M .J. O’Neill et al. Brain Research 888 2001 138 –149
139
cell [10,22,51]. Glutamate can also act on metabotropic channel blocker that exhibits broad activity against neuro-
receptors leading to the production of diacylglycerol nal calcium channels and at 10 mg kg i.p. protected
DAG and inositol triphosphate IP3 which, lead to the against global ischaemia-induced brain damage in the
release of calcium from intracellular stores [51]. Calcium gerbil when administered 30 min after occlusion [4,7].
also enters neurones through voltage-dependent calcium More recent studies have reported that SB 201823-A
channels which open in response to cellular depolarization antagonises calcium currents in rat central neurones and
[39,40]. The net result of these various mechanisms by reduces the infarct volume in rat and mouse models of
which calcium concentrations are elevated is a calcium focal ischaemia [4] and that another neuronal calcium
‘overload’ which leads to activation of proteases, nu- antagonist, NS-649, has provided protective effects in the
cleases, phospholipases, NO synthase and other degrada- mouse MCAO model [49].
tive enzymes that lead to free radical production, mito- At Eli Lilly Co. Ltd. we ran a high throughput screen
chondrial degeneration and cell death [39,40]. In addition, using HEK293 cells transfected with human a1A, a1B and
recent studies have shown that apoptotic mechanisms a1E calcium channel subunits representing N, P Q and
contribute to cell death in vitro and damage following R-type calcium channels. The aim of this screen was to
cerebral ischaemia in vivo [17]. Therefore, strategies discover small organic molecules that block neuronal
aimed at inhibiting apoptosis caspases inhibitors, etc. calcium channels and thereby modulate neurotransmitter
may also be useful interventions in ischaemic situations release in disease situations. We identified a compound that
for review see [25,50]. the medicinal chemistry department modified to the more
In line with the above mechanisms, several studies have active compound LY393615 NCC1048, which inhibited
reported that compounds acting as antagonists of excitatory N, P and Q-type calcium channels. At a concentration of
amino acid receptors have beneficial effects against is- 10 mM, LY393615 produced 75616, 8668 and
chaemic insults [11,12,22,28,36]. However, several early 9563 inhibition of calcium flux in HEK 293 cells
studies also reported neuroprotection with compounds such transfected with human a1A, a1B or a1E calcium channel
21
as S -emopamil, which belong to the phenylalkylamine subunits. The compound was was also evaluated on Ca
class of calcium channel inhibitors, in animal models of currents in HEK 293 cells transfected with human a1B or
21
cerebral ischaemia [18,23,24]. In addition, other L-type a1E Ca
channel subunits and on P-type calcium chan- calcium antagonists such as nimodipine and nicardipine
nels in isolated Purkinje cells using voltage patch clamp
21
have been shown to display neuroprotective effects in techniques. LY393165 inhibited peak Ca
currents dose- some animal models of ceebral ischaemia [1,15,33].
dependently with IC values of 1.960.1 mM in a1B cells,
50
At least 5 subtypes of high threshold HVA denoted L, 5.261.1 mM in a1E cells and 4.061.0 mM in Purkinje
N, P, Q and R and one type of low threshold LVA, the cells. This report summarises the neuroprotective effects of
T-type, voltage-dependent calcium channels have been LY393615 in an in vitro hypoxia–hypoglycemic slice
described [47]. The availability of several synthetic model and in gerbil global ischaemia and two models of
conopeptides has provided an opportunity to evaluate the rat focal cerebral ischaemia in vivo.
therapeutic potential of selective blockade of N-type calcium channels in a variety of pathological conditions
including cerebral ischaemia. A single bolus intravenous
2. Materials and methods
administration of v-conotoxin MVIIA SNX-111 pro- vided protection even when administered 24 h after the
2.1. In vitro hypoxia–hypoglycemia ischaemic insult [48]. Similar effects were observed in two
other studies [42,55]. SNX-111 has also been found to be Male Wistar rats 150650 g were sacrificed by cervical
highly effective in reducing the neocortical infarct volume dislocation. Coronal brain slices 350 mm thick at the
in rat models of focal ischaemia, both when administered level of the striatum typically bregma 60.5 mm: Paxinos
during the occlusion [43] and after the ischaemic episode and Watson [30] were incubated in chambers containing
[9]. Buchan et al. [9] administered SNX-111 at 5 mg kg 500 ml oxygenated artificial cerebrospinal fluid aCSF at
i.v. either 6 or 24 h after 10 min of 4-vessel occlusion 348C for 30 min. Slices were then subjected to ‘ischaemia’
4VO and found protection in both cases [9]. The group by transfer to a chamber containing 500 ml deoxygenated,
also showed protection with the same dose in a rat model hypoglycaemic aCSF for 10 min [45] followed by a further
of focal ischemia. 30 min of reincubation in oxygenated aCSF. Control slices
More recent studies have reported neuroprotective ef- were maintained in oxygenated aCSF throughout. In a
fects with smaller non-peptide compounds that inhibit proportion of cases, LY393615 10 mM was included in
neuronal calcium channels. It has been reported that NNC the aCSF throughout the pre-incubation, ischaemia and
09-0026 inhibits neuronal voltage-dependent calcium chan- post-ischaemic periods.
nels without cardiovascular effects and provides neuro- Slices were then incubated for 30 min at 378C with
protective effects in animal models of cerebral ischaemia 2,3,5-triphenyltetrazolium chloride TTC, a colorimetric
[3,37]. SB 201823-A is another non-peptide calcium index of mitochondrial enzyme activity, fixed in 10
140 M
formalin and transferred to coverslips. Stained slices were face mask throughout the operation. Through a midline
initially scanned in true colour using an AGFA ‘Studios- cervical incision, both common carotid arteries were
can’ desktop scanner at 60 mm resolution. The ensuing exposed and freed from surrounding connective tissue. In
images were converted to 8-bit greyscale 0–255 for animals to be rendered ischaemic, both common carotid
mathematical analysis of staining [21]. The intensity of arteries were clamped for 5 min. At the end of the
striatal and cortical TTC staining was analysed by Osiris E
occlusion period, blood flow was re-established. In sham- University
Hospital of
Geneva, Switzerland
or operated animals, the arteries were exposed but not
ScionImage E Scion Corporation software and expressed
occluded. The wound was then sutured and the animals as a mean percentage greyscale value over the whole
allowed to recover. Throughout surgery, body temperature region of interest striatum or surrounding cortex.
was maintained at 378C using a ‘K-TEMP’ temperature Statistical comparisons between groups were made by
controller heating pad International Market Supply, Che- One Way ANOVA with posthoc application of the Stu-
shire, UK. After surgery, the animals were placed in a dent–Newman–Keuls test.
four compartmental thermacage Beta Medical and Sci- entific, UK which maintained the environmental tempera-
2.2. Pharmacokinetics ture at 288C and rectal temperatures were measured for a 6
h period after occlusion. LY393615 was administered at To assess exposure of LY393615, gerbils were dosed
10, 12.5 or 15 mg kg i.p. 30 min before and 2 h 30 min with 15 mg kg i.p. and brains harvested at 15, 30, 45 min,
post-occlusion in the first experiment. LY393615 was 1, 2, 4, 6 and 8 h later. We also evaluated exposure in rats
administered at 15 mg kg i.p. 30 or 60 min post-occlusion at the same time points after 1 mg kg i.v. In both cases
followed by two further doses of 5 mg kg i.p. 2 and 3 h brains were weighed and then homogenised with two
after the initial dose in the second and third experiments. volumes of distilled water. A 20 mM aqueous solution of
Five days after surgery, the animals were perfused LY393613
N-Butyl-2-[bis-4-fluorophenylmethox- transcardially with 30 ml of 0.9 saline followed by 100
y]ethylamine hydrochloride was prepared for use as an ml of 10 buffered formalin solution. The brains were
internal standard. 300 ml aliquots of brain homogenate and removed and placed in 10 formalin for 3 days, processed
10 ml internal standard solution were transferred to 1.5 ml and embedded in paraffin wax. Coronal sections 5 mm
eppendorf centrifuge tubes, mixed thoroughly and ex- were taken 1.5, 1.7 and 1.9 mm caudal to bregma using a
tracted with 10 ml trifluoroacetic acid TFA and 100 ml microtome Leitz 1400 sledge microtome. The slices were
acetonitrile. Tubes were spun, in a microcentrifuge, for 5 stained with haematoxylin and eosin and the neuronal
min at 13,000 r.p.m. and the supernatant transferred to density in the CA1 subfield of the hippocampus was
HPLC microvials for assay. LY393615 standards of 50, 20, measured using a microscope with grid lines 0.0530.05
5, 2, 0.5 and 0.1 mM concentration were prepared in mm as described previously [28]. The neuronal density is
homogenised control rat brain and then treated in the same expressed as number of viable cells per mm CA1 hip-
manner as the samples. pocampus. Statistical analysis of histological data was
Samples and standards were analysed by HPLC MS assessed using a 2-tailed unpaired Students t-test, with P
using a Hewlett Packard 1100 series autosampler and values ,0.05 being considered statistically significant.
binary pump Hewlett Packard, Bracknell, UK coupled to a Micromass Platform II mass spectrometer Micromass,
2.4. Focal ischaemia Manchester, UK. Chromatography was performed on a 25
cm32.1 mm Hichrom 5 C column Hichrom, Theale,
2.4.1. Endothelin-1 model. Rats were anaesthetised with
18
UK using acetonitrile:water 60:40 containing 5 mM inhalation anaesthetic and placed on a thermostatically
ammonium formate with the pH adjusted to 2.8 with TFA. controlled heating blanket to maintain body temperature
Twenty ml volumes were injected. MS analysis was within the range 37–388C. The rat was then placed in a
performed in the electrospray mode with a source tempera- Kopf stereotaxic frame and the scalp incised so as to
ture of 1208C and cone voltage of 25 V. Ions at m z 346 uncover the parietal bones. A 28-guage steel cannula was
LY393615 and m z 320 LY393613 were monitored. then inserted stereotaxically at the following co-ordinates
from bregma: AP5 10.9 mm, L5 25.2 mm and 28.7 2.3. Global ischaemia
mm below skull, Sharkey et al. [35]. Endothelin-1 200 pmol in 3 ml was infused over a 3 min period. The
Male Mongolian gerbils Bantin and Kingman, Hull, cannula was left in situ for a further 5 min and then
UK at least 3 months old and weighing in excess of withdrawn. The wounds muscles and skin were sutured
60–80 g were used. The animals were maintained in and the rat allowed to recover.
standard lighting conditions and food and water were LY393615 was administered at 15 mg kg i.p. immedi-
available ad libitum. The animals were anaesthetised with ately or 1 h post-occlusion followed by additional doses of
a 5 halothane oxygen mixture and maintained using 2 5 mg kg at 2 and 3 h after the initial injection. 3 days after
halothane delivered with oxygen at 1 litre per min via a surgery, the rats were perfused with heparinised saline
M .J. O’Neill et al. Brain Research 888 2001 138 –149
141
follow by 10 buffered formalin via the heart. The brains ‘ischaemia’ in both striatum P ,0.01 and cortex P ,
were removed for histology, stained with cresyl violet and 0.001.
the area of ischaemic damage at 8 stereotaxic levels was For global ischaemia studies, 5 mm sections taken 1.5–
measured using Optimus 5.2 software and from this an 1.9 mm caudal to the bregma in the anterior hippocampus
infarct volume was calculated. Statistical analysis of were examined under a microscope with grid lines. The
histological data was carried out using ANOVA followed pyramidal cell density was counted at three different
by Students t-test using P ,0.05 as the level of signifi- stereotaxic levels in the CA1 region of the hippocampus
cance. and the results expressed as mean6S.E.M. neuronal den-
sity per 1 mm CA1. The results indicated that there was 2.4.2. Monofilament model. In the monofilament model, a
severe loss of neurones in the CA1 region of the hip- segment of 220 mm diameter nylon monofilament coated
pocampus of 5 min occluded animals. The neuronal death with nail polish at the tip 370 mm, was inserted into the
involved nearly all the pyramidal neurones and this internal carotid artery of each male Wistar rat 295–315
neurodegeneration was not evident in any other forebrain g, and advanced until it blocked the origin of the middle
region. LY393615 provided dose-dependent neuroprotec- cerebral artery Belayev et al. [6]; Zea Longa et al. [54].
tion against the ischaemia-induced cell death in the CA1. After 2 h, the monofilament was retracted and reperfusion
Thus, LY393615 provided very good protection when occurred. LY393615 was administered at 15 mg kg i.p. 15
administered at 15 mg kg i.p. 30 min before and 2 h 30 min after reperfusion followed by 2 mg kg h i.v. infusion
min after occlusion Figs. 4 and 5. We also evaluated the for 6 h. Twenty-four hours later the brains were removed,
effects of lower doses of LY393615 at 10 mg kg or 12.5 frozen, sliced into 30 mm sections and stained with cresyl
mg kg i.p. 30 min before and 2 h 30 min post-occlusion. violet. The infarct area of each slice was measured with
Results indicated that 10 mg kg provided a small, but imaging system software ImagePro Plus, and infarct
significant neuroprotection Fig. 4. The intermediate dose volumes were calculated. Statistical analysis of histological
12.5 mg kg i.p. also provided a significant neuroprotec- data was carried out using ANOVA followed by Students
tive effect Fig. 4. t-test using P ,0.05 as the level of significance.
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
3. Results compound crossed the blood–brain barrier quickly and was
