Note: Descriptions are shown in the official language in which they were submitted.
CA 02282236 1999-09-16
THE USE OF EXCITATORY AMINO ACID TRANSPORTER
INHIBITORS TO PROTECT AGAINST CNS WHITE MATTER INJURY
FIELD OF THE INVENTION
This invention relates to a method of reducing the damaging effect of CNS
white matter injury
(including traumatic CNS injury, anoxia, and ischemia) to mammalian CNS
tissue, particularly
spinal cord tissue, by zn vivo treatment thereof with inhibitors of excitatory
amino acid
transporters, such as the Na+-dependent glutamate transporter and
pharmaceutical compositions
comprising said inhibitors.
BACKGROUND TO THE INVENTION
The central nervous system consists of gray matter and white matter. Gray
matter contains the
cell bodies of neurons, embedded in a neuropil made up predominantly of
delicate neuronal and
glial processes. White matter, consists mainly of long processes of neurons,
the majority being
surrounded by myelin sheaths, and nerve cell bodies are lacking. Both gray and
white matter
contain large numbers of neuroglial cells and a network of blood capillaries.
In some parts of the
central nervous system, notably the brain stem (medulla, pons, and midbrain),
there are regions
that contain both nerve cell bodies and numerous myelinated fibers. These
regions are therefore
an admixture of gray matter and white matter.
There are a number of conditions that result in disruption of white matter
integrity and function
in the CNS. In addition to traumatic CNS injury, pathological states that
involve disruption of
white matter are: anoxia, ischemia, and demyelination. Stroke is a very common
condition
affecting both gray and white matter structures (Pantoni, et al., Stroke
(1996) 27:1641-1646).
The mode of injury of white matter fibers includes Ca 2+ overload mainly
through reverse
operation of the Na+-Ca2+ exchanger {for a review see: Stys, J. Cereb. Blood
Flow Metab. (1998)
18:2-25). More recent data implicate a component of glutamate excitotoxicity
in anoxic white
matter. Blocking AMPA receptors is significantly neuroprotective against
anoxic injury in
CA 02282236 1999-09-16
isolated spinal cord white matter (Li & Stys, J. Neurotrauma (1998)
15(10):880), and broad
spectrum inhibition of glutamate receptors with kynurenic acid protects optic
nerves
(representative CNS white matter tracts) against in vitro anoxia as well (Sakr
& Stys,
unpublished).
Traumatic brain injury is known to cause significant non-disruptive axonal
injury that contributes
to the neuropsychological deficits frequently encountered in this condition.
While the precise
mechanisms of such axonal injury are not known, injury cascades seen in
anoxic/ischemic and
traumatic spinal cord injury are likely to contribute to white matter damage
following head
1o trauma as well (Maxwell, et al., J. Neurotrauma (1997) 14:419-440;
Obrenovitch & Urenjak, J.
Neurotrauma (1997) 14:677-698).
Acute, traumatic spinal cord injury (SCI) is a devastating clinical condition
for which current
treatment is only modestly effective. It usually results in lifelong
disability for the patient and its
15 effect is enormous in terms of the psychological, social, and financial
costs to the patient, the
family and society. The neurological deficits resulting from SCI are due
primarily to damage to
the nerve fibres that carry messages up and down the spinal cord.
Despite the clinical diagnosis of "neurologically complete SCI", the spinal
cord itself is rarely
2o transected, as demonstrated by routine post-mortem histopathological
investigations that have
characterized the anatomical integrity of the lesion site. These observations
have been
corroborated by more recent noninvasive magnetic resonance imaging techniques,
which have
correlated changes in spinal cord tissue in the living patient with the gross
clinical histopathology
obtained post-mortem.
In this regard, there is increasing clinical and experimental evidence for
significant preservation
of descending tracts in neurologically complete SCI. In fact, use of the
potassium channel
Mocker, 4-aminopyridine, in chronic spinal-injured cats and more recently in
patients, provides
strong evidence for the persistence of anatomically intact but physiologically
dysfunctional
2
CA 02282236 1999-09-16
descending supraspinal pathways. These drug studies in humans and in animals
suggest that even
though these remaining intact nerve fibres are dysfunctional, they may be
induced to regain some
physiologically significant function with the appropriate pharmacological
intervention. More
importantly, increasing the survival of spinal cord axons following the acute
injury using
pharmacological intervention as proposed here, may significantly improve
clinical outcome;
even a small number of remaining functional fibers can support significant
clinical function
(Blight AR, 1983, J. Neuroscience)
Identification of the factors or mechanisms that cause nerve fiber injury,
particularly during the
l0 early post-injury period, could enable specific drug therapies to be
targeted to maximize
neurologic recovery. A large body of work exists dealing with the
pathophysiology of gray
matter anoxic and ischemic injury (for reviews see Seisjo, B.K., (1986) Eur
Neurol 25:45-56;
Choi, D.W., (1990) J. Neurosci 10:2493-2501; Haddad, G.G. and Jiang, C.,
(1993) Prog
Neurobiol 40:277-318; Seisjo, B.K. and Wieloch, T., Cellular and Molecular
Mechanisms of
15 Ischemic Brain Drainage (New York: Raven Press, 1996), pp.527). An emerging
theme
implicates cellular overload of Ca2+, occurring largely through glutamate-
gated receptors and
possibly voltage-gated Ca2+ channels, cell swelling as a result of excessive
Na+ and Cl-- influx,
free radical production, and delayed apoptotic neuronal death. In contrast,
much less is known
about the fundamental mechanisms of anoxic and ischemic injury to CNS
myelinated axons,
2o despite the fact that white matter has been shown to be very vulnerable to
this type of injury
(Follis, F., et al., (1993) J Cereb Blood Flow Metab 13:170-178; Kochhar, A.,
et al., (1991a)
Brain Res 542:141-146; Kochhar, A., et al., (1991b) JNeurotrauma 8:175-186;
van Euler, M., et
al., (1994) Exp Neurol 129:163-168).
25 Transporter proteins play a particularly important role in uptake of
extracellular amino acids in
the vertebrate brain (see Nicholls & Attwell, 1990, TIPS 11: 462-468). Amino
acids that function
as neurotransmitters must be scavenged from the synaptic cleft between neurons
to enable
continuous repetitive synaptic transmission. More importantly, it has been
found that high
extracellular concentrations of certain amino acids (including glutamate and
cysteine) can cause
3
CA 02282236 1999-09-16
neuronal cell death. High extracellular amino acid concentrations are
associated with a number
of pathological conditions, including ischemia, anoxia and hypoglycemia, as
well as chronic
illnesses such as Huntington's disease, Parkinson's disease, Alzheimer's
disease, epilepsy and
amyotrophic lateral sclerosis (see Pines et al., 1992, Nature 360: 464-467).
Glutamate is one example of such an amino acid. When present in excess (>about
300
pM; Bouvier et al., 1992, Nature 360: 471-474; Nicholls & Attwell, ibid.; >5
~,M for 5 min.;
Choi et al., 1987, J. Neurosci. 7: 357-358), extracellular glutamate causes
neuronal cell death.
Glutamate transporters play a pivotal role in maintaining non-toxic
extracellular concentrations
of glutamate in the brain.
During anoxic conditions (such as occur during ischemia), the amount of
extracellular glutamate
in the brain rises dramatically. This is in part due to the fact that, under
anoxic conditions,
glutamate transporters work in reverse, thereby increasing rather than
decreasing the amount of
extracellular glutamate found in the brain. The resultantly high extracellular
concentration of
glutamate causes neuron death, with extremely deleterious consequences for
motor and other
brain functions.
It has been considered that there are at least three glutamate pools that can
contribute to
glutamate release during CNS insults. One is Ca2+ independent reversal of the
glutamate
transporter (Nicholls and Atwell, 1990; Szatkowski et al., 1990; Attwell et
al., 1993). Another
mechanism seen in primary astrocyte cultures is a swelling-induced Ca2+
independent release
(Kimelberg et al., 1990). It has been proposed that inhibition of uptake
and/or reversal of the
glutamate transporter can occur when the electrochemical gradients for Na+ and
K+ are disrupted
during CNS insults; these effects can contribute significantly to the
increased [glu]o seen during
pathological states (Hansen, 1985; Ikeda et al., Attwell et al., 1993; Wahl et
al., 1994).
Ordinarily there is tight regulation of extracellular glutamate levels, which
are normally
measured to be approximately 1-2 ~M (Erecinska and Silver, 1990). High-
affinity Na+
4
CA 02282236 1999-09-16
dependent glutamate transporters are thought to be primarily responsible for
maintaining this low
extracellular glutamate concentration and are present on both neurons and
astrocytes. There are
now known to be at least three different subtypes of glutamate transporters
(GLAST-1, GLT-1
and EEAC1) in the rat, as well as an EAAT4 isoform in human cerebellum (Pines
et al., 1992;
Kanai and Hediger, 1992; Storck et al., 1992; Wadiche et al., 1995). Recent
work has suggested
that GLAST-1 and GLT-1 are primarily responsible for maintaining low [glu-]o
(Nicholls and
Attwell, 1990; Rothestein et al., 1994).
GLAST-1 and GLT-1, appear to be specific to brain and are expressed
predominantly in glia.
1o GLAST-1 mRNA is expressed diffusely throughout the cerebrum, but is
restricted to the
Purkinje cell layer of cerebellar cortex. EAAC-1 is expressed abundantly in
brain, intestine, and
kidney and at lower levels in liver and heart. Within brain, EAAC-1 mRNA is
found
predominantly in neurons, especially in certain neuronal subsets of
hippocampus, cerebellar
cortex, and cerebral cortex. These three transporters have about 50 percent
sequence similarity
15 and an inferred topology of 6 to 10 transmembrane segments. They all
energize amino acid
uptake by Na+ symport coupled to K+ antiport and are Cl--independent. While
they are all
specific for glutamate and aspartate, the details of glutamate transport may
vary among them.
2o Many studies have been performed over the years on the effects of anoxia
and ischemia in
peripheral axons (for a review see Stys, P.K., et al., (1995) in Waxman, S.G.,
et al., eds., The
Axon: Structure, Function and Pathophysiology (New York: Oxford University
Press, 1995),
pp. 462-479). Despite the strong similarity in structure and function between
central and
peripheral fibers, their responses to energy failure are quite different, and
therefore findings in
25 the peripheral nervous system cannot be extrapolated to central axons.
For example, in the case of hypoxia, an elegant study by Utzschneider, D.A.,
et al., ((1991)
Brain Res 551:136-141), emphasized the dramatically different effects of
hypoxia on central
(dorsal root ganglion cell processes in spinal dorsal columns) versus
peripheral (dorsal roots)
CA 02282236 1999-09-16
axons. The central component is very sensitive to hypoxia, whereas the
peripheral projections
originating from the same cell are completely unaffected by a 30 min exposure.
Even more
striking is the observation that although longer hypoxic exposures result in
significant
deregulation of elemental content (including large accumulations of Ca2+) in
the axoplasm of
peripheral myelinated fibers, reoxygenation promotes complete recovery
(Lehning, E.J., et al.,
(1966b) Brain Res 715:189-196); this is in stark contrast to central axons, in
that not only does
reoxygenation fail to correct the pathologic translocation of ions caused by
the hypoxic insult,
but many axons continue to deteriorate, accumulating more Ca2+ and presumably
suffering more
damage (Stys, P.K. and LoPachin, R.M., (1996) Neuroscience 73:1081-1090).
Figure 1 summarizes the current understanding regarding the unique as well as
overlapping
events responsible for gray and white matter anoxic and ischemic injury (Stys,
PK., (1998) J.
Cereb. Blood Flow Metab 18:2-25). Both gray and white matter are vulnerable to
interruption of
energy supply, which leads to impairment of ion transport and collapse of
transmembrane ion
gradients. Glutamate-gated channels play a central role in gray matter
pathophysiology,
allowing Na+ and Caz+ entry, whereas Na+ channels are the main Na+ influx
pathway in
myelinated axons. The resultant cellular Na+ overload and membrane
depolarization may further
activate certain subtypes of voltage-sensitive Ca2+ channels (VSCC) in gray
matter, while
promoting Ca2+ overload largely through reverse Na+-Ca2+ exchange in white
matter.
2o Downstream injury mechanisms likely then converge, involving excess
activation of Ca2+-
dependent biochemical pathways, generation of free radicals, and mitochondrial
injury,
culminating in cell death. Optimal protection of the CNS as a whole will
therefore require
combination therapy aimed at unique steps in gray and white matter regions, or
intervention at
common points in the injury cascades.
There is substantial evidence in the literature that following the initial
mechanical impact of
traumatic CNS and spinal cord injuries, sequential and progressive tissue
damage occurs at the
injury site. These observations have given rise to the secondary injury
hypothesis which
implicates a cascade of neuropathological mechanisms in the post-traumatic
destruction of spinal
6
CA 02282236 1999-09-16
cord tissue. Included in the list of secondary injury mechanisms are post-
traumatic ischemia and
the release of excitotoxic amino acids.
Death of neurons following traumatic or ischemic disorders has been related to
excess
intracellular calcium which occurs, for example through excessive activation
of post-synaptic
glutamate receptors (Choi, D.W. and Rothman, S.M., (1990) Ann Rev Neurol
13:171-182;
Young, W. (1992) JNeurotrauma 9(suppl)a9-s25). Considerably less is known
about glial cell
death, although electrophysiological and pharmacological studies indicate that
glial cells
probably do not have the same complement of glutamate receptors as do neurons.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide the use of excitatory
amino acid transporter
inhibitors to protect against CNS white matter injury. In accordance with an
aspect of the
present invention there is provided an in vivo method of reducing the
deleterious effect of
traumatic CNS tissue injury by applying to said tissue a therapeutically
effective amount of an
inhibitor to Na+ dependent glutamate transporter.
It is an object of the present invention to provide in its broadest aspect a
method of reducing the
damaging effect of traumatic CNS injury, anoxia, and/or ischemia to mammalian
CNS white
matter.
It is a further object of the invention to provide pharmaceutical compositions
for use in treating
mammals to reduce the damaging effect of traumatic CNS injury, anoxia, and/or
ischemia to
mammalian CNS white matter.
The present invention is based on a determination of a neuroprotective effect
against acute,
7
CA 02282236 1999-09-16
anoxic or traumatic CNS and spinal cord injury by the manipulation of Na+-
dependent glutamate
transporter. This determination is applicable to all myelinated nerve tracts
in the CNS and
further addresses the possibility that neurotoxicity results from the
accumulation of glutamate
molecules in the external environment of myelinated axons.
Accordingly, the invention provides in one aspect an in vivo method of
reducing the deleterious
effect of traumatic CNS injury, anoxia, and/or ischemia to mammalian CNS white
matter by
applying to said tissue a therapeutically effective amount of an inhibitor to
a Na+-dependent
glutamate transporter.
Preferred compounds are dihydrokainate (DKA), L-trans-2,4-pyrrolidine
dicarboxylate (L-trans-
PDC), isomers of L-anti-endo-3,4-methanopyrrolidine dicarboxylate and closely
related analogs.
In a further aspect the invention provides a therapeutic composition for
reducing the deleterious
effect of in vivo traumatic CNS tissue injury comprising a therapeutically
effective amount of an
inhibitor to a Na+-dependent glutamate transporter and a pharmaceutically
acceptable carrier,
diluent or adjuvant therefore.
BRIEF DESCRIPTION OF THE FIGURES
2o Table 1 present results of anoxia studies using: lA) GYKI 52466 and anoxia -
anoxia applied
from 60 to 120 min; 1B) DKA & PDC and anoxia - anoxia applied from 60 to 120
min;
Table 2 presents the results of spinal cord injury (SCI) studies: 2A) GYKI
52466 & SCI: SCI
applied briefly right after 60 min mark; 2B) PDC & SCI: SCI applied briefly
right after 60 min
mark.
Figure 1 presents a comparison of events in gray and white matter leading to
anoxic / ischemic
injury of CNS tissue.
8
CA 02282236 1999-09-16
Figure 2 presents a demonstration of the protective effects against anoxia of
a selective AMPA
receptor antagonist, GYKI 52466. GYKI52466 was applied beginning 1 hour before
in vitro
anoxia of spinal dorsal column slice. Normalized compound action potential
(CAP) amplitudes
are plotted against time, in the absence and presence of GYKI52466 (panel A)
(* P <0.05).
Panel B shows representative CAP tracings. These results indicate that
glutamate, acting largely
via AMPA receptors, participated in anoxic spinal dorsal column injury. (From
Li, Mewling,
Morley and Stys, J. Neurosci., 1999)
Figure 3 presents a demonstration of protective effects of GYKI 52466 in in
vitro traumatic
spinal cord injury. In particular, these results demonstrate that glutamate,
acting largely via
AMPA receptors, participates in traumatic spinal dorsal column injury. GYKI
52466, a selective
AMPA antagonist was applied beginning 1 hour before in vitro clip compression
of spinal dorsal
column slice, and demonstrated a highly protective effect against SCI (panel
A) (* P <0.05).
Representative CAP tracings are shown in panel B. (From Li, Mewling, Morley
and Stys, J.
Neurosci., 1999)
Figure 4 shows protective effects of Na+-dependent glutamate transporter
inhibitors:
dihydrokainate (DHK) or L-trans-PDC (PDC). In separate preparations, these
inhibitors were
applied beginning 1 hour before in vitro anoxia of spinal dorsal column slice.
Bar graph shows
significant protective effect measured by compound action potential (CAP)
amplitudes. These
results indicate highly protective effect against anoxia. Panel B shows
fluorescent measurements
of intracellular glutamate in axons and oligodendrocytes. Anoxia causes
significant depletion of
this excitatory neurotransmitter from both compartments, which is completely
reversed by
inhibition of Na+-dependent glutamate transport. These results directly
demonstrate that
glutamate is released by reverse transport during anoxia, and that inhibition
of reverse transport
is significantly neuroprotective (* P <0.05). (From Li, Mewling, Morley and
Stys, J. Neurosci.,
1999)
Figure 5 shows the protective effects of an exemplary Na+-dependent glutamate
transporter
9
CA 02282236 1999-09-16
inhibitor, L-trans-PDC, in a model of traumatic spinal cord injury. L-trans-
PDC was applied 1
hour before invitro SCI of spinal dorsal column slice induced by clip
compression. These results
show a highly protective effect against SCI and indicate that glutamate is
likely released by
reverse transport during SCI (* P <0.05).
DETAILED DESCRIPTION OF THE INVENTION
This invention is based on the determination that glutamate released by
reversal of the
Na+dependent glutamate transporter in the non-synaptic white matter tract is
an important
to mediator of anoxic and traumatic spinal cord injury. Thus, pharmacological
agents aimed at
inhibiting (completely or incompletely) reverse glutamate transport can be
used as
neuroprotectants against traumatic and ischemic CNS white matter injury.
The term, Na+dependent glutamate transporter, means any protein or isoform
determined to be a
15 member of the family of the Na+ dependent transporters of glutamate. Some
of these
transporters may be known to also transport other amino acids or
neurotransmitters, so the
transport activity need not be exclusive to glutamate. Common examples are the
GLAST
isoforms, the GLT isoforms and the EAAC isoforms. Newly identified Na+
dependent
transporters of glutamate that function in the manner described herein, also
fall within the scope
20 of this invention.
The term, inhibition, means complete or incomplete inhibition of Na+ dependent
transporter
activity.
25 Compounds that inhibit (completely or incompletely) Na+ dependent glutamate
transport are
used in the methods of this invention. A series of qualifying criteria are
provided to determine
whether a compound can be used in the method of this invention.
CA 02282236 1999-09-16
Tests to Establish Qualifying Criteria
There are a number of assays known in the art that are useful for determining
whether a
compound inhibits Na+ dependent transport activity.
Test No. 1: In Vitro Anoxia Assay
This assay determines the ability of the test compound to block the reduction
of electrical
activity following anoxia. In one example, 60 min of N2/C02 exposure in slices
of rat dorsal
column is used as an in vitro model of anoxia. Functional integrity is
measured
electrophysiologically by recording propagated extracellular compound action
potentials. The
tissue is rendered anoxic by N2/C02 exposure followed by reoxygenation. Post-
anoxic CAPs are
measured at 1 and 2 hrs of reoxygenation and compared to pre-anoxic responses
thus yielding a
percent recovery. Glutamate transport blockers are typically applied
beginnning 30-60 min
before the start of anoxia. Improvement in post-anoxic recovery compared to
drug-free controls
is an indicator of protective efficacy of putative glutamate transport
inhibitors. In addition, a
direct measure of glutamate release from axons and glia is performed by
quantitative
immunohistochemistry for glutamate. This allows a direct relative measure of
cellular glutamate
changes during anoxia in the absence or presence of glutamate transport
inhibitors (Li, Mealing,
Money and Stys, J. Neurosci., 1999). A reduction of cellular glutamate loss
during anoxia in the
presence of inhibitor is a strong indication of said agent's ability to reduce
toxic glutamate
release.
Test No. 2: In Vitro Mechanical Injury Assay
This assay determines the ability of the test compound to block the reduction
of electrical
activity following in vitro mechanical injury. In one example 15 sec. of clip
compression @ 2
grams in slices of rat dorsal column is used as an in vitro model of
mechanical injury. As in Test
11
CA 02282236 1999-09-16
No. 1, the compound action potential is used as a measure of functional
integrity, and post-
traumatic compared to pre-traumatic responses give an indication of recovery
after in vitro clip
compression. Glutamate transport blockers are typically applied beginnning 30-
60 min before
compression. Improvement in CAP amplitude in the presence of glutamate
transport inhibitor
reflects this agent's neurprotective activity as it relates to reduced
glutamate transporter activity
and consequently diminished release.
Test No. 3: Measurement of Labelled Glutamate or Aspartate Uptake
l0 Compounds can be screened to identify those which specifically interact
with a Na+ dependent
glutamate transporter and decrease its ability to take up a neurotransmitter,
e.g., an
antagonist/inhibitor. One method comprises transforming host cells with a
vector encoding a Na+
dependent glutamate transporter, such that a transporter polypeptide is
expressed in that host,
incubating the host cells with glutamate, aspartate or analog thereof which
has been labelled by a
detectable marker sequence (e.g., radiolabel or a non-isotopic label such as
biotin) and the
potential compound and determining whether translocation of the
neurotransmitter into the cell is
either inhibited or increased. By measuring the amount of neurotransmitter
inside the cell, one
skilled in the art could determine whether the compound is an effective
antagonist.
2o Test No. 4: Measurement of Labelled D-Aspartate Efflux
Primary astrocyte cultures can be used to assay the efflux of preloaded
tritiated D-aspartate in the
presence of the test compound. [3H]-D-aspartate is used as a nonmetabolizable
marker for the
intracellular glutamate and aspartate pools. Both of these amino acids are
transported on the
same carrier protein and label the nonvesicular pool of excitatory amino acids
(Erecinska and
Silver, Prog Neurobiol (1990) 35:245-296; Barbour et aL, JPhysiol (Lond)
(1993) 466:573-597.
In one example of this test, astrocytes grown on coverslips are incubated
overnight in 2.5 ml of
MEM containing 10% horse serum, together with 4 p,Ci/ml of [3H]-D-aspartate (1
mCi/ml;
12
CA 02282236 1999-09-16
specific activity 86.4 mCi/mg aspartate). Optionally, 8 ~Ci/ml Na2siCr04 is
added to the
incubation medium (1 mCi/ml; specific activity, SOmCi/mg Cr). The appearance
of S1 Cr in the
perfusate during release experiments can be used to determine whether an
increase in [3H]-D-
aspartate release is a result of cell detachment or lysis (Kimelberg et al.,
Brain Res (1993)
622:237-242. The loaded coverslips are inserted into a Lucite perfusion
chamber with a cut out
depression in the bottom for the 18 x 18 mm glass coverslips. The chamber has
a screw top and
when screwed down leaves a space above the cells of around 100 p.m. This
perfusion chamber is
well suited for measuring the release of [3H]-D-aspartate from astrocytes in
response to KCl
buffer because the volume in the chamber is relatively small (18 x 18 x 0.1 mm
= 32.4 ~1). This
chamber allows a complete change of the perfusing buffer within 2 min, as
determined by
removal of a trypan blue solution.
The cells are perfused with HEPES-buffered solution consisting of 140 mM NaCI,
3.3 mM KCI,
0.4 mM MgS04, 1.3 mM CaCIZ, 1.2 mM KH2P04, 10 mM (+)D-glucose, 25 mM HEPES.
NaOH (10 N) was used to pH the buffers to 7.4. Increased KCI buffers were made
by replacing
NaCI with KCI. The osrnolarity of all buffers are measured by a freezing point
osmometer
(Advanced Instruments, Neeham Heights, MA); the osmolarities are 285-290 mOsm.
Sucrose is
added to make any adjustments in osmolarity to exactly 290.
The Lucite chamber and a fraction collector are placed in an incubator set at
37°C, and the
perfusate is collected in 1 min intervals. At the end of the experiment, the
cells are digested off
the coverslip with 1N NaOH. The radioactivity is counted using a liquid
scintillation analyzer.
Percent fractional release for each point is calculated by summing the
radioactive counts from
the end time point to the beginning of each minute plus the radioactivity left
in the cell digest and
dividing the dpms released in each minute by these summed dpms.
Preferred compounds work directly, such dihydrokainate or L-trans-PDC Recently
a series of
L-3,4-methanopyrrolidine dicarboxylate (MPDC) isomers were investigated as
potential
inhibitors of the high affinity Na+-dependent glutamate transporter, wherein
kinetic analysis
13
CA 02282236 1999-09-16
demonstrated that L-anti-endo-MPDC is a potent competitive inhibitor
comparable to that of L-
glutamate and L-trans-2,4-pyrrolidine dicarboxylate (L-trans-PDC) {Bridges, et
al., (1994),
Neurosci Lett, 174(2):193-7). Another preferred compound is DL-TBOA
(Shimamoto, K. et al.,
(1998) Mol. Pharmacol. )
One skilled in the art would appreciate that the multitude of pharmacological
effects of the
candidate compounds could have to be taken into account in determining
usefulness and efficacy
for in vivo utility.
1o Means of delivering the Na+-dependent glutamate transport inhibitors to the
site of injury are
known in the art. Administration may be by injection, including intravenous
injections.
Catheters are another preferred mode of administration, potentially into the
intrathecal space.
Formulations may be any that are appropriate to the route of administration,
and will be
15 apparent to those skilled in the art. The formulations may contain a
suitable carrier, such as
saline, and may also comprise bulking agents, other medicinal preparations,
adjuvants and any
other suitable pharmaceutical ingredients.
EXAMPLES
EXAMPLE L~ Demonstration That Glutamate Applied Exogenously is Injurious to
Axons
An in vitro model of spinal white matter anoxia and trauma demonstrates that
glutamate applied
exogenously is highly injurious, probably acting through AMPA/kainate receptor
subtypes. For
example, using an electrophysiological assay, the electrical response is
attenuated to about 50%
of control amplitude after 120 minutes of glutamate exposure; 60 min of wash
did not reverse
this injury. In contrast, glutamate applied with antagonist such as NBQX or
kynurenic acid
results in more than 80% activity after a similar exposure. Evidence suggests
that much of the
glutamate dependent injury is directed at the myelin sheath.
14
CA 02282236 1999-09-16
EXAMPLE IL~ Demonstration of glutamate release via reverse of Na+-dependent
transporters.
This study demonstrates the effect of glutamate on the compound action
potentials (CAP)
recorded from isolated dorsal column segments in vitro at 37°C. CAP
amplitude decreased
significantly (eg. to 54% of control after 120 min) after perfusion with 1 mM
glutamate for 90 -
180 minutes in comparison with the time zero or time-matched controls in
glutamate-free
perfusate (P<0.01). These results indicate that glutamate impaired signal
conduction in dorsal
columns, and may induce functional injury in these white matter tracts. It is
not known where
1o glutamate originates from, if indeed it contributes to axonal injury in
spinal white matter tracts
which are devoid of synaptic machinery. Thus, the immunocytochemical
distribution of high
affinity Na+-dependent glutamate transporters, GLTl, GLAST and EAAC1, in
dorsal columns
was studied with confocal microscopy. It was found that GLT1 and EAAC1
displayed similar
patterns and were seen on the outer myelin sheath and at the nodal gap. Faint
axoplasmic signal
was also detected, accentuated at the nodal constriction. Although GLAST
staining was found in
similar regions, in contrast to GLT-1 and EAAC1, the myelin sheath appeared to
be stained
throughout its entire thickness with GLAST label.
Therefore it is concluded that in vitro application of glutamate results in
the functional
2o impairment of myelinated axons in dorsal spinal cord. Under pathological
conditions such as
trauma or ischemia, glutamate may be released in a non-vesicular fashion from
axons and/or glia
via reverse operation of Na+-dependent transporters, leading to damage to
nearby myelin,
supporting glia and possibly the axon cylinder itself.
EXAMPLE IIL~ Demonstration of the Role of Endogenous Glutamate in Two Injury
Paradigms
Two injury paradigms were used to demonstrate the role of endogenous
glutamate: in vitro
anoxia (60 min of N2/COZ exposure) and in vitro mechanical injury (15 sec. of
clip compression
2 grams), in slices of rat dorsal column. Both paradigms result in a reduction
of electrical
activity to about 25% (anoxia) and 35% (trauma) of control Applying the
selective AMPA
CA 02282236 1999-09-16
receptor blocker GYKI 52466 allowed recovery to more than 55% after anoxia vs.
25% control
(Figure 2), and to 65% after trauma vs. 35% control (Figure 3). This provides
evidence that
endogenous glutamate is released to cause significant injury, acting largely
(although not
necessarily exclusively) at the AMPA subtype of glutamate receptor.
EXAMPLE IV.' Demonstration that Glutamate is Released from the Axon Cylinder
and/or Glia
The axon cylinder is known to possess high concentrations of glutamate in the
axoplasm. Both
anoxia (LoPachin & Stys (1995) J. Neurosci., 15:6735-6746) and mechanical
trauma (Blight &
to LoPachin, Soc. Neurosci Abstr (1998)24:251) cause ionic perturbations that
would lead to
glutamate release via reverse Na+-glutamate transporters which are present in
these axons.
These studies and direct measurements from our laboratory (Li, Mealing, Morley
and Stys, J.
Neurosci., 1999; see Fig. 4B) support the hypothesis that the source of
glutamate is the axon
cylinder and glia. In particular, these studies demonstrate that the release
mechanism is reversal
15 of the Na+-dependent glutamate transporter that normally takes up glutamate
into the cell. Under
abnormal conditions, with high internal Na+ and membrane depolarization,
transport is reversed
and glutamate can be released instead, as has been described in gray matter
ischemia. This has
never been demonstrated in white matter anoxia or trauma.
20 Spinal cord slices in vitro were treated with Na+-dependent glutamate
transport blockers
(dihydrokainate or L-trans-PDC). As demonstrated in Figure 4, both were highly
neuroprotective against anoxia (dihydrokainate: 65% and L-trans-PDC; 75%, vs.
25% without
Mockers), and Figure 5 shows the results in trauma (L-trans-PDC; 70% vs. 35%
without
inhibitor).
These studies demonstrate that glutamate is an important mediator of anoxic
and traumatic spinal
cord injury. Glutamate is released by reversal of the Na+-dependent glutamate
transporter in this
non-synaptic white matter tract. Pharmacological agents aimed at blocking
glutamate transport
can be used as neuroprotectants against traumatic and ischemic CNS white
matter injury.
16
CA 02282236 1999-09-16
While the invention has been described in detail and with reference to
specific embodiments
thereof, it will be apparent to one skilled in the art that various changes
and modifications can be
made therein without departing from the spirit and scope of the invention.
17
CA 02282236 1999-09-16
1A Amplitude means means SD SD
Ctrl no GYKI Ctrl GYKI
drug
Om 100 100 0 0
60m 100 90,41309520 10.2149852
120m
180m. 26.666379656.04880954.4896560316.527948
240m 25.262833756.86309526.6288658713.1974253
1B Amplitudemeans means means SD SD SD
Ctrl DKA PDC Ctri DKA PDC
Om 100 100 100 0 ~ 0 0
60m 100 99.600153694.65452160 13.631153715.3966918
120m
180m 26.666379664.134408669.30668414.4896560312.08205412.604088
240m 25.262833760.516743576.56448236.6288658712.23133912.234225
TABLE 1
CA 02282236 1999-09-16
2A Amplitude means means SD SD
Ctrl GYKI Ctrl GYKI
Om 100 100 0 0
60m 100 100.9213350 16.9880136
90m 37.021308648.305975713.104677416.9843585
120m 32.629266164.589495811.626389616.4124141
180m 35.279221965.634407114.231423414.5902904
2B Amplitude means means SD SD
Ctrl PDC Ctrl PDC
Om 100 100 0 0
60m 100 96.7262150 16.8568686
90m 37.021308646.852871913.104677414.3666811
120m 32.629266163.02459511.626389613.3041156
180m 35.279221970.621207714.23142347,62929473
TABLE 2
