LY393615, a novel neuronal Ca2+ and Na+ channel blocker with neuroprotective effects in models of in vitro and in vivo cerebral ischemia

Brain Research 888 (2001) 138–149 www.elsevier.com / locate / bres

Research report

LY393615, a novel neuronal Ca 21 and Na 1 channel blocker with neuroprotective effects in models of in vitro and in vivo cerebral ischemia Michael J. O’Neill a , *, Caroline A. Hicks a , Mark A. Ward a , David J. Osborne a , Graham Wishart a , Kusum S. Mathews c , Daniel P. McLaughlin c , Jonathan A. Stamford c , Deborah R. McCarty b , Kelly E. Patrick b , Carlos Roman b , Jerome H. Fleisch b , Jeremy Gilmore a , John R. Boot a a

Lilly Research Centre Ltd., Erl Wood Manor, Windlesham, Surrey GU20 6 PH, UK b Lilly Corporate Center, Indianapolis, IN 46285 -0814, USA c Neurotransmission Laboratory, Academic Department of Anaesthesia and Intensive Care, St. Bartholomew’ s and the Royal London School of Medicine and Dentistry, Whitechapel, London E1 1 BB, UK Accepted 26 September 2000

Abstract In the present studies we have examined the effects of a new calcium channel blocker, LY393615 ((N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, NCC1048) in a model of hypoxia–hypoglycaemia in vitro and in a gerbil model of global and in two rat models of focal cerebral ischaemia in vivo. Results indicated that LY393615 protected against hypoxia– hypoglycaemic insults in brain slices and also provided significant protection against ischaemia-induced hippocampal damage in gerbil global cerebral ischaemia when dosed at 10, 12.5 (P,0.05) or 15 mg / kg i.p. (P,0.01) 30 min before and 2 h 30 min after occlusion. The compound penetrated the brain well after a 15 mg / kg i.p. dose and had a half-life of 2.5 h. In further studies LY393615 was protective 1 h post-occlusion when administered at 15 mg / kg i.p. followed by 2 doses of 5 mg / kg i.p. 2 and 3 h later. LY393615 dosed at 15 mg / kg i.p. followed by 2 further doses of 5 mg / kg i.p. (2 and 3 h later) also produced a significant reduction in the infarct volume following Endothelin-1 (Et-1) middle cerebral artery occlusion in the rat when administration was initiated immediately (P,0.01) or 1 h (P,0.05) after occlusion. The compound was also evaluated in the intraluminal monofilament model of focal ischaemia. The animals had the middle cerebral artery occluded for 2 h, and 15 min after reperfusion LY393615 was administered at 15 mg / kg i.p. followed by 2 mg / kg / h i.v. infusion for 6 h. There was no reduction in infarct volume using this dosing protocol. In conclusion, in the present studies we have reported that a novel calcium channel blocker, LY393615, with good bioavailability protects against neuronal damage caused by hypoxia–hypoglycaemia in vitro and both global and focal cerebral ischaemia in vivo. The compound is neuroprotective when administered post-occlusion and may therefore be a useful anti-ischaemic agent.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of nervous system Topic: Ischaemia Keywords: Cerebral ischemia; Neuroprotection; Neuronal calcium channel; LY393615

1. Introduction Although cerebral ischaemia is of major clinical importance in stroke and cerebrovascular disorders, the exact *Corresponding author. Tel.: 144-1276-853-547; fax: 144-1276-853525. E-mail address: oneill michael [email protected] (M.J. O’Neill). ] ]

mechanism of ischaemia-induced neuronal cell death remains to be elucidated. It is thought that during ischaemia, the lack of energy to the brain may depolarize neurones and result in large increases in neurotransmitters such as glutamate, aspartate, dopamine and serotonin [13,39,40]. Glutamate, through an action on N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, allows calcium to enter the

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03043-2

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cell [10,22,51]. Glutamate can also act on metabotropic receptors leading to the production of diacylglycerol (DAG) and inositol triphosphate (IP3) which, lead to the release of calcium from intracellular stores [51]. Calcium also enters neurones through voltage-dependent calcium channels which open in response to cellular depolarization [39,40]. The net result of these various mechanisms by which calcium concentrations are elevated is a calcium ‘overload’ which leads to activation of proteases, nucleases, phospholipases, NO synthase and other degradative enzymes that lead to free radical production, mitochondrial degeneration and cell death [39,40]. In addition, recent studies have shown that apoptotic mechanisms contribute to cell death in vitro and damage following cerebral ischaemia in vivo [17]. Therefore, strategies aimed at inhibiting apoptosis (caspases inhibitors, etc.) may also be useful interventions in ischaemic situations (for review see [25,50]). In line with the above mechanisms, several studies have reported that compounds acting as antagonists of excitatory amino acid receptors have beneficial effects against ischaemic insults [11,12,22,28,36]. However, several early studies also reported neuroprotection with compounds such as (S)-emopamil, which belong to the phenylalkylamine class of calcium channel inhibitors, in animal models of cerebral ischaemia [18,23,24]. In addition, other L-type calcium antagonists such as nimodipine and nicardipine have been shown to display neuroprotective effects in some animal models of ceebral ischaemia [1,15,33]. At least 5 subtypes of high threshold (HVA) denoted L, N, P, Q and R and one type of low threshold (LVA), the T-type, voltage-dependent calcium channels have been described [47]. The availability of several synthetic conopeptides has provided an opportunity to evaluate the therapeutic potential of selective blockade of N-type calcium channels in a variety of pathological conditions including cerebral ischaemia. A single bolus intravenous administration of v-conotoxin MVIIA (SNX-111) provided protection even when administered 24 h after the ischaemic insult [48]. Similar effects were observed in two other studies [42,55]. SNX-111 has also been found to be highly effective in reducing the neocortical infarct volume in rat models of focal ischaemia, both when administered during the occlusion [43] and after the ischaemic episode [9]. Buchan et al. [9] administered SNX-111 at 5 mg / kg i.v. either 6 or 24 h after 10 min of 4-vessel occlusion (4VO) and found protection in both cases [9]. The group also showed protection with the same dose in a rat model of focal ischemia. More recent studies have reported neuroprotective effects with smaller non-peptide compounds that inhibit neuronal calcium channels. It has been reported that NNC 09-0026 inhibits neuronal voltage-dependent calcium channels without cardiovascular effects and provides neuroprotective effects in animal models of cerebral ischaemia [3,37]. SB 201823-A is another non-peptide calcium

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channel blocker that exhibits broad activity against neuronal calcium channels and at 10 mg / kg i.p. protected against global ischaemia-induced brain damage in the gerbil when administered 30 min after occlusion [4,7]. More recent studies have reported that SB 201823-A antagonises calcium currents in rat central neurones and reduces the infarct volume in rat and mouse models of focal ischaemia [4] and that another neuronal calcium antagonist, NS-649, has provided protective effects in the mouse MCAO model [49]. At Eli Lilly & Co. Ltd. we ran a high throughput screen using HEK293 cells transfected with human a1A, a1B and a1E calcium channel subunits (representing N, P/ Q and R-type calcium channels). The aim of this screen was to discover small organic molecules that block neuronal calcium channels and thereby modulate neurotransmitter release in disease situations. We identified a compound that the medicinal chemistry department modified to the more active compound LY393615 (NCC1048), which inhibited N, P and Q-type calcium channels. At a concentration of 10 mM, LY393615 produced 75616%, 8668% and 9563% inhibition of calcium flux in HEK 293 cells transfected with human a1A, a1B or a1E calcium channel subunits. The compound was was also evaluated on Ca 21 currents in HEK 293 cells transfected with human a1B or a1E Ca 21 channel subunits and on P-type calcium channels in isolated Purkinje cells using voltage patch clamp techniques. LY393165 inhibited peak Ca 21 currents dosedependently with IC 50 values of 1.960.1 mM in a1B cells, 5.261.1 mM in a1E cells and 4.061.0 mM in Purkinje cells. This report summarises the neuroprotective effects of LY393615 in an in vitro hypoxia–hypoglycemic slice model and in gerbil global ischaemia and two models of rat focal cerebral ischaemia in vivo.

2. Materials and methods

2.1. In vitro hypoxia–hypoglycemia Male Wistar rats (150650 g) were sacrificed by cervical dislocation. Coronal brain slices (350 mm thick) at the level of the striatum (typically bregma 60.5 mm: Paxinos and Watson [30]) were incubated in chambers containing 500 ml oxygenated artificial cerebrospinal fluid (aCSF) at 348C for 30 min. Slices were then subjected to ‘ischaemia’ by transfer to a chamber containing 500 ml deoxygenated, hypoglycaemic aCSF for 10 min [45] followed by a further 30 min of reincubation in oxygenated aCSF. Control slices were maintained in oxygenated aCSF throughout. In a proportion of cases, LY393615 (10 mM) was included in the aCSF throughout the pre-incubation, ischaemia and post-ischaemic periods. Slices were then incubated for 30 min at 378C with 2,3,5-triphenyltetrazolium chloride (TTC, a colorimetric index of mitochondrial enzyme activity), fixed in 10%

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formalin and transferred to coverslips. Stained slices were initially scanned in true colour using an AGFA ‘Studioscan’ desktop scanner at 60 mm resolution. The ensuing images were converted to 8-bit greyscale (0–255) for mathematical analysis of staining [21]. The intensity of striatal and cortical TTC staining was analysed by OsirisE (University Hospital of Geneva, Switzerland) or ScionImageE (Scion Corporation) software and expressed as a mean percentage greyscale value over the whole region of interest (striatum or surrounding cortex). Statistical comparisons between groups were made by One Way ANOVA with posthoc application of the Student–Newman–Keuls test.

2.2. Pharmacokinetics To assess exposure of LY393615, gerbils were dosed with 15 mg / kg i.p. and brains harvested at 15, 30, 45 min, 1, 2, 4, 6 and 8 h later. We also evaluated exposure in rats at the same time points after 1 mg / kg i.v. In both cases brains were weighed and then homogenised with two volumes of distilled water. A 20 mM aqueous solution of LY393613 (N-Butyl-2-[bis-(4-fluorophenyl)methoxy]ethylamine hydrochloride) was prepared for use as an internal standard. 300 ml aliquots of brain homogenate and 10 ml internal standard solution were transferred to 1.5 ml eppendorf centrifuge tubes, mixed thoroughly and extracted with 10 ml trifluoroacetic acid (TFA) and 100 ml acetonitrile. Tubes were spun, in a microcentrifuge, for 5 min at 13,000 r.p.m. and the supernatant transferred to HPLC microvials for assay. LY393615 standards of 50, 20, 5, 2, 0.5 and 0.1 mM concentration were prepared in homogenised control rat brain and then treated in the same manner as the samples. Samples and standards were analysed by HPLC / MS using a Hewlett Packard 1100 series autosampler and binary pump (Hewlett Packard, Bracknell, UK) coupled to a Micromass Platform II mass spectrometer (Micromass, Manchester, UK). Chromatography was performed on a 25 cm32.1 mm Hichrom 5 C 18 column (Hichrom, Theale, UK) using acetonitrile:water (60:40) containing 5 mM ammonium formate with the pH adjusted to 2.8 with TFA. Twenty ml volumes were injected. MS analysis was performed in the electrospray mode with a source temperature of 1208C and cone voltage of 25 V. Ions at m /z 346 (LY393615) and m /z 320 (LY393613) were monitored.

2.3. Global ischaemia Male Mongolian gerbils (Bantin and Kingman, Hull, UK) at least 3 months old and weighing in excess of 60–80 g were used. The animals were maintained in standard lighting conditions and food and water were available ad libitum. The animals were anaesthetised with a 5% halothane / oxygen mixture and maintained using 2% halothane delivered with oxygen at 1 litre per min via a

face mask throughout the operation. Through a midline cervical incision, both common carotid arteries were exposed and freed from surrounding connective tissue. In animals to be rendered ischaemic, both common carotid arteries were clamped for 5 min. At the end of the occlusion period, blood flow was re-established. In shamoperated 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 378C 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 environmental temperature at 288C and rectal temperatures were measured for a 6 h period after occlusion. LY393615 was administered at 10, 12.5 or 15 mg / kg i.p. 30 min before and 2 h 30 min post-occlusion in the first experiment. LY393615 was administered at 15 mg / kg i.p. 30 or 60 min post-occlusion followed by two further doses of 5 mg / kg i.p. 2 and 3 h after the initial dose in the second and third experiments. 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 mm) 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 (0.0530.05 mm) as described previously [28]. The neuronal density is expressed as number of viable cells per mm CA1 hippocampus. Statistical analysis of histological data was assessed using a 2-tailed unpaired Students t-test, with P values ,0.05 being considered statistically significant.

2.4. Focal ischaemia 2.4.1. Endothelin-1 model. Rats were anaesthetised with inhalation anaesthetic and placed on a thermostatically controlled heating blanket to maintain body temperature within the range 37–388C. The rat was then placed in a Kopf stereotaxic frame and the scalp incised so as to uncover the parietal bones. A 28-guage steel cannula was then inserted stereotaxically at the following co-ordinates (from bregma: AP5 10.9 mm, L5 25.2 mm and 28.7 mm below skull, Sharkey et al. [35]). Endothelin-1 (200 pmol in 3 ml) was infused over a 3 min period. The cannula was left in situ for a further 5 min and then withdrawn. The wounds (muscles and skin) were sutured and the rat allowed to recover. LY393615 was administered at 15 mg / kg i.p. immediately or 1 h post-occlusion followed by additional doses of 5 mg / kg at 2 and 3 h after the initial injection. 3 days after surgery, the rats were perfused with heparinised saline

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follow by 10% buffered formalin via the heart. The brains were removed for histology, stained with cresyl violet and the area of ischaemic damage at 8 stereotaxic levels was measured using Optimus 5.2 software and from this an infarct volume was calculated. Statistical analysis of histological data was carried out using ANOVA followed by Students t-test using P,0.05 as the level of significance.

2.4.2. Monofilament model. In the monofilament model, a segment of 220 mm diameter nylon monofilament coated with nail polish at the tip (370 mm), was inserted into the internal carotid artery of each male Wistar rat (295–315 g), and advanced until it blocked the origin of the middle cerebral artery (Belayev et al. [6]; Zea Longa et al. [54]). After 2 h, the monofilament was retracted and reperfusion occurred. LY393615 was administered at 15 mg / kg i.p. 15 min after reperfusion followed by 2 mg / kg / h i.v. infusion for 6 h. Twenty-four hours later the brains were removed, frozen, sliced into 30 mm sections and stained with cresyl violet. The infarct area of each slice was measured with imaging system software ImagePro Plus, and infarct volumes were calculated. Statistical analysis of histological data was carried out using ANOVA followed by Students t-test using P,0.05 as the level of significance.

3. Results The structure of (N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, LY393615) is illustrated in Fig. 1. Figs. 2 and 3 show the effects of LY 393615 against ischaemic insult in vitro. Control slices mostly showed uniform staining throughout the cortex and striatum following TTC. Fig. 2 consists of representative pseudocoloured corticostriatal sections showing the effect of ischaemia and LY393615. Ischaemia (10 min) caused a significant (P,0.05) reduction in mean TTC staining intensity in both striatum and cerebral cortex (Fig. 3). LY393615 had no significant effect on TTC staining intensity in control slices (if anything it appeared to protect slice) but prevented the reduction in staining induced by

Fig. 1. Illustrates the structure of (N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, LY393615).

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‘ischaemia’ in both striatum (P,0.01) and cortex (P, 0.001). For global ischaemia studies, 5 mm sections taken 1.5– 1.9 mm caudal to the bregma in the anterior hippocampus were examined under a microscope with grid lines. The pyramidal cell density was counted at three different stereotaxic levels in the CA1 region of the hippocampus and the results expressed as mean6S.E.M. neuronal density per 1 mm CA1. The results indicated that there was severe loss of neurones in the CA1 region of the hippocampus of 5 min occluded animals. The neuronal death involved nearly all the pyramidal neurones and this neurodegeneration was not evident in any other forebrain region. LY393615 provided dose-dependent neuroprotection against the ischaemia-induced cell death in the CA1. Thus, LY393615 provided very good protection when administered at 15 mg / kg i.p. 30 min before and 2 h 30 min after occlusion (Figs. 4 and 5). We also evaluated the effects of lower doses of LY393615 at 10 mg / kg or 12.5 mg / kg i.p. 30 min before and 2 h 30 min post-occlusion. Results indicated that 10 mg / kg provided a small, but significant neuroprotection (Fig. 4). The intermediate dose (12.5 mg / kg i.p.) also provided a significant neuroprotective effect (Fig. 4). We then went on to study the pharmacokinetics (at 15, 30, 45 min, 1, 2, 4, 6 and 8 h) of LY393615 in the gerbil brain after 15 mg / kg i.p. The results indicated that the compound crossed the blood–brain barrier quickly and was detectable in the brain as early as 15 min after injection (Fig. 6). Maximal levels were observed at 1–2 h after injection and the half-life was approximately 2.5 h. Based on the pharmacokinetic studies we postulated that a loading dose (15 mg / kg i.p.) followed by two further ‘top-up’ doses (5 mg / kg i.p.) would give relatively consistent high brain levels for several hours after injection. Therefore, in the next study we evaluated the effects of 15 mg / kg i.p. 30 min after occlusion followed by 2 further doses of 5 mg / kg i.p. at 2 h 30 min and 3 h 30 min post-occlusion. Histological results indicated that this dosing protocol also provided good (P,0.01) neuroprotection (Fig. 7). We then determined if the time window could be extended using the above protocol by administering the LY393615 at 15 mg / kg 1 h post-occlusion followed by 5 mg / kg at 2 and 3 h post-occlusion. Results indicated that this dosing protocol also provided significant (P,0.05) neuroprotection (Fig. 8). Two models of focal cerebral ischaemia were carried out; the endothelin-1 model and the monofilament model. In the endothelin-1 model LY393615 was administered at 15 mg / kg i.p. either immediately or 1 h after endothelin-1 injection onto the middle cerebral artery in the rat and followed by two further doses at 3 and 4 h after the initial injection injection. The area of damage in mm 2 was measured at 8 stereotaxic levels and the infarct volume calculated in mm 3 . LY393615 produced a reduction (P, 0.01) in infarct volume when administration was initiated

142 M. J. O’ Neill et al. / Brain Research 888 (2001) 138 – 149 Fig. 2. Representative greyscale scans showing TTC staining of the striatum and cortex in control and ischaemia-treated slices, alone and in the presence of LY393615 (10 mM). Pinker tissue is an indicator of higher mitochondrial enzyme activity.

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4. Discussion

Fig. 3. Effect of LY393615 (10 mM) on ischaemia-induced reduction in TTC staining (expressed as percent greyscale). All values are means6S.E.M. (n511 / 15).*P,0.05 vs. Control, **P,0.01,***P, 0.001 vs. Ischaemia, $ P,0.001 vs. Control 1LY393615.

immediately after occlusion (Fig. 9). The compound also provided some protection (P,0.05) when administration was initiated 1 h post-occlusion (Fig. 9). Pharamcokinetic studies in rats indicated that LY393615 penetrated the brain rapidly after a 1 mg / kg i.v. dose (Fig. 10). The half-life was approximately 2 h and therefore an infusion would give most consistent brain levels for further focal ischaemia studies. LY393615 was also examined in the monofilament model of cerebral ischaemia. The animals had the middle cerebral artery occluded for 2 h. Fifteen minutes after reperfusion LY393615 was administered at 15 mg / kg i.p. followed by 2 mg / kg / h i.v. infusion for 6 h. The results indicated that there was no significant reduction in infarct volume (Fig. 11).

Fig. 4. The effects of LY393615 on the neuronal density in the CA1 region of the hippocampus 5 days after 5 min bilateral carotid artery occlusion. LY393615 was administered at 10, 12.5 or 15 mg / kg i.p. 30 min pre and 2 h 30 min post-occlusion. Results are expressed as mean6S.E.M. viable cells / mm CA1 hippocampus (n58 animals per group). 5 min BCAO caused a severe loss in neuronal cells in the CA1 region (P,0.001). LY393615 provided significant neuroprotection against the ischaemia-induced cell death. *P,0.05 vs. 5 min BCAO control, ***P,0.001 vs. 5 min BCAO control. Student’s t-test.

The in vitro profile of LY33165 is summarised in Table 1. At a concentration of 10 mM, LY393615 produced 75616%, 8668% and 9563% inhibition of calcium flux in HEK 293 cells transfected with a1A, a1B or a1E calcium channel subunits. In vitro electrophysiological studies using patch clamp techniques indicated that LY393165 inhibited peak Ca 21 currents dose-dependently with IC 50 values of 1.960.1 mM in a1B cells, 5.261.1 mM in a1E cells and 4.061.0 mM in Purkinje cells. In the present studies we have evaluated the effects of LY393615 in models of in vitro and in vivo cerebral ischaemia. The results indicate that LY393615 was protective in vitro and provided excellent protection when administration was initiated prior to occlusion and good protection when administered 30 min post-occlusion in global ischaemia. The compound also had significant protective effects when delayed until 1 h post-occlusion in both global and focal cerebral ischaemia.

4.1. In vitro ischaemia Several studies have reported that depolarization following cerebral ischaemia opens voltage-gated calcium channels and this in turn produces massive neurotransmitter release. The large release in neurotransmitters is thought to produce excitotoxicity, free radical production, membrane degeneration and ultimately lead to cell death. We have previously show that v-conotoxin GVIA and v-agatoxin IVA delay neurotransmitter release in striatal slices, while in contrast nimodipine and nicardipine do not [46]. Therefore, N-and P/ Q-, but not L-type calcium channels seem to control this ischaemia-induced neurotransmitter release. In the present studies we have used a selective neuronal calcium channel blocker, LY393615, and investigated if this compound provides protection against hypoxia-hypoglycaemia damage in vitro. LY393615 has been shown to block calcium currents in HEK293 cells transfected with human a1A, a1B and a1E Ca 21 channel subunits, P-type calcium channels in dissociated Purkinje cells and inhibit synaptic transmission in hippocampal slices [44]. The results of the present studies indicate that LY393615 provided significant protection against hypoxic–hypoglycaemic insult in striatal slices using 2,3,5-triphenyltetrazolium chloride staining [21]. Other recent studies have reported that v-conotoxin (selective N-type blocker) protects against hypoxia induced neurodegeneration in organotypic hippocampal-slice cultures [32]. While further studies have used combinations of compounds in an attempt to identify the calcium channel subtypes contributing to neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation [41]. These authors found that nimodipine (L-type) and daurisoline (P-type) provided less than 20% protection, v-conotoxin MVIIA (N-type) provided 40% protection and 200 nM v-agatoxin

144 M. J. O’ Neill et al. / Brain Research 888 (2001) 138 – 149 Fig. 5. The effect of LY393615 administered at 15 mg / kg i.p. 30 min before and 2 h 30 min after 5 min bilateral carotid artery occlusion (BCAO). Representative sections stained with haematoxylin and eosin of whole hippocampus from (a) sham operated, (b) 5 min BCAO control and (c) 5 min BCAO and LY393615 and animals and high power magnification from the same animals (d, e and f).

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Fig. 6. Brain levels of LY393615 (15 mg / kg i.p.) at 15, 30, 45 min, 1, 2, 4, 6 and 8 h after dosing in gerbils. Results indicate that LY393615 penetrates the brain and attains levels that are above the IC 50 for calcium channel blockade. The compound had a half-life of approximately 2.5 h.

IVA (P1Q-type) provided 75% protection. However, vconotoxin MVIIC (P1Q1N-type) provided complete protection, suggesting that all three neuronal calcium channels contribute to ischaemic brain injury in the hippocampus.

4.2. Global ischaemia Earlier studies had also reported neuroprotection with v-conotoxin GVIA (SNX-111) in models of transient global ischaemia in rats and gerbils [9,48]. However, these peptides had poor blood brain barrier penetration and have to be administered centrally or intravenously at high doses, which effected blood pressure. Therefore, a small molecular calcium channel blocker with good brain penetration

Fig. 7. The effects of LY393615 on the neuronal density in the CA1 region of the hippocampus 5 days after 5 min bilateral carotid artery occlusion. LY393615 was administered at 15 mg / kg i.p. 30 min postocclusion followed by 5 mg / kg at 2 h 30 min and 3 h 30 min post-occlusion. Results are expressed as mean6S.E.M. viable cells / mm CA1 hippocampus (n58 animals per group). Five minutes BCAO caused a severe loss in neuronal cells in the CA1 region (P,0.001). LY393615 provided significant neuroprotection against the ischaemia-induced cell death. **P,0.01 vs. 5 min CAO control. Student’s t-test.

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Fig. 8. The effects of LY393615 on the neuronal density in the CA1 region of the hippocampus 5 days after 5 min bilateral carotid artery occlusion. LY393615 was administered at 15 mg / kg i.p. 1 h postocclusion followed by 5 mg / kg at 3 and 4 h post-occlusion. Results are expressed as mean6S.E.M. viable cells / mm CA1 hippocampus (n58 animals per group). Five minutes BCAO caused a severe loss in neuronal cells in the CA1 region (P,0.001). LY393615 provided significant neuroprotection against the ischaemia-induced cell death. *P,0.05 vs. 5 min BCAO control. Student’s t-test.

would be a better candidate for treatment of ischaemic conditions. We have demonstrated that LY393615 provided good neuroprotection when dosed at 15 mg / kg i.p., 30 min before occlusion in the gerbil model. This protection was dose-dependent, with the 15 mg / kg dose providing much greater protection than the lower 10 and 12.5 mg / kg doses. Pharmacokinetic studies indicated that the compound penetrated into the brain and that the half-life was approximately 2.5 h. Based on these studies, we calculated that 15 mg / kg i.p. followed by two further doses of 5 mg / kg 2 and 3 h later would give consistent brain levels that were several fold higher than the IC 50 for LY393615 on

Fig. 9. Effects of LY393615 on infarct volume (mm 3 ) 72 h after endothelin-induced middle cerebral artery occlusion in the rat. LY393615 was administered at 15 mg / kg i.p. either immediately or 1 h postocclusion followed by 5 mg / kg i.p. 2 and 3 h after the initial dose (n510–15 animals per group). LY393615 produced a significant reduction in the infarct volume when administered immediately (**P,0.01) or 1 h (*P,0.05) post-occlusion. Students t-test.

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M. J. O’ Neill et al. / Brain Research 888 (2001) 138 – 149 Table 1 The in vitro profile of LY393615 a

Fig. 10. Brain levels of LY393615 (1 mg / kg i.v.) at 15, 30, 45 min, 1, 2, 4, 6 and 8 h after dosing in rats. Results indicate that LY393615 penetrates the brain rapidly has a half-life of approximately 2 h.

neuronal calcium channel subunits in vitro. Using this protocol we went on to demonstrate that LY393615 provided significant protection in this model when the first injection was delayed until 1 h post-occlusion. Several other small molecules; NNC 09-0026 ((2)trans-1-butyl-4-(4-dimethylaminophenyl) - 3 - [(4-trifluoro methylphenoxy)methyl]piperidine dihydro chloride); SB 201823-A (4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride); NS 649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole); CNS 1237 (N-acenaphthyl-N9-4-methoxynaphth-1-yl guanidine) have also been reported to have neuroprotective effects in models of cerebral ischaemia. For example, Sheardown and co-workers [38] have reported that NNC 09-0026 (30 mg / kg i.p.) protected in the gerbil when administered 30 min, 24 and 48 h post-occlusion [37], but no neuroprotec-

Fig. 11. Effects of LY393615 on infarct volume (mm 3 ) after 2 h of middle cerebral artery occlusion in the rat using the monofilament method. LY393615 was administered at a loading dose of 15 mg / kg i.p. 15 min after reperfusion followed by an i.v. infusion of 2 mg / kg / h for 6 h and infarct volume measured at 1 day post-surgery (n512–16 animals per group). LY393615 failed to reduce the infarct volume under this dosing protocol. Students t-test.

(A) Inhibition of Ca 21 flux at 10 mm using Fluoroscan: HEK 293 cells transfected with human a1A HEK 293 cells transfected with human a1B HEK 293 cells transfected with human a1E

75616 8668 9563

(B) Inhibition of Glutamate release at 10 mm: KC1-induced glutamate release Electrical-induced glutamate release Veratradine-induced glutamate release

7666 63 (n52) 81 (n52)

(C) Patch Clamp IC 50 values (mm) for inhibition of: HEK 293 cells transfected with human a1B HEK 293 cells transfected with human a1E P-type calcium channels in isolated Purkinje cells

1.960.1 5.261.1 4.061.0

a

Effects of LY393615 ON (A) calcium flux in HEK 293 cells transfected with human a1A, a1B and a1E calcium channel subunits (B) inhibition of DC1 Electrical and Veratradine-induced glutamate release in symaptosomes and (C) IC 50 values for inhibition of calcium channels in transferred cell limes and isolated Purkinje cells using whole cell patch clamp electrophysiological techniques.

tion when the compound (30 mg / kg i.p.) was administered once 30 min post-occlusion. NNC 09-0026 (30 mg / kg i.v.) administered slowly over a 1 h period beginning 30 min post-ischaemia has also been reported to reduce the infarct volume in a rat model of focal ischaemia [3]. CNS 1237 is a use-dependent Na 1 and Ca 21 channel blocker that produced a reduction in infarct volume when dosed as a bolus of 3 mg / kg i.v. followed by an infusion of 0.75 mg / kg / h for 4 h [14]. It has also been reported that SB 201823-A administered at 10 mg / kg i.p. at 30 min postocclusion attenuated ischaemia-induced hyperactivity and protected the CA1 pyramidal cells in the hippocampus following 8 min of BCAO in the gerbil [7]. Other studies have indicated that SB 201823-A reduces the infarct volume in rat and mouse models of focal ischaemia [4]. NS 649 is another compound that has been reported to have protective effects in the mouse MCAO model [49]. We have previously evaluated all of these small molecules as reference neuronal calcium channel antagonists in the gerbil model of cerebral ischaemia [26]. The doses selected were based on previously published work and tolerability in animals. NNC 09-0026 (30 mg / kg i.p.) provided 22% neuroprotection when administration was initiated 30 min before occlusion followed by two further doses at 24 and 48 h post-occlusion. CNS 1237 provided some neuroprotection (21%) against the ischaemia-induced cell death in the CA1 region of the hippocampus. SB 201823-A, when administered at 10 mg / kg 30 min before and 2 h 30 min after occlusion failed to provide any neuroprotection and NS 649 at 50 mg / kg i.p. (dosed 30 min before and again at 2 h 30 min after occlusion) also failed to provide any protection against the ischaemiainduced damage in the CA1 hippocampal region of the gerbil brain. These results clearly indicate LY393615 is far superior to the other small molecule neuronal calcium

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channel blockers as neuroprotective agents in global cerebral ischaemia. The compound exhibited good solubility and penetrated the brain well and this probably accounts for the good neuroprotection we observed. In the past 5 years we have evaluated several pharmacological interventions in cerebral ischaemia. As mentioned above, the level of protection observed with LY393615 in this model is much greater than that we have previously observed, using the same protocol, with other calcium channel blockers (NCC 09-0026, CNS1237 [26]). Interestingly, the protection with LY393615 is also much greater than that provided by competitive NMDA antagonists (CGS19755, CPP [28]; polyamine site antagonists of the NMDA receptor [5] and glycine site antagonists (ACEA1021, GV150526A [16]), non-competitive AMPA antagonists (GYKI52466, LY300164 [20]), nitric oxide inhibitors ( L-NAME and 7-nitroindazole [27]) and similar to what we have seen with the competitive AMPA antagonists (LY302679; LY293558 [28]), the iGluR5 antagonist (LY377770 [29]) and the mGluR2 / 3 agonist (LY379268 [8]). Therefore, LY393615 has comparable neuroprotection to some of the best compounds we have evaluated to date in the gerbil model of cerebral ischaemia.

4.3. Focal ischaemia In focal ischaemia studies we evaluated the neuroprotective potential of LY393615 in the endothelin-1 and monofilament models of middle cerebral artery occlusion (MCAO) in rats. The results indicated that LY393615 provided a significant reduction in infarct volume when administered immediately or 1 h post-occlusion in the endothelin-1 model of focal cerebral ischaemia. The degree of protection was similar to that previously reported with MK-801 in this model [29,34]. LY393615 failed to protect when administered at 15 mg / kg i.p. 15 min after 2 h of MCAO and followed by a 6 h infusion of 2 mg / kg i.v. for 6 h in the monofilament model. However, the compound was administered 2 h 15 min after ischaemia and further pharmacokinetic studies indicated that although LY393615 penetrated the brain following i.v. administration, it has a short half-life (2.04 h) and, therefore, a higher infusion dose (3.5 mg / kg / h) may have been more optimal to achieve maximal chances of protection. Other studies have reported that the conotoxin SNX-111, which blocks N-type calcium channels is effective at reducing infarct volume in rat models of focal cerebral ischaemia when administered during [43] or after the occlusion (Buchan et al. [9]; Yenari et al. [53]). The compound was also reported to have neuroprotective effects in a rabbit model of focal ischaemia [31]. Other recent studies have demonstrated that i.c.v. administration of v-agatoxin IVA, which blocks P/ Q-type calcium channels, protects against focal ischaemia in rats [2]. However, other investigators have demonstrated that another P-type calcium antagonist, daurisoline (a non-peptide) failed to protect in a rat model

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of focal ischaemia [19]. More recent work has indicated that small organic molecules such as SB 201823A are protective in models of focal ischaemia [3,4]. The newer compound SB 206284A has been reported to protect when administered 4 h post-occlusion in a photochemical model of focal ischaemia [52]. However, many of the studies to date have used short occlusion times i.e. 90 min whereas in our studies we have used the standard 2 h occlusion, which is a more severe model.

4.4. Mechanism of action of LY393615 As mentioned above, LY393615 blocks several types of neuronal calcium channels and also inhibits sodium channels. Therefore, the exact mechanism of neuroprotection is not clear. In theory all of the activities (i.e. inhibition of N-type, P/ Q-type of calcium channels or sodium channels) could be neuroprotective. On reviewing the literature it is clear that selective N-type calcium channel blockers and sodium channel blockers have provided consistent protection in a variety of models. However, results are more variable with P/ Q-type calcium blockers. Therefore, it is most likely that the N-type calcium channel blockade and the sodium channel effects account for the observed neuroprotection. It is interesting to note that there is an increasing tendency to screen for selective molecules using transfected cell lines, but there are multiple receptors contributing to ischaemia-induced brain damage. The pharmacological profile of LY393615 may make it more suitable as a neuroprotectant than a compound that only blocked one receptor subtype. In the present studies we did not measure brain temperature and it is well established that hypothermia is neuroprotective. We did monitor rectal temperatures at 30 min intervals and this is thought to be a good reflection of brain temperature. The compound did not alter rectal temperature at the neuroprotective doses. In a similar way the protection observed could be due to effect on blood pressure. In parallel studies we found that there was no changes in blood pressure using a dose of 15 mg / kg i.p. Therefore, the neuroprotective actions of LY393615 appear to be mediated by calcium and / or sodium channel blockade. In summary, in the present studies we have reported neuroprotective effects with LY393615 a model of in vitro ischaemia in striatal slices and in models of global and focal cerebral ischaemia in vivo. The compound had good solubility and pharmacokinetic studies demonstrated that the compound had good brain penetration with a half-life of 2–2.5 h. LY393615 was active post-occlusion and may therefore be useful as an anti-ischaemic agent.

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