchan, 1990). Thus ARL 15896AR was similarly shown to be protective in the rat model of malonate-induced striatal lesions (Greene et al., 1996). In our study using the 2-hr MCAO model in the SHR, ARL 15896AR was effective in reducing resultant cortical damage when initial doses were administered up to 1 hr after MCAO. This encouraging result led to the design of the collaborative investigation that is discussed in the following paragraphs.