Brain Research 888 2001 138–149 www.elsevier.com locate bres
Research report
21 1
LY393615, a novel neuronal Ca and Na
channel blocker with neuroprotective effects in models of in vitro and in vivo cerebral
ischemia
a , a
a a
Michael J. O’Neill , Caroline A. Hicks , Mark A. Ward , David J. Osborne ,
a c
c c
Graham Wishart , Kusum S. Mathews , Daniel P. McLaughlin , Jonathan A. Stamford ,
b b
b b
Deborah R. McCarty , Kelly E. Patrick , Carlos Roman , Jerome H. Fleisch ,
a a
Jeremy Gilmore , John R. Boot
a
Lilly Research Centre Ltd ., Erl Wood Manor, Windlesham, Surrey GU20 6PH, 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 1BB, 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-fluoro- phenyltetrahydrofuran-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 mechanism of ischaemia-induced neuronal cell death re-
mains to be elucidated. It is thought that during ischaemia, Although cerebral ischaemia is of major clinical impor-
the lack of energy to the brain may depolarize neurones tance in stroke and cerebrovascular disorders, the exact
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
Corresponding author. Tel.: 144-1276-853-547; fax: 144-1276-853-
NMDA and
a-amino-3-hydroxy-5-methyl-4-isoxazole
525. E-mail address
: oneill michael jlilly.com M.J. O’Neill.
propionate AMPA receptors, allows calcium to enter the
] ]
0006-8993 01 – see front matter
2001 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 4 3 - 2
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
