LY377770, a novel iGlu5 kainate receptor antagonist with neuroprotective effects in global and focal cerebral ischaemia

Neuropharmacology 39 (2000) 1575–1588 www.elsevier.com/locate/neuropharm

LY377770, a novel iGlu5 kainate receptor antagonist with neuroprotective effects in global and focal cerebral ischaemia Michael J. O’Neill a,*, Liesbeth Bogaert b, Caroline A. Hicks a, Ann Bond a, Mark A. Ward a, Guy Ebinger c, Paul L. Ornstein d, Yvette Michotte b, David Lodge b

d

a Eli Lilly & Co. Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium c Department of Neurology, University Hospital, Laarbeeklaan 101, 1090 Brussels, Belgium d Lilly Corporate Center, Indianapolis, IN 46285-0814, USA

Accepted 22 November 1999

Abstract We have evaluated the neuroprotective effects of the decahydroisoquinoline LY377770, a novel iGlu5 kainate receptor antagonist, in two models of cerebral ischaemia. Global ischaemia, induced in gerbils by bilateral carotid artery occlusion (BCAO) for 5 min, produced a large increase in locomotor activity at 96 hr post-occlusion and a severe loss of CA1 cells in the hippocampus histologically at 120 hr post-occlusion. LY377770 (80 mg/kg i.p. 30 min before or 30 min after BCAO followed by 40 mg/kg i.p. administered at 3 and 6 hr after the initial dose) attenuated the ischaemia-induced hyperactivity and provided (92%) and (29%) protection in the CA1 cells respectively. This protection was greater than that seen with maximally tolerated doses of other glutamate receptor antagonists (CGS19755, CPP, MK-801, ifenprodil, eliprodil, HA-966, ACEA1021, L701,324, NBQX, LY293558, GYKI52466 and LY300164). Focal ischaemia was induced by infusing 200 pmol of endothelin-1 (Et-1) adjacent to the middle cerebral artery and LY377770 was administered at 80 mg/kg i.p. immediately, 1 or 2 hr post-occlusion followed by 40 mg/kg i.p. 3 and 6 hr after the first dose. The infarct volume, measured 72 hr later, was reduced by LY377770 when given immediately (P⬍0.01), at 1 hr (P⬍0.05) but not significantly at 2 hr post-occlusion. Reference compounds, LY293558 (20 mg/kg i.p. and then 10 mg/kg as above) and MK-801 (2.5 mg/kg i.p.), both administered immediately post-occlusion produced significant (P⬍0.05) but somewhat less neuroprotection. In parallel microdialysis studies, LY377770 (75 mg/kg i.p.) attenuated ischaemia-induced increases in extracellular levels of glutamate, but not of dopamine. In conclusion, these results indicated that iGlu5 kainate receptors play a central role in ischaemic brain damage following global and focal cerebral ischaemia. LY377770 is a novel, soluble, systemically active iGlu5 antagonist with efficacy in global and focal ischaemia, even when administered post-occlusion. LY377770 may therefore be useful as a neuroprotectant in man.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Cerebral ischaemia; Kainate receptors; iGlu5 receptor; Glutamate antagonists; LY377770; Neuroprotection; Glutamate; Microdialysis

1. Introduction Glutamate and glutamate receptors play a central role in acute neurodegeneration following cerebral ischaemia (Meldrum and Garthwaite, 1990; Siesjo¨, 1992a,b). Ischaemia results in a loss of energy supply to the brain

* Corresponding author. Tel.: +44-(0)1276-853547; fax: +44(0)1276-853525. E-mail address: [email protected] (M.J. O’Neill).

leading to neuronal depolarization and massive release of excitatory amino acids and other neurotransmitters (Benveniste et al., 1984; Globus et al., 1988). The energy failure will also prevent the reuptake of glutamate by its transporter systems and reversal of the transporter may further increase extracellular glutamate levels (see Billups et al., 1998). This glutamate acts at ionotropic glutamate receptors (NMDA, AMPA and kainate) increasing calcium entry into the cell (see Gill and Lodge, 1997) and on phospholipase C-coupled metabotropic receptors increasing release of calcium from

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intracellular stores (see Kristian and Siesjo¨, 1998). This results in activation of calcium-dependent enzymes (phospholipases, proteases, nucleases, etc.) which ultimately leads to cell death (see Boxer and Bigge, 1997; del Zoppo et al., 1997). The neuroprotective potential of several excitatory amino acid antagonists has been extensively evaluated in animal models of global (e.g. bilateral carotid artery occlusion (BCAO) in the gerbil) and focal (e.g. middle cerebral artery occlusion (MCAO) in the rat) cerebral ischaemia. Early studies focused on the neuroprotective effects of competitive and non-competitive NMDA receptor antagonists (e.g. Gill et al., 1987; Park et al., 1988a; Park et al., 1988b; Bullock et al., 1990). However, the side-effect profile (psychotomimetic, learning and memory impairment; Morris et al., 1986; Koek et al., 1988) and the production of vacuoles and neuronal damage in rat neocortical areas (Olney et al. 1990, 1991) reduced interest in developing several of these compounds. Further problems (solubility, dose-limiting adverse effects, attaining neuroprotective concentrations, etc.) have plagued clinical trials with NMDA antagonists in ischaemic stroke (see Lees, 1997). More recent attention has switched to the AMPA receptor antagonists (Gill and Lodge, 1997). Neuroprotective effects in global ischaemia following pretreatment with the competitive AMPA receptor antagonist, NBQX, were first reported by Sheardown et al. (1990) and subsequently delayed treatment also proved to be effective (Li and Buchan, 1993; Sheardown et al., 1993). NBQX also reduced infarct volume following focal ischaemia in rats (Gill et al., 1992, Graham et al., 1996). Older quinoxalinedione AMPA antagonists, such as NBQX, however, suffered from poor solubility, precipitation in kidneys and nephrotoxicity (Xue et al., 1994), although some newer ones appear to have overcome this problem and retained neuroprotective activity (e.g. YM872; Kawasaki-Yatsugi et al., 1998; Shimizu-Sasamata et al., 1998). The selective and non-competitive 2,3-benzodiazepine class of AMPA antagonists also provided neuroprotection in both global (Le Peillet et al., 1992; Lodge et al., 1996) and focal (Smith and Meldrum, 1992: Xue et al., 1994) models of cerebral ischaemia in the rat. A distinct series of decahydroisoquinolines included highly soluble, parenteral and competitive AMPA receptor antagonists (Ornstein et al. 1993, 1995, 1996a,b; Schoepp et al., 1995). Among them, (3SR,4aRS,6RS,8aRS) -6-[2-(1H-tetrazol-5-yl)-ethyl]-1,2,3,4a,5,6,7,8a-decahydroisoquinoline-3-carboxylic acid (LY215490, or its active isomer LY293558), was neuroprotective in focal ischaemia in both the cat (Bullock et al., 1994) and rat (Gill and Lodge, 1995) and in global ischaemia in the gerbil (O’Neill et al. 1996b, 1998). Subsequent pharmacological studies showed that LY293558 was a mixed AMPA and iGlu5 kainate receptor antagonist (Bleakman et al., 1996) and other compounds in the series, e.g.

LY377770, were more selective iGlu5 antagonists (Bleakman and Lodge, 1998; O’Neill et al., 1998). Thus, on rat neurones, LY377770 had IC50s of approximately 0.3, 13, 50 and ⬎100 µM on iGlu5, AMPA NMDA and iGlu6 subtypes of glutamate receptor respectively (David Bleakman, personal communication). When members of this series of decahydroisoquinolines, with various antagonistic profiles at NMDA, AMPA and iGlu5 receptors, were studied in the gerbil global ischaemia model, LY377770 provided the greatest neuroprotection (O’Neill et al., 1998), raising the prospect that iGlu5 antagonists may be effective in other models of cerebral ischaemia. In the present studies we have attempted to replicate and extend the neuroprotective effects of LY377770 following global ischaemia in the gerbil by counting viable cells and TUNEL staining in the hippocampus and by assessing locomotor effects. We have also compared the effects of LY377770 in this global model with a panel of other glutamate receptor antagonists. We also evaluated LY377770, for the first time, in focal cerebral ischaemia using endothelin-1 (Et-1) to occlude the middle cerebral artery and compared the effects of LY377770 with those of LY293558 and MK-801. In parallel microdialysis studies, we investigated the effects of LY377770 on extracellular glutamate and dopamine levels in the striatum in this Et-1 focal model.

2. Materials and methods 2.1. Global ischaemia Global ischaemia was produced in gerbils by 5 min bilateral carotid artery occlusion (BCAO). 2.1.1. Animals and surgery Male Mongolian gerbils (Bantin and Kingman, Hull, UK) at least 3 months old and weighing in excess of 60 g were maintained in standard lighting conditions with food and water available ad libitum. The animals were anaesthetised with a 5% halothane/oxygen mixture and maintained using 2% halothane delivered with oxygen at 1 litre/min via a face mask throughout the operation. Through a midline cervical incision, both common carotid arteries were exposed, freed from surrounding connective tissue and clamped for 5 min. At the end of the occlusion period, blood flow was re-established. In sham-operated animals the arteries were exposed but not occluded. The wound was then sutured and the animals allowed to recover. Throughout surgery, body temperature was maintained at 37°C using a “K-TEMP” temperature controller/heating pad (International Market Supply, Cheshire, UK). After surgery, the animals were placed in a four-compartmental Thermacage (Beta Medical and Scientific, UK) which maintained the environ-

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mental temperature at 28°C and rectal temperatures were measured for a 6 hr period after occlusion. LY377770 was administered intraperitoneally (i.p) at a dose of 80 mg/kg followed by two subsequent i.p. doses of 40 mg/kg 3 and 6 hr later. In most experiments (see Results), this 80/40 dose was commenced 30 min before or 30 min after BCAO. 2.1.2. Locomotor activity 96 hr post-occlusion locomotor activity was measured in clear Perspex boxes (30×30×30 cm) with a metal base with a 2 cm covering of fine sawdust. Each of the 16 cages had five, equally spaced horizontal photocell beams 5.0 cm above the cage floor. Each beam break was recorded as a photocell count. All the boxes were individually connected to a Compaq PC and the photocell interruptions were recorded as the number of counts, using software provided by Greenacre Instruments Ltd. All experiments were videotaped for subsequent analysis. The animals were placed in individual boxes for 40 min and photocell interruptions recorded every minute. Statistical analysis of locomotor data was assessed using an ANOVA followed by an appropriate post hoc t-test, with P values ⬍0.05 being considered statistically significant. 2.1.3. Histology Five days after surgery the animals were perfused transcardially with 30 ml of 0.9% saline followed by 100 ml of 10% buffered formalin solution. The brains were removed and placed in 10% formalin for 3 days, processed and embedded in paraffin wax. Coronal sections (5 µm) were taken 1.5, 1.7 and 1.9 mm caudal to bregma using a microtome (Leitz 1400 sledge microtome). The slices were stained with haematoxylin and eosin and the neuronal density in the CA1 subfield of the hippocampus was measured using a microscope with grid lines (0.05 mm×0.05 mm) as described previously (O’Neill et al., 1998). The neuronal density was expressed as number of viable cells per mm CA1 hippocampus. Statistical analysis of histological data was assessed using a twotailed unpaired Student’s t-test, with P values ⬍0.05 being considered statistically significant. 2.1.4. TdT fragment end labelling of DNA Apoptotic endonucleases affect cellular DNA by producing classical DNA laddering and also generate free 3⬘-OH groups at the ends of these DNA fragments. Adjacent hippocampal sections were end-labelled using FragEL DNA framentation kits (Calbiochem-Novabiochem, UK). Briefly, sections were deparaffinised with washes of xylene before rehydration using decreasing concentrations of industrial methylated spirits (IMS). The sections were then permeabilized by incubating with 20 µg/ml proteinase K and endogenous peroxidases inactivated with 0.3% H2O2. Following a 10–20 min incu-

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bation with TdT equilibration buffer, the labelling reaction was performed. TdT labelling reaction mixture was prepared, applied to each section and incubated for 1.5 hr at 37°C. After termination of the labelling reaction, the labelled DNA was detected by incubating with a streptavidin horse radish peroxidase (HRP) conjugate. The reaction product was visualised using diaminobenzine (DAB) and counterstained with methyl green. Sections were dehydrated and prepared for microscopic examination and results expressed as the number of TUNEL positive cells per mm CA1 hippocampus. Statistical analysis of histological data was assessed using a two-tailed unpaired Student’s t-test, with P values ⬍0.05 being considered statistically significant. 2.1.5. Dosing protocols for other EAA antagonists For comparison, we examined the neuroprotective effects of a range of excitatory amino acid (EAA) antagonists in the gerbil model of cerebral ischaemia when treatment was initiated either 30 min before occlusion (“pre-occlusion”) or immediately after reperfusion (“post-occlusion”). The doses used were selected based on maximum tolerated dose following a gerbil behaviour. The compounds evaluated were: (1) competitive NMDA receptor antagonists cis-4-(phosphonomethyl)-2piperidine-carboxylic acid (CGS19755; dosed at 20 mg/kg i.p. 30 min before occlusion and followed by four further doses of 10 mg/kg i.p. at 2 hr intervals) and 4(3-phophono-propyl)piperazine-2-carboxylic acid (CPP; dosed at 10 mg/kg i.p. 30 min before occlusion and followed by four further doses of 5 mg/kg i.p. at 2 hr intervals); (2) non-competitive NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801; dosed at 2.5 mg/kg i.p. 30 min before occlusion); (3) glycine site antagonists of the NMDA receptor, (+)-3-amino-1-hydroxy-2-pyrrolidone (HA-966; dosed at 15 mg/kg i.p. 30 min before at 2 hr 30 min after occlusion), 5-nitro-6,7-dichloro-2,3quinoxalinedione (ACEA 1021; dosed at 25 mg/kg i.p. 30 min before and 2 hr 30 min after occlusion) and 7chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinolinone (L701,324; dosed at 40 mg/kg i.p. 30 min before at 2 hr 30 min after occlusion) (see Hicks et al., 1999); (4) polyamine site antagonists of the NMDA receptor, ifenprodil and eliprodil (both dosed at 20 mg/kg immediately after reperfusion followed by two further doses of 10 mg/kg i.p. at 2 hr intervals) (see Bath et al., 1996); (5) non-competitive AMPA receptor antagonist 1-(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) and a related substituted 2,3-benzodiazepine (+)3-N-acetyl-1(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (LY 300164), both dosed at 10 mg/kg i.p. immediately, 1, 2 and 4 hr after reperfusion (see Lodge et al., 1996); and (6) competitive AMPA/kainate receptor antagonists 2,3-dihydroxy-6nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX; dosed at

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30 mg/kg i.p. immediately, 3 and 6 hr after reperfusion) and (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)]decahydroisoquinoline-3-carboxylic acid (LY293558; dosed at 20 mg/kg i.p. eiher 30 min before occlusion or immediately after reperfusion followed by two further doses of 10 mg/kg i.p. at 3 hr intervals). In all experiments n=8–10 animals per group. 2.2. Focal ischaemia for histological studies Male Sprague Dawley rats (280–320 g) were anaesthetised with a 5% halothane/oxygen mixture and maintained using 2% halothane delivered with oxygen at 1 litre/min via a face mask throughout the operation. The rats were then placed on a thermostatically controlled heating blanket to maintain body temperature at 37– 38°C and positioned in a stereotaxic head holder. The scalp was incised over the parietal bones and a 28-gauge steel cannula inserted stereotaxically at the following coordinates from bregma: AP=0.9 mm, L=⫺5.2 mm and ⫺8.7 mm below skull surface. Endothelin-1 (200 pmol in 3 µl) was injected over a 2–5-min period. The cannula was withdrawn, the wound (muscles and skin) sutured and the rat allowed to recover. In the first experiment, dosing commenced immediately after Et-1 injection with LY377770 (80 mg/kg i.p. followed by 40 mg/kg i.p. 3 and 6 hr later (80/40 dose)), LY293558 (20 mg/kg i.p. followed by 10 mg/kg 3 and 6 hr later (20/10 dose)) or MK-801 (2mg/kg i.p.). In the second experiment, the same dosing regime for LY377770 was started either 1 or 2 hr post-occlusion. Three days after surgery the rats were given an overdose of anaesthetic and the thorax opened to permit perfusion with heparinised saline and formalin or triphenyltetrazolium chloride (TTC) via the heart or vena cava. The brains were removed for histology, the area of ischaemic damage was measured at eight stereotaxic levels and from this an infarct volume was calculated.

sates, which were split for the analysis of dopamine 25 µl, and glutamate 15 µl. Dopamine samples were protected from oxidation by adding 10 µl of antioxidant (0.02 M HCl, 0.2% Na metabisulfite, 0.02% Na2EDTA) to the vial. Effects of LY377770 in non-ischaemic animals were assessed by taking six baseline samples before administering a single dose of LY377770 (75 mg/kg i.p.) and further samples every 20 min for 3 hr. Effects of pretreatment or post-treatment were assessed by giving LY377770 (75 mg/kg i.p.) 30 min before or after 120 pmol/6 µl of Et-1 was infused (1 µl/min) via the MCA cannula. Sampling continued every 20 min for 6 hr. 2.3.1. Materials and electrochemical analysis of neurotransmitters Dopamine, glutamate and o-phthalaldehyde were obtained from Sigma (St. Louis, MO, USA), β-mercaptoethanol from Janssen Chimica (Beerse, Belgium) and Na2EDTA and solvents from Merck (Darmstadt, Germany). All aqueous solutions were prepared in fresh water purified by the Seralpur Pro 90 CN system (Belgolabo, Overijse, Belgium) and filtered through a membrane filter (pore size 0.2 µm). For analysis of dopamine, reversed-phase ion-pair microbore liquid chomatography was used with electrochemical detection (Smolders et al., 1996). Chromatographic conditions and precolumn derivatisation procedures for the analysis of glutamate and GABA have been described in detail (Smolders et al., 1995). Briefly, precolumn derivatisation was performed with o-phthalaldehyde/β-mercaptoethanol and then reversed-phase micobore liquid chroma-

2.3. Focal ischaemia for microdialysis studies Male Wistar rats (250-280 g), anaesthetised with a mixture of ketamine and diazepam (50 mg:5 mg/kg i.p.), were placed in a stereotaxic head holder. A cannula (CMA 12 probe with 3 mm of membrane removed) (CMA, Stockholm, Sweden) was positioned close to the middle cerebral artery (MCA) at AP +0.3 mm, L +5.6 mm and V +4.0 mm relative to bregma. A CMA 12 dialysis probe (3 mm) was also stereotaxically implanted in the ipsilateral striatum at AP +1.2 mm, L +2.4 mm and V +2.8 mm relative to bregma. The rats were then given 4 mg/kg 1% ketoprofene i.p. and allowed to recover. The probe in the striatum was perfused continuously with Ringers solution (1.1 mM CaCl2) at a flow rate of 2 µl/min. Sampling was started 24 hr after surgery. Sampling periods of 20 min resulted in 40 µl dialy-

Fig. 1. The effects of LY377770 administered 30 min before or 30 min after 5 min BCAO on rectal temperatures (mean±SEM; n=8 animals per group) measured at 30 min intervals for 6 hr post-occlusion. The dose of LY377770 was 80 mg/kg i.p. 30 min before or 30 min after 5 min BCAO followed by two further doses of 40 mg/kg i.p., 3 and 6 hr later (80/40). In this and the following three figures, this 80/40 dose was administered 30 min before or 30 min after the occlusion. LY377770 had no significant effect on rectal temperature (Student’s t-test).

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Fig. 3. The effects of LY377770 on the neuronal density at three stereotaxic levels in the CA1 region of the hippocampus 5 days after surgery. Histological results are expressed as mean±SEM viable cells/mm CA1 hippocampal region (n=8 animals per group). BCAO for 5 min produced severe damage to the CA1 region (P⬍0.0001) while LY377770 (80/40 see Fig. 1) produced significant protection when dosing was initiated 30 min prior (P⬍0.001) or 30 min after (P⬍0.05) occlusion (Student’s t-test).

3. Results 3.1. Global ischaemia in the gerbil

Fig. 2. The effects of LY377770 on ischaemia-induced hyperactivity 96 hr post-occlusion. Locomotor activity was measured for a 40-min period and is illustrated as the number of counts (mean±SEM; n=8 animals per group) in 10 min bins at 10, 20, 30 and 40 min (Fig. 2a) or total locomotor activity counts for a 40-min period (Fig. 2b). Shamoperated animals habituated rapidly to the apparatus and maintained low locomotor activity counts. In contrast, 5 min of BCAO caused a large increase in locomotor activity counts which was sustained for the 60-min period. LY377770 (80/40 see Fig. 1), commenced 30 min before (P⬍0.01) or 30 min after (P⬍0.05) the BCAO, attenuated the ischaemia-induced hyperactivity (Student’s t-test). ** P⬍0.01 vs Sham control, + P⬍0.05, ++ P⬍0.01 vs BCAO control.

tography with gradient elution and fluorescence was used to measure glutamate. Striatal dopamine and glutamate dialysate concentrations were expressed as percentages of basal values. Data were not corrected for the recovery across the membrane of the microdialysis probes. The basal value was taken as the mean of six stable dialysate concentrations (basal conditions). Data were analysed with oneway analysis of variance (ANOVA) for repeated measures, and Fisher’s protected least significance difference (Fisher’s PLSD) post hoc test.

In these experiments LY377770 (80 mg/kg followed at 3 and 6 hr by 40 mg/kg i.p.) was administered 30 min before or 30 min after 5 min BCAO. Rectal temperatures, measured before and after BCAO treatment with LY377770, indicated that global ischaemia raised rectal temperatures but that LY377770 had no significant effect on rectal temperature in the 6 hr post-occlusion (Fig. 1). Locomotor activity, assessed 96 hr post-occlusion, showed a large increase following 5 min BCAO in untreated ischaemic animals compared with sham-operated animals (Fig. 2). LY377770 produced a marked attenuation in the ischaemia-induced hyperactivity whether administered 30 min before occlusion (P⬍0.01) or 30 min post-occlusion (P⬍0.05; Fig. 2). Histological results indicated that there was a severe loss of CA1 hippocampal cells 120 hr post-occlusion. LY377770 protected against this ischaemia-induced cell death when administered pre- or post-occlusion. The protection (92%; P⬍0.001) was largest when administration was initiated 30 min before occlusion, but significant protection (29%; P⬍0.01) was also observed when LY377770 was administered 30 min postocclusion (Fig. 3). Adjacent sections were stained for TUNEL-positive nuclei using the TUNEL stain. Results indicated that 5 min BCAO produced a large increase in TUNEL-positive cells in the CA1 hippocampal region (Fig. 4). LY377770 prevented the ischaemia-induced increase in

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3.2. Focal cerebral ischaemia

Fig. 4. The effects of LY377770 on the number of TUNEL positive cells in the CA1 region of the hippocampus 120 hr post-occlusion. BCAO for 5 min produced a large increase in the number of apoptotic cells in the CA1 hippocampal region (P⬍0.001). LY377770 (80/40 Fig. 1) blocked the ischaemia-induced increase in TUNEL positive cells when administered prior to occlusion (P⬍0.001) and also provided a significant attenuation when administered post-occlusion (P⬍0.05). Results are expressed as mean±SEM. TUNEL-positive cells/mm CA1 hippocampal region (n=8 animals per group; Student’s t-test).

TUNEL positive cells when administered 30 min prior to occlusion. The compound also attenuated the number of ischaemia-induced apoptotic cells when administered 30 min after occlusion (Fig. 4). In a large series of studies we investigated the effects of several other excitatory amino acid antagonists (Fig. 5), administration being initiated either 30 min before (pre-occlusion) or immediately after (post-occlusion) BCAO. Compounds were dosed at near maximally tolerated doses as judged in preliminary studies on control animals. Results indicated that competitive (CGS19755, CPP) and glycine site (HA-966, ACEA1021, L701,324) NMDA receptor antagonists given pre-occlusion failed to provide any neuroprotection, whereas the channel blocking (MK-801) and polyamine site (ifenprodil, eliprodil) NMDA antagonists given immediately postocclusion offered about 10% protection (P⬍0.05). The selective and non-competitive AMPA antagonists, GYKI52466 and LY300164, given immediately postocclusion provided no significant and about 10% protection (P⬍0.05) respectively, whereas a competitive and mixed AMPA/kainate receptor antagonist (NBQX) given at the same time-point provided about 25% protection (P⬍0.01). The mixed AMPA/iGlu5 antagonist, LY293558, given pre- or post- occlusion showed about 55% (P⬍0.01) and 25% (P⬍0.01) protection respectively. This compares with 92% (P⬍0.001) and 45% (P⬍0.01) protection showed by the selective iGlu5 antagonist, LY377770.

In focal ischaemia studies in the rat, Et-1 injection near the MCA produced a large area of damage in the striatum and cortical regions of untreated animals. In the first experiment, LY377770 (80 mg/kg i.p.) and LY293558 (20 mg/kg i.p.) administered immediately after occlusion, followed by two further injections at half the dose 3 and 6 hours later, were compared with MK801 (2.5 mg/kg i.p.). Results indicated that LY293558 (P⬍0.05), LY377770 (P⬍0.01) and MK-801 (P⬍0.05) all produced significant reductions in the infarct volume. LY377770 was, however, the most effective (Figs. 6 and 7). In the second experiment we evaluated the time window of protection with LY377770. LY377770 was administered at 80 mg/kg i.p. at either 1 or 2 hr postocclusion followed by two further doses of 40 mg/kg at 3 hr intervals after the initial injection (n=10–12 animals per group). LY377770 produced a significant reduction in the area of damage at four stereotaxic levels when administered 1 hr after the occlusion (Fig. 8a). The compound also produced some attenuation of the area of damage when administered 2 hr after occlusion, but this only reached significance at one stereotaxic level (Fig. 8b). When the areas were integrated, LY377770 produced a significant reduction in the infarct volume (P⬍0.05) when administered 1 hr after occlusion. The compound also tended to reduce the infarct volume when administered 2 hr after occlusion, but this failed to reach significance (Fig. 8c; P=0.064). 3.3. Microdialysis studies in focal ischaemia LY377770 (75 mg/kg) produced an approximate 30% decrease in extracellular glutamate levels in the striatum of non-ischaemic control animals (Fig. 9a). Infusion of Et-1 near the MCA produced a large 50–60-fold increase in extracellular glutamate levels in the striatum. This increase was slightly delayed and was not apparent until the fraction collected one after that in which the Et-1 infusion was started. The increase was most marked in the first hour after Et-1 infusion, but was still present to some extent 3 hr later (Fig. 9b). LY377770 administered at 75 mg/kg i.p. 30 min before Et-1 infusion completely blocked the ischaemia-induced increase in extracellular glutamate at all time-points (Fig. 9b). When administered 40 min after Et-1 infusion, LY377770 still attenuated the ischaemia-induced glutamate levels at later time-points (Fig. 9c). In contrast to glutamate, LY377770 produced a transient increase in extracellular dopamine levels in control animals (Fig. 10a). Infusion of Et-1 near the MCA produced a 25–30-fold increase in extracellular dopamine levels in the striatum. This increase was rapid and appeared in the same fraction as the Et-1 infusion was

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Fig. 5. A comparison of the neuroprotective effects of a range of excitatory amino acid antagonists in the gerbil model of cerebral ischaemia when treatment was initiated either 30 min before occlusion (“pre-occlusion”) or immediately after reperfusion (“post-occlusion”). The compounds evaluated were: (1) competitive NMDA receptor antagonists, CGS19755 and CPP; (2) non-competitive NMDA receptor antagonist, MK-801; (3) glycine site antagonists of the NMDA receptor, HA-966, ACEA 1021, L701,324 (see Hicks et al., 1999); (4) polyamine site antagonists of the NMDA receptor, ifenprodil and eliprodil; (5) non-competitive AMPA receptor antagonists, GYKI 52466 and LY300164; (6) competitive AMPA/kainate receptor antagonists, NBQX and LY293558; and (7) the iGlu5 antagonist, LY377770. The results clearly indicate that both competitive AMPA antagonists and iGlu5 antagonists provide greater neuroprotection than NMDA receptor antagonists or non-competitive AMPA receptor antagonists based on the 2,3-benzodiazepine structure. In all experiments n=8–10 animals per group (Student’s t-test: for details of exact experimental conditions see Materials and methods). * P⬍0.05, ** P⬍0.01, *** P⬍0.001 vs 5 min BCAO control.

infusion, LY377770 also failed to have any effect on the ischaemia-induced dopamine levels (Fig. 10c).

4. Discussion Using histological, behavioural and biochemical measures, the present studies show that a new iGlu5 kainate receptor antagonist, LY377770, is neuroprotective in models of global and focal cerebral ischaemia. 4.1. Global ischaemia studies

Fig. 6. Effects of LY377770 and LY293558 on cerebral ischaemia assessed 72 hr after Et-1-induced MCAO in the rat. LY377770 (80/40; see Fig. 1) and LY293558 (20/10—same protocol as for LY377770) dosing was initiated immediately after completion of the Et-1 injection. Infarct volume (mm3; mean±SEM; n=10–12 animals per group) Both LY293558 and LY377770 produced a significant reduction in the infarct volume (P⬍0.05 and P⬍0.01, respectively; Student’s t-test).

started. The increase was transient and only present for 1 hr after Et-1 infusion (Fig. 10b). LY377770 administered at 75 mg/kg i.p. 30 min before Et-1 infusion had no effect on the ischaemia-induced increase in dopamine levels (Fig. 10b). When administered 40 min after Et-1

The present data confirm the previous report (O’Neill et al., 1998) that LY377770 is neuroprotective in the gerbil using a different dosing protocol. The core body temperature data suggest that this neuroprotection with LY377770 is unlikely to be due to pharmacological induction of hypothermia as has been suggested with other glutamate antagonists (Buchan and Pulsinelli, 1990; Corbett et al., 1990; Nurse and Corbett, 1996). However, further studies may be required to confirm this as it has been suggested that some excitatory amino acid ligands only provide protection in models of global ischaemia when histological evaluation is carried out 1 week after occlusion and that the protection is lost or much smaller if examined 4 weeks later. For example it

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has been demonstrated that NBQX produces prolonged mild hypothermia which may delay rather than protect against cell death (Nurse and Corbett, 1996). Apoptosis has been suggested to account for the delayed cell death in global ischaemia (Green and Reed, 1998; Thornberry and Lazebnik, 1998) and the present data with TUNEL staining, in as far as this signals apoptosis, suggest that LY377770 protects cells from this form of cell death at 5 days after BCAO. The present results also extend the histological results into the behavioural realm. Several previous studies have also shown BCAO in gerbils produces an increase in locomotor activity (e.g. Mileson and Schwartz, 1991; Yamamoto et al., 1993) and it has been suggested that this correlates with loss of hippocampal neurones (Anderson et al., 1997). In agreement with this, our results indicate that 5 min of global cerebral ischaemia cause a large increase in locomotor activity which can be prevented or attenuated when LY377770 is administered 30 min prior to or 30 min after BCAO respectively. Hyperactivity scores appear to correlate inversely with the number of remaining CA1 neurones in each of the groups of animals. Other studies have also demonstrated that pharmacological neuroprotection attenuates the hyperactivity (Judge et al., 1991; Benham et al., 1993; Schwartz et al., 1994; O’Neill et al., 1996a). However, this may not be true for all pharmacological interventions and we have observed previously that some compounds provide some neuroprotection without reducing the ischaemia-induced hyperactivity. For example, some free-radical scavengers and antioxidants provided protection without attenuating the hyperactivity (O’Neill et al., unpublished results). It is now well established that compounds acting as excitatory amino acid receptors have neuroprotective

effects in several forms of cerebral ischaemia, but have had side-effect issues which have limited their clinical usefulness (see Introduction). Such preclinical studies demonstrated the importance of NMDA and AMPA receptors in the neuronal damage following global ischaemia but the role of kainate receptors in this process has not been elucidated. Only recently have selective kainate receptor antagonists been available for comparison (Bleakman and Lodge, 1998; Chittajallu et al., 1999). In the last four years we have profiled the effects of several excitatory amino acid antagonists in our model of global ischaemia (Fig. 5). Our studies indicate that various NMDA antagonists (MK-801, CGS19755, CPP, HA-966, ACEA1021, L701,324, ifenprodil, eliprodil; Bath et al., 1996; O’Neill et al., 1996a,b; Hicks et al., 1999) and selective AMPA antagonists (GYKI52466, LY300164; Lodge et al., 1996; O’Neill et al., 1998) provide at most modest protection. The degree of protection in our model with these compounds is, in general, somewhat less than reported in the literature (see Introduction). This may be explained by the severity of our gerbil BCAO model. This is indicated by the occlusion time–response curve, the near complete loss of CA1 neurones and the core temperature control achieved by the use of thermacages (O’Neill et al., 1998; Hicks et al., 1999). In contrast to the poor to modest protection seen with the selective AMPA antagonists, GYKI52466 and LY300164, the mixed AMPA/kainate antagonists, NBQX and LY293558, provided good protection (O’Neill et al. 1997, 1998; Hicks et al., 1999). Since LY293558 only displaces binding at iGlu5, and not at iGlu6, iGlu7 or KA1/2 subunits, antagonism at iGlu5containing kainate receptors would appear to be the

Fig. 7. Effects of MK-801 on the area (mm2) and volume (mm3) of damage 72 hr after Et-1-induced MCAO in the rat. MK-801 (2.5 mg/kg i.p.) was administered immediately Et-1 injection (n=10 animals per group). MK-801 produced a significant reduction in the area (a) of damage at four stereotaxic levels and in the integrated infarct volume (b, P⬍0.05; Student’s t-test).

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Fig. 8. Effects of LY377770 on the area of damage (mm2) and infarct volume (mm3) 72 hr after middle cerebral artery occlusion in the rat. LY377770 was administered at 80 mg/kg i.p. at 1 or 2 hr postocclusion followed by two further doses of 40 mg/kg at 3 hr intervals after the initial injection (n=10–12 animals per group). LY377770 produced a significant reduction in the area of damage at four stereotaxic levels when administered 1 hr after the occlusion (a). The compound also produced some attenuation of the area of damage when administered 2 hr after occlusion, but this only reached significance at one stereotaxic level (b). LY377770 also produced a significant reduction in the infarct volume (P⬍0.05) when administered 1 hr after the occlusion (c) and attenuated the infarct volume when administered 2 hr after occlusion, but this failed to reach significance (Student’s t-test).

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Fig. 9.

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important factor explaining their increased efficacy as neuroprotectants. It is possible, however, that the short duration of action of the 2,3-benzodiazepines may limit their effectiveness. The present results with the selective iGlu5 antagonist, LY377770, further support the importance of iGlu5-containing kainate receptors in the hippocampal damage following global ischaemia. Interestingly, however, another more selective iGlu5 antagonist, LY382884, was less effective than LY377770 but the reasons for this remain unclear (O’Neill et al., 1998). The possible explanations include some other pharmacological activity of LY377770, e.g. the weak AMPA and NMDA receptor activity (see Introduction), or possibly pharmacodynamics. 4.2. Focal ischaemia studies As with global ischaemia, because of the unavailability of selective kainate antagonists, no previous studies have directly assessed the role of these receptors in the damage following focal ischaemia. For these focal ischaemia studies, we used the relatively new Et-1 method introduced by Sharkey et al. (1993) and so we included MK-801 as a positive control with which to compare LY377770 with LY293558. MK801 (2.5 mg/kg) proved to be effective in reducing the infarct volume by approximately 30% (P⬍0.05) as previously reported in this model (Sharkey et al., 1994) and in several other focal ischaemia models (see Introduction). Although the infarct volume is somewhat smaller with this Et-1 model than some other MCAO models, it does have the advantage of less invasive surgery, less vascular damage and slower reperfusion. Additionally, the level of protection with MK-801 remains similar between the various models (Sharkey et al., 1994). Administered immediately after the Et-1 injection, the mixed AMPA/iGlu5 receptor antagonist LY293558 (20/10) also reduced the infarct volume by approximately 35% (P⬍0.05) whereas the selective iGlu5 receptor antagonist LY377770 reduced the infarct by some 45% (P⬍0.01). When LY377770 treatment was delayed by 1 and 2 hr the level of protection fell to

Fig. 9. Effects of LY377770 (75 mg/kg i.p.) on extracellular glutamate levels in the striatum. In control non-ischaemic rats, LY377770 produced a significant decrease in glutamate levels 20–180 min after injection (a). Infusion of Et-1 produced a large increase in extracellular glutamate levels 20 min after ischaemia (b and c). This increase was maximum in fractions 5 (5600%) and 6 (4800%), and was still present 2 hr later (2000%). Pre-treatment with LY377770 prevented the ischaemia-induced increase in extracellular glutamate at all time-points (b). When treatment was delayed 40 min, LY377770 still attenuated the ischaemia-induced increase in extracellular glutamate at later timepoints (80–160 min post-occlusion). Results are expressed as mean±SEM % of control extracellular glutamate levels. n=5–7 animals per group. * P⬍0.05, ** P⬍0.01, *** P⬍0.001 vs basal levels, + P⬍0.05, ++ P⬍0.01 vs Et-1 control.

Fig. 10. Effects of LY377770 (75 mg/kg i.p.) on extracellular dopamine levels in the striatum. In control rats (a), LY377770 produced a small transient increase in DA levels 20–60 min after injection. Infusion of Et-1 produced a large transient increase in extracellular dopamine levels 20 min after ischaemia (b and c). This increase was maximum in fractions 4 (1800%) and 5 (2800%) but was not present 2 hr later. LY377770, given before (b) or after (c), had no effect on the ischaemia-induced increase in extracellular dopamine at any timepoints. Results are expressed as mean±SEM % of control extracellular dopamine levels. n=5–7 animals per group. * P⬍0.05, ** P⬍0.01 vs basal levels.

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approximately 37% and 25% respectively, but still remained significant at the 1 hr time-point (P⬍0.05). This level of protection with iGlu5 antagonists is similar to that reported earlier in rat MCAO models with the racemic parent of LY293558 (Gill and Lodge, 1995) and with mixed AMPA/kainate antagonists such as NBQX, YM90K and YM872 (Gill et al., 1992; Yao et al., 1997; Kawasaki-Yatsugi et al., 1998). It is somewhat greater than that reported for GYKI52466 (Xue et al., 1994). Thus, these earlier studies are consistent with a role for kainate receptors over and above that of AMPA receptors in acute neurodegeneration following focal ischaemia. Furthermore, the new knowledge about the iGlu5 activity of LY293558 (Bleakman et al., 1996; Bleakman and Lodge, 1998) suggests that iGlu5 antagonism may contribute to the neuroprotection seen with this compound previously (Bullock et al., 1994; Gill and Lodge, 1995). Indeed, our present studies showing that LY377770, the selective iGlu5 antagonist, is somewhat more effective than LY293558 suggest a paramount role for this subtype of kainate receptor in focal ischaemic damage. A role of other kainate receptors, e.g. iGlu6, cannot be excluded by the present data. Indeed, recent studies have demonstrated a reduced susceptibility to kainate seizures in GluR6-deficent mice (Mulle et al., 1998). The relative contribution of iGlu5 and other glutamate receptors in focal ischaemia models may be resolved by examining other 6-substituted decahydroisoquinolines with various profiles of activity at iGlu1– iGlu5 and NMDA receptors (Ornstein et al. 1993, 1995, 1996a,b; Clarke et al., 1997; Bleakman and Lodge, 1998; O’Neill et al., 1998). 4.3. Mode of action of iGlu5 receptor antagonists? The precise mechanism by which iGlu5 antagonism provides such profound neuroprotection is uncertain but the present microdialysis results give a clear insight. As expected from the literature for glutamate (Benveniste et al., 1984; Globus et al., 1988; Butcher et al., 1990) and dopamine (Globus et al., 1988; Phebus and Clemens, 1989; Bentue´-Ferrer et al. 1993, 1994), focal cerebral ischaemia in the rat induced a huge rise in striatal extracellular glutamate (50–60-fold) and dopamine (25–30-fold) levels. Interestingly, the dopamine level rises and falls more quickly than that of glutamate suggesting that the onset of ischaemia may activate dopaminergic terminals before the onset of profound ischaemia-induced depolarisation that causes glutamate release. The possibility that release of dopamine underlies in part the glutamate release is supported by the observation that ablation of the dopaminergic substantia nigra attenuated the release of glutamate in global (Clemens and Phebus, 1988; Globus et al., 1988) and focal (Buisson et al., 1991) cerebral ischaemia. LY377770 produced a small decrease (25%) in back-

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ground extracellular glutamate levels in control animals. Furthermore, given 30 min before the MCAO, LY377770 produced a dramatic decrease (90%) in the ischaemia-induced rise and even when given 40 min after MCAO reduced the subsequent levels of extracellular glutamate. By contrast, a small increase was observed in control dopamine levels with no significant effect of LY377770 on ischaemia-induced release of this monoamine. This result on glutamate release correlates with in vitro studies on hippocampal slices that have demonstrated that iGlu5 receptors regulate excitatory (Vignes et al., 1998) synaptic transmission in rat hippocampal slices. In this simple case, however, presynaptic iGlu5 receptors would have to facilitate glutamate release (see Chittajallu et al., 1996). An alternative explanation is that by blocking iGlu5 inhibition of GABA release (Clarke et al., 1997), LY377770 increases GABA release which in turn prevents glutamate release. The data from control non-ischaemic rats further suggest that there may be tonic activation of glutamate receptors of the iGlu5 subtype. The huge magnitude of the reduction of the ischaemiainduced rise in extracellular glutamate was nevertheless a surprise, since it has been thought that much of the rise may be due to reversal of the transporter (see Billups et al., 1998). The present microdialysis result suggests that the release is from tissues expressing iGlu5 receptors and implicates iGlu5 receptor-expressing presynaptic glutamatergic terminals within the striatum. How such autoreceptors mediate release is unknown but it appears that a vicious cycle of released glutamate inducing depolarisation and further glutamate release is dependent to a large extent on iGlu5 receptors. The role of hypothermia in ischaemia has been studied extensively and indeed many earlier excitatory amino acid antagonists may have produced protection via this mechanism. Many studies have demonstrated that intraischaemic hypothermia is neuroprotective (Barone et al., 1997). Other studies have reported that the delayed hypothermia is also protective in global and focal cerebral ischaemia (Busto et al., 1989; Colbourne and Corbett, 1994). The discovery that MK-801 caused hypothermia led to the suggestion that MK-801 was providing neuroprotection by producing hypothermia (Buchan and Pulsinelli, 1990; Corbett et al., 1990), while others postulate that the protective actions of MK-801 are mediated by a small transient hypothermia that acts synergistically with the drug to yield neuroprotection (Hayward and Woodruff, 1993). In fact, studies by Green et al. (1995) demonstrated that combined hypothermia and delayed treatment with MK-801 was more effective than either alone in a rat model of global ischaemia. Other recent studies have suggested that the protective effect of NBQX may also be due to hypothermia (Nurse and Corbett, 1996). In the present studies

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to monitor temperature effects rectal temperatures were measured periodically for 6 hr after occlusion and the animals were placed in four compartmental thermacages (which maintained environmental temperatures at 28°C) immediately after surgery and remained there for a minimum of 6–8 hr. We did not monitor brain temperature so a possible contribution of brain temperature changes to the tolerance cannot be totally excluded. In addition, we cannot rule out the possibility of delayed hypothermia contributing to the observed effects, but in all cases the animals were fully recovered when returned to their home cages.

5. Concluding statements As mentioned earlier, NMDA antagonists with psychotomimetic effects in humans caused increased glucose utilisation, c-fos and hsp-70 expression and morphological changes in the rat cingulate cortex (Kurumaji et al., 1989; Olney et al. 1990, 1991; Sharkey et al., 1996). This does not appear to be the case with iGlu5 receptor antagonists, because LY377770 was devoid of any increase in c-fos expression in the present study and LY293558 has previously been shown not to increase glucose utilisation in the cingulate cortex (Browne and McCulloch, 1994). For these reasons, non-NMDA receptor antagonists may be better clinical candidates than NMDA antagonists since they do not cause the above cingulate cortex lesions but rather they prevent those induced by NMDA antagonists (Patel and McCulloch, 1995; Narita et al., 1997). However, earlier AMPA/kainate receptor antagonists of the quinoxalinedione class, such as NBQX, suffered the additional problems of low solubility and nephrotoxicity (Xue et al., 1994). This is not an issue with the decahydroisoquinolines, as we found both LY293558 and LY377770 to have good solubility and no obvious signs of precipitation or damage in the kidney. In conclusion, in the present studies we have demonstrated the neuroprotective effects in both global and focal cerebral ischaemia of LY377770, an iGlu5 antagonist based on the decahydroisoquinoline structure. Additionally we showed that LY377770 provided functional improvement in global ischaemia and prevented glutamate release in focal ischaemia. The results provided the first evidence that iGlu5 receptors contribute to the damage after focal cerebral ischaemia and suggest reduction of presynaptic glutamate release as a likely explanation. LY377770 has good solubility, few sideeffects and protects against ischaemic brain damage when administered post-occlusion and therefore may be useful for the treatment of acute neurodegenerative diseases.

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