Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

Free Radical Biology & Medicine, Vol. 33, No. 7, pp. 947–961, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter

PII S0891-5849(02)00979-6

Original Contribution EFFECTS OF METALLOPORPHYRIN CATALYTIC ANTIOXIDANTS IN EXPERIMENTAL BRAIN ISCHEMIA HUAXIN SHENG,* JAN J. ENGHILD,§ RUSSELL BOWLER,㛳 MANISHA PATEL,㛳 INES BATINIC´ -HABERLE,‡ CARLA L. CALVI,* BRIAN J. DAY,㛳 ROBERT D. PEARLSTEIN,† JAMES D. CRAPO,㛳 and DAVID S. WARNER*† *Department of Anesthesiology, †Department of Surgery (Neurosurgery), ‡Department of Biochemistry, Multidisciplinary Neuroprotection Laboratories, Duke University Medical Center, Durham, NC, USA; §Department of Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark; and 㛳Department of Medicine, National Jewish Medical and Research Center, Denver, CO, USA (Received 5 March 2002; Revised 14 May 2002; Accepted 6 June 2002)

Abstract—Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t1/2 was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O2/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects. © 2002 Elsevier Science Inc. Keywords—Brain, Ischemia, Metalloporphyrin, Catalytic antioxidant, Mouse, Rat, Proteomic, Cell culture, Aconitase, Free radicals

INTRODUCTION

clusion (MCAO) [4,5]. Second, investigations with transgenic mice have demonstrated consistent improvement of histologic/neurologic outcome from either focal or global ischemia when overexpression of either copper/ zinc (CuZn-SOD), manganese (Mn-SOD) [6] or extracellular (EC-SOD) superoxide dismutase is achieved [7–9]. Conversely, targeted deletion of the Mn-SOD [10], CuZn-SOD [11], or EC-SOD isozymes [12] worsens ischemic outcome. Third, pharmacologic advances in synthesis of catalytic antioxidants have led to evaluation of such molecules in a variety of stroke models [13–16]. Efficacy in reducing infarct size and neurologic deficit, despite a substantial delay in administration after onset of acute brain injury, has raised hope that compounds possessing this mechanism of action may find clinical utility.

Reactive oxygen and nitrogen species play critical roles in the response of brain to an ischemic insult [1]. Renewed interest in therapeutic application of antioxidants has been derived from three independent sources of information. First, hydroxyl radical production persists for at least several hours after onset of ischemia in rodent models, suggesting the potential for a clinically relevant therapeutic window [2,3]. This is consistent with the observation that superoxide (•O2⫺) production is sustained after reperfusion from middle cerebral artery ocAddress correspondence to: David S. Warner, M.D., Department of Anesthesiology, Duke University Medical Center, Box 3094, Durham, NC 27710, USA; Tel: (919) 684-6633; Fax: (919) 684-6692; E-Mail: [email protected]. 947

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Fig. 1. Structures of catalytic antioxidant meso-metalloporphyrins. Illustrated are AEOL 10150 (manganese (III) tetrakis (1,3diethylimidazolium-2-yl) porphyrin) and AEOL 10113 (manganese (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin).

A metalloporphyrin catalytic antioxidant has recently been shown to be effective in the modification of temporary focal ischemia in rodent models [16]. This metalloporphyrin, AEOL 10113 [17,18], is limited by the fact that it is composed of several stereoisomers that make a pharmaceutically acceptable formulation difficult and severely limit the ability to assess tissue levels and carry out pharmacokinetic studies. AEOL 10150 is a structurally different metalloporphyrin catalytic antioxidant than AEOL 10113, which possesses imidizole side chain substitutions (Fig. 1). AEOL 10150 provides a nearly 2-fold increase in SOD potency compared to AEOL 10113 and reduces the IC50 for in vitro lipid peroxidation by 50% (Table 1). In contrast to AEOL 10113, AEOL 10150 does not exist as a stereoisomer, is relatively easy to synthesize, and can be accurately and simply analyzed in tissues [19]. We hypothesized that AEOL 10150 would yield neuroprotection equivalent to AEOL 10113 at doses markedly less than those required to produce neurotoxicity in a rat model of focal cerebral ischemia. The work was extended to define intravenous Table 1. Activities of Catalytic Antioxidant Meso-metalloporphyrins

Compound AEOL 10150 AEOL 10113

SOD

Catalase

Lipid Peroxidation

(U SOD/mg)a 18,069 10,648

k (min⫺1)b 1.72 1.61

IC50 (␮M)c 0.5 1.0

a Unit of superoxide dismutase (SOD) activity (U SOD) is defined as the amount of compound that inhibits one half the reduction of cytochrome c by superoxide at pH 7.8. b k is the first order rate constant of hydrogen peroxide decay in the presence of varying concentrations of compound and 1 mM hydrogen peroxide at pH 7.8. c The concentration of compound that inhibits 50% (IC50) of an iron/ascorbate-mediated lipid peroxidation of a rat brain homogenate using thiobarbituric acid reactive species as an index of lipid peroxidation.

(i.v.) AEOL 10150 efficacy in a mouse MCAO model and to perform pharmacokinetic assessment of the transfer of AEOL 10150 from blood to brain under conditions of ischemia/reperfusion. We then sought to define interactions between AEOL 10150 and the proteomic response of brain to focal ischemia and probe mechanisms for the protection observed in primary mixed neuronal/ glial cultures exposed to oxygen/glucose deprivation (OGD). We hypothesized that AEOL 10150 would reduce changes in protein expression attributable to ischemia, reduce neuronal cell death induced by oxidative stress in vitro, and preserve aconitase activity as a marker of superoxide mediated cell injury. MATERIALS AND METHODS

The following studies were approved by the Duke University Animal Care and Use Committee.

Experiment One (relative behavioral neurotoxicity of AEOL 10113 vs. AEOL 10150) Rats have been shown to respond to intravenous AEOL 10113 with a major reduction in mean arterial blood pressure (MAP) [16]. Because systemic hypotension is known to exacerbate experimental brain ischemia insults [20], we first investigated the effects of i.v. AEOL 10150 on MAP in the rat. Male Wistar rats (250 –275 g; Harlan Sprague Dawley, Inc., Indianapolis, IN, USA), were anesthetized with halothane. The trachea was intubated and the lungs were mechanically ventilated. MAP was continuously monitored in the femoral artery. Animals were then given a range of doses of AEOL 10150 i.v. and the blood pressure response was recorded. In all animals given doses greater than 0.1 mg/kg of AEOL 10150, MAP dropped by values greater than 20% imme-

Metalloporphyrins and ischemic brain

diately after injection. The duration of hypotension was dose-dependent. A dose of 0.5 mg/kg resulted in irreversible profound hypotension while smaller doses caused transient effects on MAP. As a result, we elected to first examine AEOL 10150 when given to the rat via an intracerebroventricular (i.c.v.) route. Pilot studies demonstrated that this approach caused no changes in MAP. We have also previously described a behavioral syndrome caused by large doses i.c.v. AEOL 10113 [16]. Signs include rapid onset of sustained proptosis, ataxia, and hypersensitivity to sound. Severity of these findings were dose-dependent. To examine for adverse effects from AEOL 10150, a dose-escalation study was performed. Rats underwent implantation of an i.c.v. cannula (see below). Two days later, sets of 2–3 rats were briefly anesthetized with halothane. AEOL 10150 or AEOL 10113, dissolved in 10 ␮l phosphate-buffered saline (PBS), was injected into the cannula and halothane anesthesia was discontinued. The initial dose was 300 ng, which for AEOL 10113 has previously been shown to cause no adverse effects [16]. Animals were observed for 2 h after injection. If no adverse behavioral responses were observed, the experiment was repeated in new sets of rats with the dose doubled. This process was repeated until side effects were observed. Nine micrograms AEOL 10150 caused just noticeable neurotoxic effects. One half this dose (4500 ng) was chosen as the highest dose of AEOL 10150 to be used for further study. Experiment Two (rat MCAO/i.c.v. AEOL 10113 vs. AEOL 10150) Rats were anesthetized with 64 mg/kg intraperitoneal (i.p.) sodium pentobarbital and positioned in a stereotactic head frame. Using aseptic technique, the skin was infiltrated with 1.0% lidocaine and a midline scalp incision was made. A burr hole was drilled over the left hemisphere, 7.2 mm anterior to the interaural line and 1.4 mm lateral to the sagittal suture. An i.c.v. cannula (33G) was positioned with the tip in the left lateral ventricle. The cannula was fixed in place with two cranial screws and stabilized with orthodontic cement. The wound was closed with suture and animals were allowed to awaken. Rats were then returned to their cages with free access to water (with addition of 1 mg/ml tetracycline) and food. Following 2–3 d of recovery, rats were fasted but allowed free access to water for 12–16 h before MCAO. Rats were then anesthetized with halothane in O2. Following tracheal intubation, the lungs were mechanically ventilated to maintain normocapnia. A 22-gauge needle thermistor was percutaneously placed adjacent to the skull beneath the temporalis. Pericranial temperature was servoregulated at 37.5 ⫾ 0.1°C by surface heating or

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cooling throughout the anesthetic. The inspired halothane concentration was adjusted to 1.0 –1.5% in 50% O2/balance N2. The tail artery was cannulated to monitor MAP and sample blood. The animals were then prepared for MCAO using modifications of the technique described by Zea Longa et al. [21]. A midline cervical incision was made and the right common carotid artery was identified. The external carotid artery (ECA) was isolated and the occipital, superior thyroid, and external maxillary arteries were ligated and divided. The internal carotid artery (ICA) was dissected distally until the origin of the pterygopalatine artery was visualized. Following surgical preparation, a 20 min interval was allowed for physiologic stabilization. Five minutes before onset of MCAO, rats received 50 IU of heparin. A 0.25 mm diameter nylon monofilament prepared with a silicone tip was inserted into the stump of the ECA and passed distally through the ICA (20 mm from carotid bifurcation) until a slight resistance was felt [22] and the filament was secured. At MCAO onset, halothane was reduced to 0.8%. Before removal of the filament, the inspired halothane concentration was increased to 1%. After 90 min of MCAO, the occlusive filament was removed. The anesthetic state and pericranial temperature regulation were continued for an additional 100 min. The tail artery catheter was removed and the wounds were closed with suture. Halothane was discontinued. Upon recovery of the righting reflex, the tracheas were extubated and the animals were placed in an O2-enriched environment (FIO2 ⫽ 50%) for 1 h. Animals were then returned to their home cages. Ninety minutes after onset of recirculation, all rats received an injection over 5 min via the i.c.v. cannula. Rats were randomly assigned to one of four treatment groups according to the injectate: Vehicle (n ⫽ 19): PBS (10 ␮l) AEOL 10113 (n ⫽ 17): 300 ng AEOL 10113 in PBS (10 ␮l) AEOL 10150 (n ⫽ 16): 300 ng AEOL 10150 in PBS (10 ␮l) AEOL 10150 (n ⫽ 15): 4500 ng AEOL10150 in PBS (10 ␮l) Animals were awakened from anesthesia 15 min after completion of the injection. Seven days postischemia, rats underwent a standardized neurologic examination designed to evaluate sensorimotor function [23]. With the observer blinded to group assignment, this test explored six different functions (spontaneous activity, movement symmetry, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch). The performance in each test was rated with a 0 –3 point score. The score given to each

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animal at the completion of testing was the sum of all six individual scores, 3 being the minimum (worst) and 18 being the maximum (best) score. Animals were then weighed, anesthetized with 5% halothane, and decapitated. The brains were removed, frozen at ⫺40°C in 2-methylbutane, and stored at ⫺70°C. Serial quadruplicate 20 ␮m thick coronal sections were taken using a cryotome at 660 ␮m intervals over the rostral-caudal extent of the infarct. The sections were dried and stained with hematoxylin and eosin. Infarct volumes were measured by digitally sampling stained sections with a video camera controlled by an image analyzer. The image of each section was stored as a 1280 ⫻ 960 calibrated pixel matrix. The digitized image was then displayed on a video monitor. With the observer blinded to experimental conditions, infarct borders in both cortex and subcortex were individually outlined (corpus callosum excluded) using an operator-controlled cursor. The area of infarct (mm2) was determined by counting pixels contained within the outlined regions of interest. Infarct volumes (mm3) were computed as running sums of infarct area multiplied by the known interval (e.g., 660 ␮m) between sections over the extent of the infarct calculated as an orthogonal projection. Experiment Three (rat MCAO/i.c.v. AEOL 10150 6 h after onset of reperfusion) Anesthetic and surgical preparation were as described for Experiment Two. Rats were subjected to 90 min MCAO as described above. Thirty minutes after onset of reperfusion, the arterial catheter was removed, halothane was discontinued, and upon recovery of spontaneous ventilation the trachea was extubated. The animals were allowed to recover in an oxygen-enriched environment (FIO2 ⫽ 50%) until recovery of the righting reflex. Six hours after onset of reperfusion, rats were briefly anesthetized with halothane by snout cone. The animals were randomly assigned to receive by i.c.v. injection either vehicle (10 ␮l PBS; n ⫽ 16) or AEOL 10150 (300 ng in 10 ␮l PBS, n ⫽ 18). Anesthesia was immediately discontinued and the animals were allowed to awaken. A recovery interval of 7 d was allowed. Neurologic examination and measurement of cerebral infarct size were performed as described for Experiment Two. Experiment Four (rat AEOL 10150 pharmacokinetic analysis) Male Wistar rats underwent implantation of an i.c.v. cannula as described above. Three days later, the animals were briefly anesthetized with halothane. Either vehicle (10 ␮l PBS; n ⫽ 5) or AEOL 10150 (300 ng in 10 ␮l

PBS; n ⫽ 4 – 6 per time interval) was injected i.c.v. At timed intervals of 0.5, 1, 3, 6, 24, or 48 h postinjection, animals treated with AEOL 10150 were euthanized with halothane. Animals treated with PBS were studied at 1 h after PBS injection. In all animals, blood was sampled from the left cardiac ventricle. After brief transcardiac perfusion of the vasculature with saline, brain (contralateral to injection site) and liver parenchyma were sampled for high-performance liquid chromatography (HPLC) analysis of AEOL 10150 concentration. Experiment Five (rat AEOL 10150 6 h post-MCAO/ temperature measurement) Rats underwent i.c.v. cannula placement and MCAO (90 min) as described above. At the time of MCAO preparation, a radiotelemetered thermistor (Type VMFH; Minimitter Co., Inc., Sunriver, OR, USA), accuracy ⫾ 0.1°C, was implanted into the peritoneal cavity to track core temperature. The thermistor had been previously calibrated (within the range of 35.0 – 40.0°C) in a circulating water bath against a mercury thermometer. This allowed extrapolation of temperatures from calibration points in accordance with the radiofrequency emitted by the probe. Radiofrequency signals from the probe were received (Telemetry Receiver Model RA1010; Data Science, St. Paul, MN, USA), digitized, and processed through a computer with custom made software to determine temperature. Six hours postischemia, either 10 ␮l i.c.v. vehicle (n ⫽ 3) or AEOL 10150 (300 ng; n ⫽ 3) was injected. The animals were allowed to awaken. Core temperature was monitored for the subsequent 18 h. Experiment Six (mouse MCAO/i.v. AEOL 10150) Because the rat demonstrated significant hypotensive responses to AEOL 10150 when given i.v., definition of the efficacy of AEOL 10150 when given i.v. could not be performed in that species. We have previously shown that AEOL 10113 does not cause a hemodynamic response in the mouse when given i.v. [16]. As a result, we examined if this also is the case for AEOL 10150. Male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA), 8 –10 weeks of age, were anesthetized with halothane and mechanically ventilated. An arterial and venous catheter were placed. While blood pressure was continuously recorded, increasing doses of AEOL 10150 were given i.v.. Doses as great as 30 mg/kg caused no effect on blood pressure over a 1 h observation interval. We were thus assured that like AEOL 10113, the mouse would provide a species for study of AEOL 10150 efficacy without confound from hemodynamic responses to the compound.

Metalloporphyrins and ischemic brain

Male C57BL/6J mice were fasted overnight from food but allowed free access to water. Mice were then anesthetized with 1.0 –1.5% halothane in 50% O2/balance N2. The trachea was intubated and the lungs mechanically ventilated. A femoral artery was cannulated for measurement of blood pressure and arterial blood gases. Via a midline cervical skin incision, the right common carotid artery was identified. The ECA was ligated and transected. The ICA was dissected distally until the origin of the pterygopalatine artery was visualized. Following surgical preparation, a 15 min interval was allowed for physiological stabilization. Pericranial temperature was continuously monitored and servoregulated with surface heating/cooling at 37.0°C throughout the procedure. Inspired halothane concentration was maintained between 0.6 –1.0%. A 6-0 nylon monofilament, blunted at the tip in a flame and then lightly coated with silicone, was inserted into the proximal ECA stump and advanced ⬇11 mm. Pilot studies were performed to define the maximal duration of MCAO that would allow a high survival rate in the vehicle-treated group under these experimental conditions. A MCAO interval of 60 min was found to cause less than 10% mortality yet still produce a large cerebral infarct. Accordingly, all experimental groups were subjected to 60 min of MCAO, after which the occlusive filament was removed. Mice were then randomly assigned to one of three groups according to i.v. treatment regimen (n ⫽ 24 per group): Vehicle ⫽ 25 ␮l PBS bolus over 5 min followed by continuous infusion of 1 ␮l/h of PBS; Low dose ⫽ 125 ␮g/kg AEOL 10150 bolus over 5 min followed by 250 ␮g 䡠 kg⫺1 䡠 h⫺1; High dose ⫽ 500 ␮g/kg AEOL 10150 bolus over 5 min followed by 1 mg 䡠 kg⫺1 䡠 h⫺1. To provide continuous i.v. infusion, an osmotic pump was implanted. Osmotic pumps (100 ␮l), designed to provide a continuous infusion at a rate of 1 ␮l/h for 3 d (model 1003D; Alza Corporation, Palo Alto, CA, USA), were filled with 100 ␮l of AEOL 10150 6 mg/ml (low dose) or 24 mg/ml (high dose) or vehicle (PBS). The apparatus was then placed into a 15 ml tube filled with saline and incubated at 37°C overnight prior to implantation to allow priming. The pump catheter was placed in a dorsal subcutaneous skin pocket. The pump cannula was tunneled subcutaneously and inserted into the jugular vein. Time from onset of reperfusion to beginning of drug delivery was approximately 5 min. The dosing strategies defined above were derived from pilot studies performed in mice subjected to 60 min MCAO and a period of reperfusion lasting up to 24 h. A dose-escalation study was performed with mice treated in sets of three. Bolus doses and infusion rates were

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increased in a dose-escalation paradigm with infusion beginning 5 min after onset of reperfusion. Mice were observed for evidence of neurotoxicity. In no mice did neurotoxicity occur immediately after onset of i.v. infusion, in contrast to observations made in the rat with i.c.v. infusion, where neurotoxic behavioral signs were typically observed within 3–5 min after injection. In contrast, if neurotoxic behaviors were observed in the mouse, signs became evident at 1–3 h after onset of infusion. We attributed this to transfer of drug from blood to brain across the blood– brain barrier, which was assumed to have become progressively disrupted during the reperfusion interval. The lowest infusion regimen tested that did not cause signs of neurotoxicity over the 24 h observation interval was a 1 mg/kg bolus followed by 2 mg 䡠 kg⫺1 䡠 h⫺1. As a result, we decreased that dose by one half and used that as our high dose. The low dose was arbitrarily defined as being 25% of the high dose. Following reperfusion from MCAO, the arterial catheter was removed, the wounds were infiltrated with lidocaine and closed with suture. Halothane was discontinued and the mice were allowed to awaken. When spontaneous ventilation and the righting reflex recovered, the trachea was extubated. Mice were placed in an O2-enriched environment (FIO2 ⫽ 50%) for 1 h and then returned to their cages. After 24 h of reperfusion, all animals underwent neurologic evaluation. Each mouse was assigned a score of 0 – 4; where 0 ⫽ no observable neurological deficit; 1 ⫽ failure to extend the left forepaw; 2 ⫽ circling to the left; 3 ⫽ falling to the left; and 4 ⫽ cannot walk spontaneously [7]. Neurological examination was performed by one observer blinded to group assignment. Following neurological evaluation, animals were euthanized with a halothane overdose. The chest was opened and 500 ␮l of blood was withdrawn from the left ventricle for later determination of plasma AEOL 10150 concentration with HPLC. The brain was then removed and frozen at ⫺20°C. Using a cryotome, six 20 ␮m thick coronal sections were taken at 320 ␮m intervals over the rostral-caudal extent of the infarct. The sections were dried and stained with hematoxylin and eosin. Infarct volume was measured by digitally sampling stained sections as described for the rat. Infarct volumes (mm3) were computed as running sums of infarct area multiplied by the known interval (e.g., 320 ␮m) between sections over the extent of the infarct expressed as an orthogonal projection. Experiment Seven (mouse AEOL 10150 pharmacokinetic analysis) To explore the transfer of AEOL 10150 from blood to brain, mice were subjected to an identical protocol for

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surgery, ischemia, and i.v. drug delivery protocol as described for Experiment Six. For the AEOL 10150 high-dose regimen (500 ␮g/kg bolus followed by 1 mg 䡠 kg 䡠 ⫺1h⫺1), plasma and brain were sampled at 1, 5, or 24 h (n ⫽ 5– 6 mice per interval). For the low-dose regimen (125 ␮g/kg bolus followed by 250 ␮g 䡠 kg⫺1 䡠 h⫺1) and vehicle regimen (25 ␮l PBS bolus followed by 1 ␮l/h), plasma and brain were sampled at 5 h after onset of infusion (n ⫽ 5 per group). Prior to brain tissue sampling, the vasculature was perfused in situ with transcardiac saline. Brain and blood AEOL 10150 were measured by HPLC.

Experiment Eight (mouse MCAO/AEOL 10150 post-treatment/proteomic analysis) This study tested the hypothesis that a potent catalytic antioxidant would independently cause diverse changes in protein expression in normal brain, but also ameliorate changes in protein expression induced by an ischemic insult. C57BL/6J mice (The Jackson Laboratory) were anesthetized with halothane and subjected to 60 min of MCAO as described above. Ischemia was confirmed by continuous application of a laser Doppler flow probe to the ipsilateral skull. After 60 min occlusion, the filament was withdrawn, allowing reperfusion of the brain. Sham animals underwent all anesthetic and surgical procedures but were not subjected to middle cerebral artery occlusion. The mice were divided into four groups (n ⫽ 6 per group): Sham surgery followed by a PBS bolus (25 ␮) i.v. followed by a continuous i.v. infusion of PBS at 1 ␮l/h; Sham surgery followed by a 0.5 mg/kg bolus of AEOL 10150 i.v. and a continuous i.v. infusion of AEOL 10150 at 1.0 mg 䡠 kg⫺1 䡠 h⫺1 (1 ␮l/h); MCAO followed by a PBS bolus (25 ␮l) i.v. and a continuous i.v. infusion of PBS at 1 ␮l/h; MCAO followed by a 0.5 mg/kg i.v. bolus of AEOL 10150 and a continuous i.v. infusion of AEOL 10150 at 1.0 mg 䡠 kg⫺1 䡠 h⫺1 (1 ␮l/h). The respective boluses and continuous infusions commenced within 5 min after removal of the occlusive filament (or an equivalent interval in sham-operated mice) corresponding to the treatment regimen used in Experiment Six. At 6 h after removal of the filament, the mice were killed by halothane overdose and the vasculature was perfused with saline. The brains were rapidly removed and frozen immediately in liquid nitrogen. The hind brain and caudal and rostral aspects of the forebrain were discarded. Samples weighing approximately 50 mg

were dissected from the core of the ischemic region using anatomic landmarks derived from the distribution of infarcts observed in Experiment Six. For proteomic analysis, Pharmalyte 4-7, IPG buffers, Immobiline DryStrips, sodium dodecyl sulfate (SDS), TEMED, ammonium persulphate, acrylamide, N,N⬘-methylenebisacrylamide, urea, silicone oil, and agarose were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). CHAPS was obtained from Boehringer Ingelheim (Ingelheim, Germany). Glycerol, Pefabloc, and all other chemicals (analytical grade) for electrophoresis and staining were obtained from Merck (Darmstadt, Germany). Tissue samples were ground in a liquid nitrogencooled mortar. The resulting powder was then solubilized by sonication in 8M urea, 50 mM Tris-Cl, pH 8.0, 1% w/v dithiothreitol, 2% w/v CHAPS, 2% v/v carrier ampholytes, pH 4 –7 and 4 mM Pefabloc, 5 mM EDTA, 40 ␮M E-64 (lysis buffer). Following solubilization, the samples were centrifuged (40,000 ⫻ g, 60 min, 15°C) and stored in aliquots at ⫺80°C until analyzed. For two-dimensional (2D) gel electrophoresis, 1 mg total protein defined by amino acid analysis was loaded on all gels. Immobiline dryStrips (18 cm long, linear gradient between pH 4 –7 (Amersham Pharmacia Biotech) were hydrated overnight with protein sample and focused on a Multiphor II horizontal electrophoresis system (Amersham Pharmacia Biotech) for 45 kVh. The second dimension SDS polyacrylamide gel electrophoresis was carried out in a Hoefer DALT multiple vertical electrophoresis unit (Amersham Pharmacia Biotech). The samples were run on 12.5% gels (23 cm ⫻ 20 cm ⫻ 1 mm) overnight [24]. Following electrophoresis, protein spots were visualized by silver staining [25]. The silverstained gels were scanned using a UMAX PowerLook III scanner with UTA III transparence adapter (UMAX Data Systems, Inc., Willich, Germany). Spot detection, quantification, and alignment were performed using the software package PDQuest (Bio-Rad, Richmond, CA, USA). Experiment Nine (primary mixed/neuronal cortical cell cultures exposed to oxygen/glucose deprivation) This study was performed to confirm that under conditions of oxidative stress, AEOL 10150 reduces cell death in association with specific inhibition of superoxide-mediated injury as defined by preservation of aconitase activity [26]. Mixed neuronal and glial cultures were prepared from embryonic day 18 rat cerebral hemispheres as described previously [27,28]. Cells were plated in 24-well dishes at a density of 1.2 ⫻ 106 cells/well. Mature cells (14–16 d in vitro) were used for experiments. Cells were washed twice in deoxygenated glucose-free Hank’s balanced salt solution (HBSS) and placed in a sealed incubator containing 0%O2/ 95% argon/5% CO2 for a period of 2 h. Chamber O2

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Table 2. Physiologic Values For Experiment Two (Rat Focal Ischemia/Intracerebroventricular Treatment 90 min After Reperfusion)

Preischemia body weight (g) Day 7 body weight (g) 10 min preischemia MAP (mmHg) Arterial pH PaCO2 (mmHg) PaO2 (mmHg) Glucose (mg/dl) Hematocrit (%) Temperature 45 min after onset of ischemia MAP (mmHg) Arterial pH PaCO2 (mmHg) PaO2 (mmHg) Temperature 10 min after termination of ischemia MAP (mmHg) Arterial pH PaCO2 (mmHg) PaO2 (mmHg) Temperature

Vehicle (n ⫽ 19)

AEOL 10113 300 ng (n ⫽ 17)

AEOL 10150 300 ng (n ⫽ 16)

AEOL 10150 4500 ␮g (n ⫽ 15)

277 ⫾ 11 229 ⫾ 52

275 ⫾ 10 257 ⫾ 49

281 ⫾ 15 289 ⫾ 36

275 ⫾ 11 266 ⫾ 45

81 ⫾ 9 7.36 ⫾ 0.04 38 ⫾ 3 175 ⫾ 27 118 ⫾ 18 41 ⫾ 2 37.5 ⫾ 0.1

83 ⫾ 15 7.38 ⫾ 0.04 37 ⫾ 3 177 ⫾ 26 120 ⫾ 13 42 ⫾ 1 37.5 ⫾ 0.1

78 ⫾ 11 7.37 ⫾ 0.04 39 ⫾ 4 175 ⫾ 31 115 ⫾ 9 42 ⫾ 2 37.5 ⫾ 0.2

82 ⫾ 12 7.37 ⫾ 0.05 38 ⫾ 4 176 ⫾ 21 128 ⫾ 18 42 ⫾ 1 37.5 ⫾ 0.1

83 ⫾ 11 7.32 ⫾ 0.04 39 ⫾ 4 161 ⫾ 27 37.5 ⫾ 0.1

83 ⫾ 13 7.33 ⫾ 0.04 38 ⫾ 4 156 ⫾ 25 37.5 ⫾ 0.1

80 ⫾ 11 7.32 ⫾ 0.05 39 ⫾ 4 150 ⫾ 30 37.5 ⫾ 0.1

78 ⫾ 13 7.32 ⫾ 0.04 41 ⫾ 4 156 ⫾ 25 37.5 ⫾ 0.1

75 ⫾ 11 7.34 ⫾ 0.05 38 ⫾ 5 162 ⫾ 26 37.5 ⫾ 0.1

80 ⫾ 15 7.35 ⫾ 0.04 39 ⫾ 5 166 ⫾ 23 37.5 ⫾ 0.1

80 ⫾ 12 7.34 ⫾ 0.04 38 ⫾ 5 153 ⫾ 28 37.5 ⫾ 0.1

78 ⫾ 12 7.34 ⫾ 0.05 40 ⫾ 4 155 ⫾ 32 37.5 ⫾ 0.1

All values are mean ⫾ SD; MAP ⫽ mean arterial pressure; PaCO2 ⫽ arterial carbon dioxide partial pressure; PaO2 ⫽ arterial oxygen partial pressure.

concentrations were continuously monitored with an O2 analyzer. AEOL 10150 or MK-801 were added to the cultures 30 min prior to placement in O2/glucose deprivation (OGD) conditions. Following 2 h of OGD, glucosefree HBSS was replaced with minimum essential medium (MEM) containing experimental drugs and the dishes were returned to normoxia for 18 h. Lactate dehydrogenase (LDH) activity was then measured in the supernatant media as described previously [27,28]. In sister cultures, aconitase and fumarase activity measurements were made 8 h after OGD. Mitochondrial fractionation was performed as previously described [27]. Resuspended mitochondrial fractions were sonicated for 2 s. Aconitase activity was measured spectrophotometrically by monitoring the formation of cisaconitate from isocitrate at 240 nm in 50 mM Tris/HCl, pH 7.4, containing 0.6 mM MnCl2 and 20 mM isocitrate at 25°C [28,29]. Fumarase activity was measured by monitoring the increase in absorbance at 240 nm at 25°C in a 1 ml reaction mixture containing 30 mM potassium phosphate, pH 7.4 [30]. For the aconitase/fumarase experiments, a concentration of 10 ␮M AEOL 10150 was studied. Statistical analysis Subcortical, cortical, and total infarct volumes, physiologic values, and aconitase/fumarase activities were compared by one-way analysis of variance. Post-hoc testing was performed with Fisher’s protected least squares differences test when indicated by a significant F

ratio. Neurologic scores were compared among groups by the Kruskal-Wallis H or Mann-Whitney U statistics as appropriate. Peritoneal temperature values were compared qualitatively. Non-parametric data are reported as median ⫾ interquartile range. Parametric values are reported as mean ⫾ SD. Statistical significance was assumed when p ⬍ 0.05. RESULTS

Experiment One (relative behavioral neurotoxicity of AEOL 10113 vs. AEOL 10150) For AEOL 10113, an i.c.v. dose of 600 ng was found to cause just noticeable behavioral side effects. For AEOL 10150, 9 ␮g was found to produce just noticeable behavioral effects. Thus, toxicity from AEOL 10150 was 1/15th that of AEOL 10113. We, therefore, studied an AEOL 10150 dose of 300 ng to provide direct comparison to AEOL 10113 and an AEOL 10150 dose of 4500 ng as a maximal dose clearly devoid of adverse behavioral effects. Experiment Two (rat MCAO/i.c.v. AEOL 10113 vs. AEOL 10150) Physiologic values are given in Table 2. There were no differences among groups. Neurologic scores 7 d postischemia are depicted in Fig. 2. A main effect for intergroup differences was present (vehicle ⫽ 11 ⫾ 2,

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Fig. 2. Rats were subjected to 90 min of middle cerebral artery occlusion. Ninety minutes after onset of reperfusion, rats were given an intracerebroventricular injection of vehicle, AEOL 10113 300 ng, AEOL 10150 300 ng, or AEOL 10150 4500 ␮g. Neurologic function (18 ⫽ no deficit) was examined 7 d later. All treatment groups were different from the vehicle group (p ⱕ 0.02) but there were no differences among treatment groups. Open circles indicate values for individual rats. Horizontal bars indicate group median values.

AEOL 10113 300 ng ⫽ 14 ⫾ 5, AEOL 10150 300 ng ⫽ 15 ⫾ 2.5, AEOL 10150 4500 ng ⫽ 14 ⫾ 4, p ⫽ 0.01). All treatment groups were different from the vehicle group (AEOL 10113 p ⫽ 0.02; AEOL 10150 300 ng p ⫽ 0.002; AEOL 10150 4500 ng p ⫽ 0.01) but there were no differences among treatment groups. In the cortex, a main effect for intergroup infarct volumes differences was present (vehicle ⫽ 75 ⫾ 48 mm3, AEOL 10113 300 ng ⫽ 35 ⫾ 34 mm3, AEOL 10150 300 ng ⫽ 44 ⫾ 35 mm3, AEOL 10150 4500 ng ⫽ 50 ⫾ 37 mm3, p ⫽ 0.02). Post-hoc analysis revealed differences between AEOL 10113 300 ng and vehicle (p ⫽ 0.01) and between AEOL 10150 300 ng and vehicle (p ⫽ 0.02). In the subcortex, there was no difference among groups (vehicle ⫽ 78 ⫾ 24 mm3, AEOL 10113 300 ng ⫽ 64 ⫾ 30 mm3, AEOL 10150 300 ng ⫽ 59 ⫾ 15 mm3, AEOL 10150 4500 ng ⫽ 65 ⫾ 20 mm3, p ⫽ 0.06). For total infarct volume, a main effect for intergroup differences was present (vehicle ⫽ 153 ⫾ 67 mm3, AEOL 10113 300 ng ⫽ 100 ⫾ 51 mm3, AEOL 10150 300 ng ⫽ 102 ⫾ 48 mm3, AEOL 10150 4500 ng ⫽ 115 ⫾ 54 mm3, p ⫽ 0.02). All treatment groups were different from vehicle (AEOL 10113 300 ng, p ⫽ 0.01; AEOL 10150 300 ng, p ⫽ 0.01; AEOL 10150 4500 ng, p ⫽ 0.049). There were no differences among treatment groups. See Fig. 3. Experiment Three (rat MCAO/i.c.v. AEOL 10150 6 h after onset of reperfusion) Physiologic values during the peri-ischemic interval were similar to those recorded during Experiment Two

Fig. 3. Rats were subjected to 90 min of middle cerebral artery occlusion. Ninety minutes after onset of reperfusion rats were given an intracerebroventricular injection of vehicle, AEOL 10113 300 ng, AEOL 10150 300 ng, or AEOL 10150 4500 ng. Cerebral infarct volumes were measured 7 d later. Differences in cortical infarct volume were present between AEOL 10113 300 ng and vehicle (p ⫽ 0.01) and between AEOL 10150 300 ng and vehicle (p ⫽ 0.02). In the subcortex, there was no difference among groups (p ⫽ 0.06). Total infarct volumes were different between all treatment groups and vehicle (p ⱕ 0.049). There were no differences amongst treatment groups. Open circles indicate values for individual rats. Horizontal lines indicate group mean values.

(data not shown). There were no differences between groups. Infarct size remained markedly reduced in both cortex and subcortex by AEOL 10150 when given at 6 h after onset of reperfusion (Table 3). Despite a 43% reduction in total infarct volume by AEOL 10150, neu-

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Table 3. Cerebral Infarct Sizes (mm3) and Neurologic Scores in Rats Given Intracerebroventricular AEOL 10150 (300 ng) or Vehicle 6 h After Reperfusion from 90 min MCAO Vehicle (n ⫽ 16) AEOL 10150 (n ⫽ 18) p Value Cortex Subcortex Total Neurologic Score

84 ⫾ 52 76 ⫾ 25 160 ⫾ 74 9⫾3

36 ⫾ 53 53 ⫾ 31 92 ⫾ 80 9⫾1

0.011 0.051 0.015 0.670

Infarct values ⫽ mean ⫾ SD. Neurologic scores ⫽ median ⫾ interquartile range (18 ⫽ normal).

rologic scores were not different between groups (Table 3). Although total infarct volume correlated with neurologic score (p ⫽ 0.02), visual inspection of the data suggests that the preponderance of association occurred at total infarct volume values ⬍100 mm3 (Fig. 4). Experiment Four (rat AEOL 10150 pharmacokinetic analysis) At all intervals after i.c.v. injection of 300 ng AEOL 10150, no compound was detected in liver. Plasma AEOL 10150 was detected in two rats at negligible concentrations (4.6 and 3.2 ng/ml). Brain parenchymal AEOL 10150 concentration peaked within 1 h after injection. The tissue half-life was calculated to be approximately 10 h (Fig. 5).

Fig. 5. Brain parenchymal AEOL 10150 concentration in the hemisphere contralateral to intracerebroventricular injection of 300 ng as a function of time after injection in normal rats. The tissue half-life of AEOL 10150 was calculated to be approximately 10 h. Values ⫽ mean ⫾ SD.

between groups when averaged over 6 h (vehicle ⫽ 37.9 ⫾ 0.6°C; AEOL 10150 ⫽ 38.4 ⫾ 0.9°C) or 18 h (vehicle ⫽ ⫾ 37.8 ⫾ 0.4°C; AEOL 10150 ⫽ 38.1 ⫾ 0.5°C) after treatment. Experiment Six (mouse MCAO/i.v. AEOL 10150)

Experiment Five (rat AEOL 10150 6 h post-MCAO/ temperature measurement) AEOL 10150 (300 ng i.c.v.) had no effect on body temperature. Peritoneal temperature values were similar

Fig. 4. Neurologic score (18 ⫽ no deficit) versus total infarct volume in individual rats subjected to 90 min of middle cerebral artery occlusion and treatment 6 h later with intracerebroventricular AEOL 10150 or vehicle. Values were recorded 7 d after the ischemic insult. Neurologic score correlated with total infarct volume (p ⫽ 0.02).

Physiologic values are given in Table 4. There were no differences among groups. Plasma AEOL 10150 concentrations measured 24 h after onset of infusion had large coefficients of variation, but concentrations were dependent upon dose of drug given (vehicle ⫽ below limit of detection; low dose ⫽ 2.1 ⫾ 1.9 ␮g/ml; high dose ⫽ 10.6 ⫾ 9.1 ␮g/ml; p ⬍ 0.0001). A main effect was present for neurologic scores (p ⫽ 0.012). Scores were improved in the high-dose AEOL 10150 (2 ⫾ 2; p ⫽ 0.01) and low-dose AEOL 10150 (2.5 ⫾ 2; p ⫽ 0.03) groups vs. vehicle (3 ⫾ 0.5). There was no difference in scores between the AEOL 10150treated groups (p ⫽ 0.56). See Fig. 6. In the cortex, despite a 28% reduction in infarct size with AEOL 10150, a main effect for intergroup differences was absent (vehicle ⫽ 37 ⫾ 16 mm3, AEOL 10150 low dose ⫽ 28 ⫾ 16 mm3, AEOL 10150 high dose ⫽ 27 ⫾ 16 mm3, p ⫽ 0.10). In the subcortex, a main effect for intergroup differences was present (vehicle ⫽ 24 ⫾ 9 mm3, AEOL 10150 low dose ⫽ 16 ⫾ 8 mm3, AEOL 10150 high dose ⫽ 18 ⫾ 9 mm3, p ⫽ 0.01). Post-hoc analysis revealed differences between both AEOL 10150 treatment groups and vehicle (AEOL 10150 low dose, p ⫽ 0.004; AEOL 10150 high dose, p ⫽ 0.05). There

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H. SHENG et al. Table 4. Physiologic Values for Experiment Six (Mouse MCAO/Intravenous AEOL 10150)

Preischemia Body Weight (g) Day 1 body weight (g) 10 min preischemia MAP (mmHg) Arterial pH PaCO2 (mmHg) PaO2 (mmHg) Pericranial temperature (°C) 30 min after onset of ischemia MAP (mmHg) Pericranial temperature (°C) 10 min after termination of ischemia MAP (mmHg) Pericranial Temperature (°C)

Vehicle (n ⫽ 24)

Low Dose (n ⫽ 24)

High Dose (n ⫽ 24)

22 ⫾ 1 21 ⫾ 1

23 ⫾ 1 22 ⫾ 2

23 ⫾ 1 22 ⫾ 1

76 ⫾ 4 7.19 ⫾ 0.05 35 ⫾ 5 213 ⫾ 17 37.0 ⫾ 0.1

77 ⫾ 3 7.17 ⫾ 0.04 35 ⫾ 5 215 ⫾ 13 37.0 ⫾ 0.1

77 ⫾ 4 7.17 ⫾ 0.04 37 ⫾ 4 217 ⫾ 13 37.0 ⫾ 0.1

72 ⫾ 4 37.0 ⫾ 0.1

73 ⫾ 4 37.0 ⫾ 0.2

72 ⫾ 5 37.0 ⫾ 0.1

74 ⫾ 6 37.0 ⫾ 0.1

76 ⫾ 4 37.0 ⫾ 0.1

73 ⫾ 5 37.0 ⫾ 0.1

All values are mean ⫾ SD; MAP ⫽ mean arterial pressure; PaCO2 ⫽ arterial carbon dioxide partial pressure; PaO2 ⫽ arterial oxygen partial pressure. There were no differences among groups.

was no difference between treatment groups. For total infarct volume, a main effect was present (vehicle ⫽ 60 ⫾ 26 mm3, AEOL 10150 low dose ⫽ 45 ⫾ 22 mm3, AEOL 10150 high dose ⫽ 46 ⫾ 23 mm3, p ⫽ 0.048). Post-hoc analysis revealed differences between both treatment groups and vehicle (AEOL 10150 low dose, p ⫽ 0.03; AEOL 10150 high dose, p ⫽ 0.04). There was no difference between treatment groups. See Fig. 7. Experiment Seven (mouse AEOL 10150 pharmacokinetic analysis) Values for brain and plasma AEOL 10150 concentrations are presented in Table 5 for mice subjected to 60 min MCAO and AEOL 10150 or vehicle infusion begin-

Fig. 6. Neurologic scores assigned 24 h after 60 min middle cerebral artery occlusion in mice treated intravenously with vehicle or low- or high-dose AEOL 10150 for 24 h. Individual values are depicted as open circles. Group median values are horizontal bars. Neurologic scores were improved in the high-dose AEOL 10150 (p ⫽ 0.01) and low-dose AEOL 10150 (p ⫽ 0.03) groups vs. vehicle (3 ⫾ 0.5). There was no difference in scores between the AEOL 10150-treated groups (p ⫽ 0.56). 0 ⫽ no deficit.

ning at onset of reperfusion. Concentrations in the left (nonischemic) hemisphere, right (ischemic) hemisphere, and plasma were dose-dependent at 5 h after onset of infusion (p ⬍ 0.0001). In the high-dose group, in which plasma and tissue was sampled at all three postischemic intervals after onset of infusion (i.e., 1, 5, and 24 h), a time-dependent accumulation of AEOL 10150 was observed in the right (ischemic) hemisphere (p ⫽ 0.05). This effect did not achieve statistical significance in the left (nonischemic) hemisphere (p ⫽ 0.09) or plasma (p ⫽ 0.19).

Experiment Eight (mouse MCAO/AEOL 10150 post-treatment/proteomic analysis) All mice subjected to ischemia had a reduction of laser Doppler red blood cell flow velocity to ⬍10% of preischemia values. Physiologic values (not shown) were similar to those reported for Experiment Six. For proteomic analysis, more than 850 proteins were resolved and specific changes in the 2D gel electrophoresis protein signatures were apparent between normal brain, ischemic brain, and ischemic brain ⫹ drug. An example of these changes is illustrated in Fig. 8. Spot detection, quantification, and alignment of all proteins were distinguishable following 2D gel electrophoresis. In the sham surgery AEOL 10150 group, 40 proteins were upregulated and 6 were downregulated 2-fold or more relative to the sham vehicle group. In the MCAO plus vehicle group, 41 proteins were upregulated and 9 were downregulated. Following AEOL 10150 treatment in MCAO animals, 37 proteins were upregulated and 0 were downregulated.

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Table 5. AEOL 10150 Concentrations in Plasma and Brain at Varying Intervals After Onset of Intravenous AEOL 10150 in Mice Subjected to Middle Cerebral Artery Occlusion

1 h after onset of infusion Plasma (ng/ml) Left hemisphere (ng/g) Right hemisphere (ng/g) 5 h after onset of infusion Plasma (ng/ml) Left hemisphere (ng/g) Right hemisphere (ng/g) 24 h after onset of infusion Plasma (ng/ml) Left hemisphere (ng/g) Right hemisphere (ng/g)

Vehicle

Low Dose

High Dose

– – –

– – –

1⫾2 0 0

642 ⫾ 325 24 ⫾ 16 34 ⫾ 21

2599 ⫾ 1022a 62 ⫾ 18a 151 ⫾ 97a,b

– – –

– – –

5353 ⫾ 4700 267 ⫾ 258 582 ⫾ 645b

2475 ⫾ 471 45 ⫾ 14 78 ⫾ 22b

Values ⫽ mean ⫾ standard deviation. n ⫽ 5– 6 mice per treatment condition at each measurement interval. Filament occlusion (60 min) was performed in the right hemisphere. Low dose ⫽ 125 ␮g/kg AEOL 10150 bolus followed by 250 ␮g 䡠 kg⫺1 䡠 hr⫺1. High dose ⫽ 500 ␮g/kg bolus followed by 1 mg 䡠 kg⫺1 䡠 hr⫺1. a Difference between all three dosage groups, within respective hemispheres or plasma, at 5 h after onset of reperfusion, p ⬍ 0.0001. b Difference over time in right hemisphere in high-dose group, p ⬍ 0.01.

ⱖ100 ␮M. The peak AEOL 10150 effect occurred at 10 ␮M, which inhibited 48% of the OGD-induced LDH release. MK-801 (10 ␮M) caused a 66% inhibition of OGD-stimulated LDH release, but this was not different from the effect of 10 ␮M AEOL 10150 (p ⫽ 0.78). For fumarase activity, there was no effect (p ⫽ 0.76) of AEOL 10150 alone vs. normoxia control, OGD alone, or OGD plus AEOL 10150. Aconitase activity was reduced by 33% versus normoxia controls (p ⫽ 0.04). AEOL 10150 alone had no effect on aconitase activity. Addition of AEOL 10150 to OGD completely restored aconitase activity (p ⫽ 0.006 vs. OGD alone). Fig. 7. Individual regional infarct volumes measured 24 h after 60 min middle cerebral artery occlusion in mice treated intravenously with vehicle or low- or high-dose intravenous AEOL 10150 for 24 h. Individual values are given as open circles. Group mean values are horizontal lines. A main effect for treatment group was present for subcortical (p ⫽ 0.01) and total (p ⫽ 0.048) infarct volumes but was absent for cortical (p ⫽ 0.10) infarct volume. For both subcortex and total infarct volumes, both treatment groups were different from vehicle (p ⬍ 0.05) but there was no difference between treatment groups.

Experiment Nine (primary mixed/neuronal cortical cell cultures exposed to OGD) AEOL 10150 had no adverse effect when given to control mixed neuronal and glial cultures not exposed to OGD (Fig. 9). Untreated cultures subjected to OGD showed a 3-fold increase in LDH release. AEOL 10150 inhibited OGD-induced cell death in a concentrationdependent manner (p ⬍ 0.0001). A neuroprotective effect of AEOL 10150 was not observed at concentrations

DISCUSSION

The principal findings of this series of studies were as follows. Intracerebroventricular AEOL 10150 had 1/15th the potency of AEOL 10113 for inducing adverse neurobehavioral side effects in the rat. AEOL 10150 and AEOL 10113 had similar potency to reduce cerebral infarct size and neurologic deficit when given i.c.v. 90 min after reperfusion from MCAO. When given 6 h after reperfusion, AEOL 10150 reduced cerebral infarct volume by 43%. AEOL 10150 also reduced infarct size and neurologic deficit when given as a postischemic i.v. treatment in mice. AEOL 10150 had a long parenchymal elimination t1/2 (i.e., approximately 10 h) when given i.c.v. When given i.v., brain uptake was slow and greater in the ischemic hemisphere. AEOL 10150 had direct effects on brain parenchymal proteomic expression. When given after MCAO, AEOL 10150 partially inhib-

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Fig. 8. Two-dimensional gel electrophoresis of mouse brain proteins from animals subjected to sham surgery or 60 min middle cerebral artery occlusion. Animals were treated with either vehicle or 0.5 mg/kg i.v. AEOL 10150 plus a continuous i.v. infusion at 1.0 mg 䡠 kg⫺1 䡠 h⫺1 beginning 5 min after onset of reperfusion. Brains were sampled 6 h after onset of therapy. Identical subsections of the second dimension SDS gels are displayed for each group. This illustrates significant detectable differences in the 2D-protein signature following cerebral artery occlusion and/or antioxidant therapy. Fiduciary lines are provided to facilitate comparison.

ited changes in proteomic expression attributable to MCAO. In mixed cultures of neurons and glia, AEOL 10150 provided protection against OGD in a magnitude similar to MK-801 and selectively inhibited aconitase inactivation [28,31] consistent with SOD mimetic effects biochemically defined for this compound (Table 1). The in vitro protective effects of AEOL 10150 were most likely due to its actions on neurons, because astrocyte cultures exposed to a 2 h period of OGD do not show increased LDH release when exposed to the same insult [27]. We have previously reported on the neuroprotective effects of AEOL 10113 [16]. Most noteworthy, administration of AEOL 10113 as late as 6 h after reperfusion from 90 min MCAO in the rat caused a 54% reduction in total infarct volume and decreased neurologic deficit measured 7 d after the insult. Brain parenchymal aconitase activity was preserved and formation of 8-hydroxy-2⬘-deoxyguanosine was decreased. The current study that showed

neuroprotective efficacy with AEOL 10150 given 6 h after onset of reperfusion supports the conclusion that metalloporphyrin catalytic antioxidants offer a therapeutic treatment window of clinical relevance. For example, in human trials of gavestinel, a glycine recognition site antagonist, only 26% of stroke patients received study drug within 4 h while by 6 h, 99% of patients were treated [32]. If this represents the requisite therapeutic window for pharmacologic efficacy, identification of compounds having windows as great as 6 h is essential. AEOL 10113 is a member of a recently synthesized family of metalloporphyrins that are potent catalytic SOD mimetics and also have potential to bind nitric oxide [17,18,33]. Substitution of the N-ethylpyridinium side chain on AEOL 10113 with a 1,3-diethylimidazolium group (AEOL 10150) served to double potency of the compound as an SOD mimetic (Table 1). This substitution had little effect on neuroprotective efficacy as shown in the current series of experiments. However,

Metalloporphyrins and ischemic brain

Fig. 9. Cortical mixed neuronal/glial cultures were exposed to 2 h O2/glucose deprivation (OGD). Vehicle or varying concentrations of AEOL 10150 were present 30 min prior to, during and after OGD. Values represent mean ⫾ SD from four separate experiments (n ⫽ 16 –20 cultures/treatment). (A) Cumulative lactate dehydrogenase (LDH) release over 18 h post-OGD. *p ⬍ 0.01 relative to 0 ␮M AEOL 10150. (B) Mitochondrial fumarase activity (activity units/g protein) 8 h post-OGD. (C) Mitochondrial aconitase activity (activity units/g protein) 8 h post-OGD. *p ⬍ 0.01 relative to all other groups.

toxicity was markedly reduced indicating a marked superiority of AEOL 10150 as a neuroprotective agent. While the therapeutic dose of AEOL 10113 is one half

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the dose required to elicit adverse neurobehavioral effects (proptosis, ataxia, and hypersensitivity to sound), the therapeutic dose of AEOL 10150 is 1/15th the dose required to produce side effects. This improved therapeutic/toxic ratio predicts better tolerability of AEOL 10150 if given to humans. The mechanism of these adverse effects, however, is currently unknown and requires definition. Aside from therapeutic considerations, the fact that delayed administration of a catalytic antioxidant can rescue approximately one half the tissue destined to infarct within 1 week after MCAO calls into question mechanisms of action, and more importantly points to the need to define events reversible as late as 6 h, which are sensitive to this class of compounds. It is clear from both the in vivo work with AEOL 10113 [16] and the current in vitro work with AEOL 10150 that dismutation of •O2⫺ resulting from oxidative stress is a potent mechanism of action of these compounds. •O2⫺ formation is markedly increased within the first few moments after onset of MCAO [4] and is sustained for many hours after various forms of acute brain injury [2,3,34]. Efficacy of AEOL 10150 as late as 6 h after reperfusion suggests that delayed •O2⫺ formation may be critical in defining tissue damage. This is consistent with prior observations that AEOL 10113 given 6 h after reperfusion from MCAO prevented mitochondrial aconitase inactivation and reduced 8-hydroxy-2⬘-deoxyguanosine formation [16]. Microglia activation and neutrophil recruitment, known to result in •O2⫺ production, are evident by 6 h after MCAO [35,36]. It is unknown what effect metalloporphyrins have on these processes but this seems worthy of investigation. It also seems important to define if the neuroprotective effect of the metalloporphyrins persists with prolonged durations of recovery. The advent of proteomic techniques offers unique opportunities to understand responses of brain to both ischemia and therapeutic intervention. It has been shown that brain exhibits massive changes in mRNA transcription as measured by gene chip technology and validated by Western blot and in situ hybridization analysis [37]. To our knowledge there have been no reports on interactions between pharmacologic intervention and these events. The purpose of our preliminary study was to examine if such responses could be measured in a model demonstrated to be both neurologically and histologically sensitive to pharmacologic intervention. Indeed, proteomic responses to ischemia were observed and partial attenuation of these changes was caused by AEOL 10150. It came as some surprise, however, to also learn that AEOL 10150 alone (in the absence of ischemia) induced independent changes in protein expression. The mechanism for this is unknown. Pilot studies that examined tissue from nonischemic rats given AEOL 10150

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provided no gross histologic evidence for neuronal necrosis. We speculate that introduction of a potent catalytic antioxidant into nonischemic brain may have caused modulation of gene expression regulated by endogenous oxidative species. Restoration of oxidative species by ischemia may have inhibited these effects, therefore reducing the drug effect noted in normal brain. The data presented here is preliminary and is currently undergoing extensive analysis to identify the precise proteins that have undergone changes. Results of that work will be reported in a subsequent publication. AEOL 10150 given 6 h after reperfusion from MCAO caused a 43% reduction in total cerebral infarct volume caused by AEOL 10150 in the absence of an observable neurologic improvement. This is inconsistent with the findings reported for AEOL 10113 where treatment at 6 h did reduce neurologic deficit with a similar magnitude of reduction in infarct size [16]. The neurologic scoring system employed is well established [23]. However, examination of Fig. 4 suggests that the preponderance of correlation between neurologic score and infarct size occurred at values ⬍100 mm3, which approximates the mean infarct size for the AEOL 1050-treated group. We suggest that in this case, the neurologic examination was insensitive within the range of size of infarct reduction, although this requires further exploration. AEOL 10150 was found to be efficacious when given intravenously to mice recovering from MCAO. The magnitude of effect, while present, was relatively small. We speculate that this can be improved upon. The small size of the mouse required implantation of the smallest longterm infusion osmotic pump commercially available. We believe that there was variability in rate of delivery from this system, despite following recommended priming procedures. This is suggested by the coefficient of variation for plasma and brain concentration measured at 24 h after onset of infusion (Table 4). Improved strategies for drug delivery might reduce this variance and produce larger overall effects on outcome. Further work with kinetic modeling of the drug, including definition of brain concentration required to achieve maximal protection and a dosing strategy able to achieve these concentrations, may be of value in optimizing preclinical outcome studies and guide design of clinical trials. The implications of the current studies are limited, in part, by the use of a relatively short-term recovery interval [38]. Rats were observed for a period of 1 week while mice were observed for a period of 24 h. Use of chronic implantation of intraventricular cannula limited the duration of recovery because of possible confounds of infection expected from use of longer outcome intervals. In the mouse, mortality from major cerebral infarction is high with extended outcome intervals. Death as a measure of outcome in a laboratory brain ischemia model is

largely unsatisfactory because the etiology can be difficult to define. To avoid this confound, a recovery duration that caused little or no mortality was used. Nevertheless, confirmation of stability of the neuroprotection observed in these studies would be of considerable value. Because we now know that a single i.c.v. injection of AEOL 10150 reduces both infarct size and neurologic deficit, it is possible to design a study where i.c.v. injection could be delivered by stereotactic microinjection. This would avoid presence of a foreign body and allow recovery intervals of many weeks or months to be studied. In summary, we examined the effects of a novel metalloporphyrin catalytic antioxidant on the response of brain to temporary focal cerebral ischemia. When given either i.c.v. to rats or i.v. to mice, AEOL 10150 reduced both neurologic deficit and infarct size. The drug slowly penetrates the blood– brain barrier but offers a sustained brain parenchymal elimination t1/2 with neuroprotective efficacy when given as late as 6 h after onset of reperfusion. AEOL 10150 alters both genetic translation in normal brain but also attenuates translational events induced by ischemia. Study of metalloporphyrin catalytic antioxidants may allow novel insight into mechanisms of ischemic brain injury. Continued investigation for therapeutic potential is warranted. Acknowledgements — This work was supported by U.S. Public Health Service grants R01 NS38944-03, U10 HL 63397, PO1 HL 31992, RO1 NS39587 and Incara Pharmaceuticals Corp., Research Triangle Park, NC, USA. Drs. Crapo, Day, and Warner hold an equity position in and serve as consultants for Incara, Inc. The authors are grateful to Ann D. Brinkhous for expert technical assistance.

REFERENCES [1] Kontos, H. A.; Wei, E. P. Superoxide production in experimental brain injury. J. Neurosurg. 64:803– 807; 1986. [2] Globus, M. Y. T.; Busto, R.; Lin, B.; Schnippering, H.; Ginsberg, M. D. Detection of free radical activity during transient global ischemia and recirculation: effects of intraischemic brain temperature modulation. J. Neurochem. 65:1250 –1256; 1995. [3] Kil, H. Y.; Zhang, J.; Piantadosi, C. A. Brain temperature alters hydroxyl radical production during cerebral ischemia reperfusion in rats. J. Cereb. Blood Flow Metab. 16:100 –106; 1996. [4] Fabian, R. H.; DeWitt, D. S.; Kent, T. A. In vivo detection of superoxide anion production by the brain using a cytochrome c electrode. J. Cereb. Blood Flow Metab. 15:242–247; 1995. [5] Peters, O.; Back, T.; Lindauer, U.; Busch, C.; Megow, D.; Dreier, J.; Dirnagl, U. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J. Cereb. Blood Flow Metab. 18:196 –205; 1998. [6] Keller, J. N.; Kindy, M. S.; Holtsberg, F. W.; St. Clair, D. K.; Yen, H. C.; Germeyer, A.; Steiner, S. M.; Bruce-Keller, A. J.; Hutchins, J. B.; Mattson, M. P. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 18:687– 697; 1998. [7] Yang, G.; Chan, P. H.; Chen, J.; Carlson, E.; Chen, S.; Weinstein, P.; Epstein, C. J.; Kamii, H. Human copper-zinc superoxide

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