U.S. patent application number 17/555921 was filed with the patent office on 2022-04-07 for method of treating or preventing neurodegeneration.
The applicant listed for this patent is Reprise Pharmaceuticals, Inc.. Invention is credited to Veit Flockerzi, Marc Freichel, Manuel Friese, Doron Merkler, Benjamin Schattling, Karin Steinbach, Rudi Vennekens.
Application Number | 20220105059 17/555921 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-07 |
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United States Patent
Application |
20220105059 |
Kind Code |
A1 |
Friese; Manuel ; et
al. |
April 7, 2022 |
METHOD OF TREATING OR PREVENTING NEURODEGENERATION
Abstract
The invention relates to a compound which is effective in
inhibiting the function of the TRPM4 ion channel and the use of
such compound in treating or preventing a neurodegenerative
disease, such as Multiple Sclerosis, Parkinson's disease,
Alzheimer's disease, or amyotrophic lateral sclerosis, in a
subject. The invention also provides a pharmaceutical composition
comprising a TRPM4 inhibitory compound. The invention further
relates to in vitro methods for identifying pharmaceutically active
compounds that are useful for treating or preventing a
neurodegenerative disease.
Inventors: |
Friese; Manuel; (Hamburg,
DE) ; Schattling; Benjamin; (Hamburg, DE) ;
Steinbach; Karin; (Geneva, CH) ; Freichel; Marc;
(Saarlouis, DE) ; Flockerzi; Veit; (Blieskastel,
DE) ; Vennekens; Rudi; (Herent, BE) ; Merkler;
Doron; (Chancy, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Reprise Pharmaceuticals, Inc. |
New York |
NY |
US |
|
|
Appl. No.: |
17/555921 |
Filed: |
December 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17081704 |
Oct 27, 2020 |
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17555921 |
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16534243 |
Aug 7, 2019 |
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17081704 |
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14369624 |
Jun 27, 2014 |
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PCT/EP2012/076773 |
Dec 21, 2012 |
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16534243 |
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International
Class: |
A61K 31/18 20060101
A61K031/18; A61K 31/132 20060101 A61K031/132; A61K 31/4453 20060101
A61K031/4453; A61K 31/566 20060101 A61K031/566; A61K 31/365
20060101 A61K031/365; A61K 31/473 20060101 A61K031/473; A61K
31/4402 20060101 A61K031/4402; A61K 45/06 20060101 A61K045/06; A61K
31/198 20060101 A61K031/198; G01N 33/68 20060101 G01N033/68; A61K
31/00 20060101 A61K031/00; A61K 31/565 20060101 A61K031/565; A61K
31/196 20060101 A61K031/196; A61K 31/352 20060101 A61K031/352; A61K
31/64 20060101 A61K031/64; A61K 31/05 20060101 A61K031/05; C12Q
1/6881 20060101 C12Q001/6881 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 29, 2011 |
EP |
11196121.5 |
Claims
1. A method of treating multiple sclerosis in a subject in need of
treatment thereof, the method consisting of orally administering to
the subject a therapeutically effective amount of gliclazide or a
pharmaceutically acceptable salt, solvate or tautomer thereof.
2. The method of claim 1, wherein the multiple sclerosis is
relapsing remitting multiple sclerosis or secondary progressive
multiple sclerosis.
3. The method of claim 2, wherein the gliclazide is administered at
a dose of between about 10 .mu.g/kg to about 2000 .mu.g/kg.
4. The method of claim 3, wherein the administration begins after
the first symptoms occur.
5. The method of claim 3, wherein the gliclazide is administered
once daily.
6. A method of treating multiple sclerosis in a subject in need of
treatment thereof, the method consisting of co-administering to the
subject a therapeutically effective amount of gliclazide or a
pharmaceutically acceptable salt, solvate or tautomer thereof and a
therapeutically effective amount of an anti-neurodegenerative
agent.
7. The method of claim 6, wherein the multiple sclerosis is
relapsing remitting multiple sclerosis or secondary progressive
multiple sclerosis.
8. The method of claim 7, wherein the gliclazide is administered at
a dose of between about 10 .mu.g/kg to about 2000 .mu.g/kg.
9. The method of claim 8, wherein the administration begins after
the first symptoms occur.
10. The method of claim 8, wherein the gliclazide is administered
once daily.
Description
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing file
entitled 058223-502C3_Seq_List.txt, with a file size of about 19.9
kilobytes in size and created on our about 16 Dec. 2021, has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Neurodegenerative diseases of the central nervous system
(CNS) which cause progressive loss of neuronal structure and
function are particularly devastating diseases for the affected
patients and their families. Among these neurodegenerative diseases
are, for example, Multiple Sclerosis (MS), Parkinson's disease,
Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and
stroke. Due to the complexity of the CNS many of these diseases are
only poorly understood to date.
[0003] One of the most common progressive neurodegenerative
diseases is Multiple Sclerosis (MS). MS is a chronic inflammatory,
demyelinating disease of the CNS and the leading cause of
neurological disability in young adults. It affects approximately
2.5 million individuals worldwide and currently no curative
treatment is available. The pathogenesis of MS has been attributed
to a breakdown of T lymphocyte tolerance to CNS self-antigens
resulting in chronic inflammation with subsequent demyelination and
neuro-axonal degeneration (Compston, A. and Coles A. (2008) Lancet.
372:1502-1517). Axonal damage arises already early in the disease,
also independent of demyelination and correlates best with clinical
disability during the progressive course of MS (Kornek et al.
(2000), Am J Pathol. 157:267-276). Similarly, neuronal damage and
atrophy of the gray matter, with a predilection for the cingulate
gyrus, the fronto-temporal cortices and the hippocampus, have also
been shown to occur from the earliest stages and are likely to play
an important role in clinical progression (Fisher et al. (2008),
Ann Neurol. 64:255-265).
[0004] While the pathophysiological mechanisms leading to
neuro-axonal injury during chronic inflammation of the CNS are
still ill defined, it has been suggested that chronic CNS
inflammation is associated with an increased oxidative stress and
release of glutamate, which results in axonal and neuronal injury
by inducing mitochondrial dysfunction and increased metabolic
demand. This creates a chronic state of virtual hypoxia with
ensuing changes in ion homeostasis (Frischer et al. (2009) Brain.
132:1175-1189). Indeed, damaged and respiratory-deficient
mitochondria as well as reduced ATP production can be detected in
neuronal cells in MS lesions (Campbell et al. (2011), Ann Neurol.
69:481-492; Dutta et al. (2006), Ann Neurol. 59:478-489; Mahad et
al. (2009), Brain. 132:1161-1174). In addition, glutamate receptor
activation by release of glutamate in MS lesions and experimental
autoimmune encephalomyelitis (EAE) inflammatory infiltrates with
subsequent Ca.sup.2+ overload have been shown to occur in axons and
neurons under inflammation-induced hypoxic conditions (Pitt et al.
(2000), Nat Med. 6:67-70). However, the downstream mechanisms,
which are initiated by ATP shortage and Ca.sup.2+ overload
culminating in a sustained influx of cations and eventually leading
to neuro-axonal degeneration, remain elusive (Stirling et al.
(2010), Trends Mol Med. 16:160-170).
[0005] A major factor by which neurodegeneration in MS occurs is
due to an imbalanced glutamate metabolism with an increased
extracellular glutamate concentration. Glutamate is released by
activated immune cells and damaged CNS cells; indeed, cerebrospinal
fluid glutamate levels are increased in relapsing-remitting MS
during relapse and during clinical progression in secondary
progressive MS (Stover et al. (1997), Eur J Clin Invest.
27:1038-1043; Sarchielli et al. (2003), Arch Neurol. 60:1082-1088).
An increase in extracellular glutamate levels causes excitotoxic
neurodegeneration through ionotropic glutamate receptors such as
NMDA- and AMPA-receptors by eliciting Ca.sup.2+ and Na.sup.+
influx. Accordingly, glutamate receptor antagonists were somewhat
efficient in reducing neuro-axonal damage in EAE (Basso et al.
(2008), J Clin Invest. 118:1532-1543).
[0006] The transient receptor potential channel of the melastatin
subfamily TRPM4 provides a persistent cation influx combined with
gating properties, which are associated with alterations of energy
metabolism and ion homeostasis (Guinamard et al. (2010), Physiology
(Bethesda). 25:155-164). TRPM4 is a voltage-dependent
Ca.sup.2+-impermeable cation channel with a unitary conductance of
25 pS that is activated by a rise in intracellular calcium (Launay
et al. (2002) Cell. 109:397-407), whereas intracellular ATP
inhibits TRPM4 activity and regulates TRPM4 Ca.sup.2+ sensitivity
(Nilius et al. (2005), J Biol Chem. 280:6423-6433). Further,
phosphatidylinositol-4,5-bisphosphate and hydrogen peroxide (Simon
et al. (2011), J Biol Chem. 285:37150-37158) remove TRPM4 channel
desensitization during Ca.sup.2+ stimulation. Channel opening leads
to the conduction of monovalent cations with Na.sup.+ as the main
charge carrier (Launay et al. (2002), Cell. 109:397-407). TRPM4 is
expressed in different tissues including the heart, arteries,
gastrointestinal tract and immune system. It controls T cell,
dendritic cell and mast cell activation or migration through
regulating membrane depolarization and Ca.sup.2+ homeostasis.
Pathologically, TRPM4 has been associated with hypertension,
secondary hemorrhage after spinal cord injury, hyper-IgE syndrome
and cardiac conduction dysfunction (Mathar et al. (2010), J Clin
Invest. 120:3267-3279; Gerzanich et al. (2009), Nat Med.
15:185-191; Kruse et al. (2009), J Clin Invest. 119:2737-2744).
Trpm4 mRNA was found to be present in brain tissue, and a
suggestive TRPM4-mediated current was recorded in the brain stem
(Mironov (2008), J Physiol. 586:2277-2291). Inhibition of TRPM4 has
been suggested to prevent progressive hemorrhagic necrosis after
spinal cord injury (US 2010/0092469 Al). Expression of the TRPM4
ion channel was up-regulated in capillary epithelial cells after
spinal cord injury, leading to capillary leakiness and failure of
capillary integrity. However, functional neuronal expression and a
contribution of TRPM4 to neurodegeneration in diseases like MS have
thus far not been shown or otherwise suggested.
[0007] The present inventors surprisingly found that TRPM4 is
expressed in neuronal somata and axons in mice suffering from
Experimental Autoimmune Encephylomyelitis (EAE) and in MS lesions
of human patients. In addition, it could be demonstrated that TRPM4
is directly involved in mediating neuronal-axonal degeneration
under neuroinflammatory conditions, such as in MS. Moreover, the
present application provides evidence that inhibitors of the TRPM4
channel protein are capable of preventing neuro-axonal injury in
the inflamed CNS without affecting the encephalitogenic immune
response.
[0008] The present invention therefore provides a new and widely
applicable therapeutic strategy for preventing neurodegeneration in
a number of different diseases and conditions which are known to be
associated with neurodegeneration, amongst others, Multiple
Sclerosis.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention relates to a compound which is effective in
inhibiting the function of the TRPM4 ion channel and the use of
such compound in treating or preventing a neurodegenerative
disease, such as Multiple Sclerosis, Parkinson's disease,
Alzheimer's disease, or amyotrophic lateral sclerosis, in a
subject. The invention also provides a pharmaceutical composition
comprising a TRPM4 inhibitory compound. The invention further
relates to in vitro methods for identifying pharmaceutically active
compounds that are useful for treating or preventing a
neurodegenerative disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows that Trpm4 deletion ameliorates disease
severity in EAE mice. Clinical scores (a) and body weight changes
(b) for groups of WT EAE (n=15) and Trpm4.sup.-/- EAE (n=10) mice
after immunization with MOG.sub.35-55 are shown as mean values
.+-.s.e.m; asterisks indicate statistical significance;
**P<0.01. Results from one representative experiment out of
three are shown.
[0011] FIG. 2 demonstrates that Trpm4.sup.-/- mice show no
EAE-relevant immune system alterations. (a) T cell proliferation
after re-stimulation in single cell suspensions of draining lymph
nodes from WT EAE (n=6) and Trpm4.sup.-/- EAE (n=6) mice as
assessed by incorporation of [methyl-.sup.3H]-thymidininfiltrating
cells after immunization in WT EAE (n=4) and Trpm4.sup.-/- EAE
(n=3) mice: (b) total number of isolated CD45.sup.+ leukocytes; (c)
CNS-infiltrating leukocyte subsets. In a, b and c, data are shown
as mean values .+-.s.e.m. of one representative experiment out of
four. (d) Representative stainings and quantifications of spinal
cord sections of diseased animals 21 days post immunization (n=8
for WT and n=5 for Trpm4.sup.-/-) for cellular infiltration (HE)
and myelin (LFB/PAS) to determine the amount of lesions and the
extent of demyelination. (e) Clinical deficits in lethally
irradiated WT and Trpm4.sup.-/- mice which received bone marrow
transplantations from either genotype (WT in Trpm4.sup.-/-, n=5;
Trpm4.sup.-/- Trpm4.sup.-/-, n=6; WT in WT, n=8; Trpm4.sup.-/- in
WT, n=6).
[0012] FIG. 3A and 3B shows that TRPM4 is neuronally expressed in
mice and humans. (a) co-localization of TRPM4 and neuronal nuclei
(NeuN) in cervical spinal cord sections of healthy WT and
Trpm4.sup.-/- mice and immunohistochemical stainings of TRPM4 as
well as control staining in human spinal cord sections. (b)
co-localizations between TRPM4 and phosphorylated and
non-phosphorylated neurofilament H (SMI 31 and SMI 32) in cervical
spinal cord sections of healthy WT and in acutely inflamed lesions
of WT-EAE mice as well as TRPM4 and neurofilaments in periplaque
white matter lesions of MS patients.
[0013] FIG. 3C shows Quantitative real-time PCR of Trpm4
transcripts from WT and Trpm4-/- whole brain homogenates and E16
hippocampal neurons after 4 weeks of culture.
[0014] FIG. 4 depicts that Trpm4-/- mice show reduced axonal and
neuronal loss during EAE. (a) Diseased WT and Trpm4-/- animals 21
days post immunization stained for amyloid precursor protein (APP).
(b) WT-control, WT-EAE, Trpm4-/- control and Trpm4-/- EAE mice were
stained for neurofilaments (SMI 31 and SMI 32) in the corticospinal
tract and dorsal column and for neuronal nuclei (NeuN) in the gray
matter of cervical spinal cord sections. Numbers of axons and
somata were counted manually and by ImageJ software. Asterisks
indicate statistical significance; *P<0.05, **P<0.01.
[0015] FIG. 5 shows that TRPM4 contributes to excitotoxic cell
death in vitro. (a) Cell integrity of hippocampal neurons as
measured by LDH concentrations in supernatant of untreated cells or
cells treated with 0.5 .mu.M antimycin A or 50 nM glutamate. (b)
ATP levels of WT and Trpm4.sup.-/- hippocampal neurons after 4 h of
antimycin A or glutamate administration. (c) Whole-cell patch-clamp
recordings in hippocampal neurons of E16 embryos from WT and
Trpm4.sup.-/- mice after 10 days in culture, under resting
conditions (untreated) and after administration of 50 nM glutamate
for 2 h. Normalized current-voltage relationship. (d) The fold
change in membrane capacity after 50 nM glutamate incubation as
compared to those of untreated controls of WT and Trpm4.sup.-/-
neurons. (e) Hippocampal neurons of E16 embryos from WT and
Trpm4.sup.-/- mice after 10 days in culture. Glutamate treated and
control cells were stained for their cytoskeleton by .beta.-tubulin
III. Cell volumes were calculated. Representative pictures of
neuronal cells from WT and Trpm4.sup.-/- mice after glutamate
incubation for 2 h. Scale bar: 30 .mu.m. In FIGS. 5a and b results
are presented as mean values .+-.s.e.m. of four independent
experiments; statistical analyses were performed by two-way ANOVA
with Bonferonni posthoc test; asterisks indicate statistical
significance; *P<0.05, **P<0.01. Results in FIG. 5 d, c and e
are shown as mean values .+-.s.e.m. of two independent experiments
each; asterisks indicate statistical significance of student's
t-test; *P<0.05, **P<0.01.
[0016] FIG. 6 shows that glibenclamide treatment reduces clinical
disability and neurodegeneration in EAE mice. (a) Mean clinical
disability scores for WT-EAE and Trpm4.sup.-/- EAE mice, which
received daily injections of glibenclamide or DMSO control.
Treatment was started when first clinical symptoms occurred (day 8
after immunization with MOG.sub.35-55). (b) Healthy WT mice (WT
control) and WT EAE mice treated either with glibenclamide or
vehicle (n=4 per group) were stained 30 days post immunization for
neurofilaments (SMI 31 and SMI 32) in the corticospinal tract and
dorsal column and for neuronal nuclei (NeuN) in the gray matter of
cervical spinal cord sections. Numbers of axons and somata were
counted by ImageJ software.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is amongst other things based on the
insight that blocking the TRPM4 ion channel confers resistance of
axons and neurons towards hostile challenges which results in
neuro-axonal preservation and less clinical disability and
neurodegeneration. Therefore, the present invention specifically
contemplates to use antagonists and/or inhibitors of the TRPM4 ion
channel for treating a neurodegenerative disease by preventing
neuronal loss in a subject. Compounds that interfere with the
function of the TRPM4 ion channel have been extensively described
in the art. However, the use of such compounds for treating a
neurodegenerative disease has not yet been suggested.
[0018] Thus, in a first aspect the invention relates to a compound
which is effective in inhibiting the function of a TRPM4 ion
channel for use in a method of treating or preventing a
neurodegenerative disease in a subject. More specifically, the
invention relates to such an inhibitory compound for use in a
method of preventing neuronal damage and/or loss in a subject
suffering from a neurodegenerative disease. As used herein, the
"function of a TRPM4 ion channel" means the capability of the
protein to regulate the influx of ions into the cell, in particular
the influx of cations such as Na.sup.+. Accordingly, a "functional"
TRPM4 ion channel refers to a channel protein that effectively
regulates the influx of ions, such as Na.sup.+, into the cell The
present invention is hence useful for treating or ameliorating the
effects of neurodegenerative diseases which are associated with
TRPM4-mediated cytotoxicity.
[0019] The compounds of the present invention effectively prevent
damage and/or loss of neurons in the nervous system (NS) of a
subject, preferably in the central nervous system (CNS) of a
subject. As used herein, the NS is to be understood as being
composed of the CNS and the peripheral nervous system. Further, as
used herein, the CNS is to be understood as containing the brain
and the spinal cord. In a preferred embodiment, the compounds of
the invention are administered for preventing damage and/or loss of
neurons in the brain. In a further embodiment, progressive damage
and/or loss of neurons may be halted by administration of the
compounds of the invention. Halting the damage and/or the loss of
neurons means that the pathological processes of the
neurodegenerative disease which finally result in damage and/or
loss of neurons are stopped or at least reduced.
[0020] The neurodegenerative disease to be treated or prevented
according to the invention may be any known neurodegenerative
disease, preferably one that is associated with inflammation. As
used herein a neurodegenerative disease is a non-traumatic, disease
which is associated with the progressive loss of functional neurons
in the NS, preferably the CNS. The neurodegenerative disease to be
treated according to the invention may be caused by a genetic
predisposition. Conditions of the NS elicited traumatic events
and/or physical shock, such as traumatic brain injury, cerebral
ischemia, hypoxia and edema are not understood as neurodegenerative
diseases in the sense of the present invention.
[0021] Further, it has been found that TRPM4 signalling contributes
to glutamate excitotoxicity which has devastating effects in a
large number of neurodegenerative diseases. Therefore, the
compounds, methods and uses of the present invention will be
particularly useful in the treatment of neurodegenerative diseases
which have been associated with glutamate excitotoxicity.
[0022] Thus, according to a preferred embodiment, the
neurodegenerative disease to be treated or prevented according to
the invention is known to be associated with glutamate
excitotoxicity. As used herein, glutamate excitotoxicity refers to
a process that results in damaging or killing neuronal and/or
axonal cells by excessive stimulation of these cells by glutamate
and other substances that are capable of activating the glutamate
receptor. Thus, glutamate excitotoxicity occurs as a result from
overactivation of glutamate receptors. For example, the
neurodegenerative disease known to be associated with glutamate
excitotoxicity may be selected from the group Multiple Sclerosis
(MS), such as relapsing remitting MS or secondary progressive MS,
Parkinson's disease, Alzheimer's disease, and amyotrophic lateral
sclerosis. In a particularly preferred embodiment, the
neurodegenerative disease is MS.
[0023] The compounds of the present invention are pharmaceutically
active compounds which are effective in decreasing the expression
and/or the activity of the TRPM4 ion channel protein. The compounds
can include all different types of organic or inorganic molecules,
including peptides, polypeptides, oligo- or polysaccharides, fatty
acids, steroids, and the like. Typically, the compounds will be
small molecules with a molecular weight of less than about 2,500
daltons, less than 2000 daltons, less than 1500 daltons, less than
1000 daltons, or less than 500 daltons. In a particularly preferred
embodiment of the invention the compounds which inhibit the
function of TRPM4 ion channel have a molecular weight of less than
about 500 daltons.
[0024] Preferably, the compounds of the present invention are able
to cross the blood-brain barrier, i.e. they are blood-brain
barrier-permeable. This means that the compounds are able to reach
a site in the brain which is affected by neuronal injury and/or
loss. Inhibitory compounds with a molecular weight of below 500
daltons are particularly suitable, since they are often able to
cross the blood-brain barrier, or their transport across the
blood-brain barrier is achieved by other means, such as by
transporters or by hydrophobic stretches in the compound which
allow diffusion across the blood-brain barrier.
[0025] The compounds contemplated by the invention affect the
function of a TRPM4 ion channel protein. In a preferred embodiment,
the inhibitory compounds interfere with the human TRPM4 ion channel
protein. In the context of the present invention, the terms "TRPM4
ion channel" or "TRPM4" refer to the calcium-activated transient
receptor potential melastatin 4 cation channel. TRPM4 belongs to
the family of transient receptor potential cation channels, and
more specifically to the subfamily M of this family of cation
channels. The human TRPM4 protein occurs in two different isoforms
that are depicted in SEQ ID NO:1 (NP_060106) and SEQ ID NO:2
(NP_001182156).
[0026] Preferably, the compounds contemplated by the invention
inhibit the function of an TRPM4 ion channel having an amino acid
sequence as depicted in SEQ ID NO:1 (NP_060106) or an amino acid
sequence having at least 90% sequence identity to the sequence of
SEQ ID NO:1 (NP_060106). In a further preferred embodiment, the
compounds contemplated by the invention inhibit the function of an
TRPM4 ion channel having an amino acid sequence as depicted in SEQ
ID NO:2 (NP_060106) or an amino acid sequence having at least 90%
sequence identity to the sequence of SEQ ID NO:2 (NP_001182156).
Further isoforms of the human TRPM4 protein which retain the ion
channel activity may also be inhibited by the compounds of the
present invention.
[0027] The compounds which are contemplated herein for treating
and/or preventing a neurodegenerative disease are effective in
inhibiting the function of the TRPM4 ion channel. This means that
the compounds effectively reduce the extent of membrane current
that occurs due to the influx of cations upon opening of the
channel. In a preferred embodiment, the compound decreases the
TRPM4 ion channel activity by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 95% compared to the activity in the absence
of the compound. In a particularly preferred embodiment, the
inhibitory compound blocks the TRPM4 ion channel, i.e. the channel
is completely deactivated so that there is no detectable influx of
cations, such as Na.sup.+. The compounds preferably act by
interacting with or binding to the TRPM4 protein, thereby
preventing the sterical changes in the channel protein that occur
upon opening of the channel, e.g. in response to an increase in the
intracellular Ca.sup.2+ concentration. Compounds that actively bind
to TRPM4 may bind to the extracellular, the intracellular or the
transmembrane part of the TRPM4 ion channel protein.
[0028] Preferably, the inhibitory compound is specific for the
TRPM4 ion channel, i.e. the compound inhibits the expression and/or
activity of the TRPM4 ion channel, while it does essentially not
inhibit the expression and/or activity of other proteins or
enzymes, e.g. other ion channels. As a result of the TRMP4
specificity, the compounds according to the invention elicit no or
only tolerable side effects when administered to a subject.
[0029] The subject to be treated with the inhibitory compound will
normally be a mammal, and preferably is a human. Generally, the
subject can be of any age. The subject preferably suffers from a
neurodegenerative disease, more preferably from a progressive
neurodegenerative disease. The subject may be in any stage of the
disease, i.e. the disease may be in an early stage wherein the
subject shows only the first pathological signs that are normally
associated with said disease, or the subject may be in a late stage
of the disease. The subject to be treated may have a genetic or
other predisposition for developing a neurodegenerative disease in
the future.
[0030] The TRPM4-inhibitory compound of the invention can be
derived from different groups of molecules. For example,
TRPM4-inhibitory compounds can include antisense polynucleotides
which bind to the gene encoding the TRPM4 ion channel and block
transcription, RNA processing and/or translation of said gene. The
antisense molecules can be RNA or DNA molecules. Also, the
TRPM4-inhibitory compound of the invention can be a RNA molecule
which exerts its effect by RNA interference. Examples for such
compounds are RNAi molecules and siRNA molecules that are capable
of blocking translation of the TRPM4-encoding mRNA. Alternatively,
the TRPM4-inhibitory compound of the invention can be a ribozyme
that cleaves the TRPM4-encoding mRNA. Other classes of molecules
which may give rise to suitable TRPM4-inhibitory compounds include
peptides, antibodies and antibody fragments. Peptides that bind and
interfere with the TRPM4 channel protein may be conveniently
screened in random peptide libraries.
[0031] A number of inhibitors of TRPM4 have been described in the
art. For example, nucleotides such as ATP, ADP, AMP, AMP-PNP, and
adenosine have been described to inhibit the TRPM4 ion channel
quickly and reversibly. Similarly, polyamines like spermine have
also been found to block TRPM4 currents (Nilius et al. (2004), Eur
J Physiol. 448:70-75).
[0032] Other compounds that have been described to inhibit the
TRPM4 ion channel are those referred to in WO 2006/034048 and WO
03/079987. These compounds include antagonists to sulfonylurea
receptor-1 (SUR1), such as 9-phenanthrol, glibenclamide,
tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,
LY397364, LY389382, glyclazide, glimepiride, estrogen, and
estrogen-related compounds (such as estradiol, estrone, estriol,
genistein, non-steroidal estrogen, phytoestrogen, zearalenone, and
the like). The above compounds can be substituted or otherwise
modified at one or more sites, as long as these modifications do
not substantially impair the TRPM4-inhibitory effect of the
compounds and do not result in any undesired toxic side
effects.
[0033] In a preferred embodiment of the invention, the
TRPM4-inhibitory compound for use in treating or preventing a
neurodegenerative disease is 9-phenanthrol or a pharmaceutically
acceptable salt, solvate, tautomer or ester thereof. Examples of
pharmaceutically acceptable salts of 9-phenanthrol include
hydrochlorides and sulphates. 9-phenanthrol is a metabolite of
phenanthrene, and the IUPAC name is phenanthren-9-ol. The structure
of 9-phenanthrol is as follows:
##STR00001##
[0034] The above structure can be substituted or otherwise modified
at one or more sites, as long as these modifications do not
substantially impair the TRPM4-inhibitory effect of 9-phe-nanthrol
and do not result in any undesired toxic side effects. For example,
one or more hydrogen atoms of the C--H bonds of the heterocyclic
ring system can be substituted with halogen atoms, such as
chlorine, bromine or iodine atoms. Further, the hydrogen of the
C--H bonds can also be replaced by an alkyl group, such as methyl,
ethyl or propyl.
[0035] In another particularly preferred embodiment of the
invention, the TRPM4-inhibitory compound for use in treating or
preventing a neurodegenerative disease is glibenclamide or a
pharmaceutically acceptable salt, solvate, tautomer or ester
thereof. Examples of pharmaceutically acceptable salts of
glibenclamide include in particular hydrochlorides and sulphates.
Glibenclamide has been described as a modulator of ATP binding
cassette proteins (ABC transporters). The IUPAC name for
glibenclamide is
N-(4[N-(cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-methoxybenzamide.
It has the following structure:
##STR00002##
[0036] The person skilled in the art will understand that the
structure described above can be substituted or otherwise modified
at one or more sites, as long as these modifications do not
substantially impair the TRPM4-inhibitory effect of glibenclamide
and do not result in any undesired toxic side effects. For example,
one or more hydrogen atoms of the C--H bonds of the heterocyclic
ring system can be substituted with halogen atoms, such as
chlorine, bromine or iodine atoms. Further, the hydrogen of the
C--H bonds can also be replaced by an alkyl group, such as methyl,
ethyl or propyl.
[0037] The TRPM4-inhibitory compound of the present invention as
described above will normally be provided in the form of a
pharmaceutical composition which also comprises one or more
excipients, carriers and/or diluents which are suitable for the
intended way of administration. Generally, the compound may be
administered in any suitable form that does not interfere with its
TRPM4-inhibitory activity. The preferred route of administration
will depend inter alia on the location of the neurodegeneration to
be treated. For example, the compound may be administered orally in
the form of tablets, capsules, granule, powder, liquids, and the
like. Alternatively, the TRPM4-inhibitory compound may be
formulated for being administered parenterally, e.g. by intravenous
injection or intravenous infusion. In a preferred aspect, the
TRPM4-inhibitory compound is administered to the subject by
intravenous infusion, more preferably by short-term infusion within
less than 60 min, e.g. within 30 min, 20 min or 15 min.
[0038] Compositions suitable for injection and/or infusion include
solutions or dispersions and powders for the extemporaneous
preparation of such injectable solutions or dispersions. The
composition for injection must be sterile and should be stable
under the conditions of manufacturing and storage. Preferably, the
compositions for injection and/or infusion also include a
preservative, such as a chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. For intravenous administration, suitable
carriers may comprise physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF) or phosphate-buffered saline (PBS). Sterile
solutions for injection and/or infusion can be prepared by
incorporating the TRPM4-inhibitory compound in the required amount
in an appropriate solvent followed by filter sterilization.
[0039] The pharmaceutical compositions of the present invention
will comprise an amount of the TRPM4-inhibitory compound that is
effective to inhibit the TRPM4 ion channel, thereby reducing the
extent of neurodegeneration by protecting the NS, preferably the
CNS, from neuronal loss. The therapeutically effective amount of
the TRPM4-inhibitory compound to be administered will depend on
several parameters, such as the mode of administration, the
particular neurodegenerative disease to be treated, the severity of
the disease, the history of the disease, the age, height, weight,
health, and physical condition of the individual to be treated, and
the like. A therapeutically effective amount of the
TRPM4-inhibitory compound can be determined by one of ordinary
skill in the art without undue experimentation given the disclosure
set forth herein.
[0040] The TRPM4-inhibitory compound will preferably be
administered to a subject in an amount that ranges from 0.1
.mu.g/kg body weight to 10,000 .mu.g/kg body weight, e.g. from 0.5
.mu.g/kg to 7,500 .mu.g/kg body weight, from 1.0 .mu.g/kg to 5,000
.mu.g/kg body weight, from 5.0 .mu.g/kg to 3,000 .mu.g/kg body
weight, from 7.5 .mu.g/kg to 2,500 .mu.g/kg body weight, from 10
.mu.g/kg to 2,000 .mu.g/kg body weight, from 25 .mu.g/kg to 1,500
.mu.g/kg body weight, from 50 .mu.g/kg to 1,000 .mu.g/kg body
weight, from 100 .mu.g/kg to 800 .mu.g/kg body weight, from 300
.mu.g/kg to 600 .mu.g/kg body weight, and more preferably from 400
.mu.g/kg to 500 .mu.g/kg body weight.
[0041] Apart from the TRPM4-inhibitory compound, the pharmaceutical
composition provided by the present invention may further comprise
other anti-neurodegenerative or anti-inflammatory compounds which
are commonly used in the treatment of neurodegenerative diseases,
such as interferon beta-1a, interferon beta-1b, fampridine,
fingolimod hydrochloride, natalizumab, glatiramer acetate, or
mitoxantrone. Where the TRPM4-inhibitory compound is used in
combination with another anti-neurodegenerative agent, the two
active ingredients can be administered to the subject in the form
of a single pharmaceutical composition comprising both agents and
pharmaceutically acceptable excipients and carriers. Administration
of such a pharmaceutical composition will automatically result in a
simultaneous administration of both agents. Alternatively, the two
therapeutic agents may also be administered separately from each
other, i.e. in the form of two separate pharmaceutical
compositions, one containing the TRPM4-inhibitory compound, and the
other containing the additional anti-neurodegenerative or
anti-inflammatory agent. The two separate compositions can be
administered simultaneously, i.e. at the same time at two distinct
sites of administration, or they may be administered sequentially
(in either order) to the same site or to different sites of
administration.
[0042] Preferably, both the composition comprising the
TRPM4-inhibitory compound and the composition comprising the second
anti-neurodegenerative or anti-inflammatory agent are administered
according to a weekly dosing regimen, more preferably a regimen in
which a single dose of the TRPM4-inhibitory compound and a single
dose of the anti-neurodegenerative or anti-inflammatory agent is
administered every week for a treatment period of 2 or more weeks,
for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or more weeks. Preferably, the treatment period
comprises at least 12 weeks. It will also be possible to administer
the overall weekly dose of the TRPM4-inhibitory compound and/or the
anti-neurodegenerative or anti-inflammatory agent in more than one
administration per week, e.g. in 2 or 3 administrations per week.
In a preferred embodiment, the amount of the TRPM4-inhibitory
compound to be administered weekly is delivered as a single
intravenous infusion per week, and the amount of the
anti-neurodegenerative or anti-inflammatory agent to be
administered weekly is also given as a single intravenous infusion
per week, either at the same day of administration of the
TRPM4-inhibitory compound, e.g. within about 10 minutes to about 6
hours after administration of the TRPM4-inhibitory compound has
been completed, more preferably within about 30, 60, 90, 120, 150,
180, 210 or 240 minutes, or at any of days 2, 3, 4, 5, 6 or 7 of
the week.
[0043] For example, a combination therapy with the above-mentioned
agents that is based on a weekly dosing regimen begins on day 1 of
a treatment period, and a first therapeutically effective dose of
the TRPM4-inhibitory compound is administered at that day. A first
therapeutically effective dose of the additional
anti-neurodegenerative or anti-inflammatory agent can be
administered either on the same day, e.g. simultaneously or within
about 30, 60, 90, 120, 150, 180, 210 or 240 minutes after
administration of the TRPM4-inhibitory compound. Alternatively, the
additional anti-neurodegenerative or anti-inflammatory agent can be
administered at any of days 2, 3, 4, 5, 6 or 7 of the first week.
At day 8, a second therapeutically effective dose of the
TRPM4-inhibitory compound is administered accompanied or followed
by the second administration of the additional
anti-neurodegenerative or anti-inflammatory agent. The skilled
person will readily be able to design other administration regimens
which are suitable for the delivery of the combined active
ingredients.
[0044] The insight that a decreased expression of the TRPM4 gene
and/or a decreased activity of the TRPM4 cation channel prevents or
halts neurodegenerative disease in the NS, preferably the CNS of a
subject allows the design of screening assays which identify
pharmaceutically active compounds which are effective in preventing
or halting neurodegenerative disease in the NS, preferably the CNS,
of a subject.
[0045] In a further aspect, the present invention therefore
provides methods for identifying a pharmaceutically active compound
that could be used for treating or preventing neurodegenerative
diseases. The methods of the present invention can therefore be
used for drug screening approaches that aim to identify new
pharmaceutically active for treating diseases, such as Alzheimer's
disease or MS.
[0046] Accordingly, the invention relates to an in vitro method for
identifying a pharmaceutically active compound for treating or
preventing an inflammatory, neurodegenerative disease in a subject,
comprising [0047] (a) contacting a candidate compound with a
functional TRPM4 ion channel; [0048] (b) detecting whether said
candidate compound interferes with the function of the TRPM4 ion
channel; wherein a compound which inhibits the function of the
TRPM4 ion channel is suitable for treating or preventing said
inflammatory, neurodegenerative disease. The neurodegenerative
disease is preferably one that is associated with glutamate
excitotoxicity. More preferably, it is selected from the group
consisting of MS, Parkinson's disease, Alzheimer's disease, and
amyotrophic lateral sclerosis, wherein MS is particularly
preferred.
[0049] A decrease in the activity of the TRPM4 ion channel, i.e. in
its capability of regulating cation influx, may be determined by
any suitable assay. For example, a pharmaceutically active compound
which inhibits the TRPM4 ion channel leads to a reduction in the
inward current that can be measured in whole-cell patch-clamp
recordings after the addition of glutamate. A method suitable for
detecting whether a given candidate compound interferes with the
function of the TRPM4 ion channel is disclosed in the below Example
7. A different method could employ the measurement of
cation-sensitive fluorescent probes, which were previously loaded
into the respective cells.
[0050] In an alternative approach, the screening can target
compounds which interfere with TRPM4 transcription and/or
translation. In these embodiments, an in vitro method is provided
for identifying a pharmaceutically active compound for treating or
preventing an inflammatory, neurodegenerative disease in a subject,
comprising [0051] (a) contacting a candidate compound with a cell
that expresses a functional TRPM4 ion channel; [0052] (b) detecting
whether said candidate compound decreases the transcription of the
TRPM4 gene and/or the translation of the TRPM4 mRNA; wherein a
compound which decreases the transcription of the TRPM4 gene and/or
the translation of the TRPM4 mRNA is suitable for treating or
preventing said inflammatory, neurodegenerative disease. Again, the
neurodegenerative disease is preferably a disease that is
associated with glutamate excitotoxicity. More preferably, it is
selected from the group consisting of MS, Parkinson's disease,
Alzheimer's disease, and amyotrophic lateral sclerosis, wherein MS
is particularly preferred.
[0053] Screening methods which monitor the transcription of the
TRPM4 gene and/or the translation of the TRPM4 mRNA are
particularly useful, e.g. for identifying compounds which inhibit
the TRPM4 ion channel by decreasing TRPM4 expression. By use of
these methods, it will for example be possible to identify
compounds that target transcription factors which are relevant for
TRPM4 expression. Also, suitable ribozymes or antisense DNA
molecules can be identified in this way.
[0054] A number of different screening methods to evaluate the
effects of candidate compounds on gene expression or enzyme
activity may be used. The methods of the invention make use of any
suitable method to detect a decrease in the expression level of the
TRPM4 ion channel gene and/or in the activity of the TRPM4 ion
channel. Suitable methods include e.g. biochemical or cellular
methods. The skilled person would be able to identify such methods
with his or her common knowledge.
[0055] For example, a decrease in the expression level of the TRPM4
ion channel gene may be detected by methods which allow the
quantification of the transcription or the translation product of
the TRPM4 ion channel gene, e.g. the quantification of TRPM4 mRNA
or protein. Such methods are well known in the art. For example,
polymerase chain reaction (PCR)-based methods, such as standard
PCR, real-time PCR and quantitative real-time PCR, or Northern
Blots may be used to determine the concentration of the TRPM4 mRNA.
The concentration will be determined relative to a sample which is
not contacted with the respective candidate compound, i.e. relative
to a negative control, or to the same sample before contacting it
with the respective compound. The concentration of TRPM4 protein
may analogously be determined with standard methods such as
SDS-PAGE, enzyme linked immunosorbent assays (ELISA), Western
Blots, mass spectrometry or any other suitable means. The TRPM4
protein concentration will then be compared to a sample which has
not been contacted with the respective compound or to the same
sample before contacting it with the respective compound.
[0056] The cell that expresses the functional TRPM4 ion channel is
preferably a mammalian cell, more preferably a human cell. The cell
can be derived from a cell line, preferably a neuron- or brain
cell-derived cell line, or from a primary cell culture, more
preferably from a primary neuronal cell culture.
[0057] The in vitro methods of the present invention may preferably
be carried out as high throughput screening techniques which allow
for the examination of thousands of different compounds in a short
period of time. Candidate compounds for use in screening methods
can be provided in the form of libraries which comprise a high
number of synthetic or natural compounds. High throughput screening
techniques are described in detail in the prior art. The compounds
identified by the screening methods may be validated in animal
models, such as mouse models to confirm their activity in vivo.
[0058] The candidate compounds used in the screening methods of the
present invention can include all different types of molecules as
described for the compounds of the present invention above.
[0059] The TRPM4 that is used in the above assays can be
recombinantly expressed. Accordingly, it will be understood by the
skilled person that the above assays can be performed not only with
the specific sequences depicted in SEQ ID NO:1 and 2, but also with
variants, derivatives and enzymatically active fragments of these
TRPM4 sequences. As used herein, variants of TRPM4 are polypeptides
that differ by one or more amino acid exchanges from the amino acid
sequence shown in SEQ ID NO:1 or 2. Generally, any amino acid
residue of the amino acid sequence shown in SEQ ID NO:1 or 2 can be
exchanged for a different amino acid, provided the resultant
sequence of the variant is still capable of forming a functional
ion channel. In particular, variants for which a total of up to 5%,
10%, 15%, or 20% of the amino acids differs from the amino acid
sequence shown in SEQ ID NO:1 or 2 are included. Polypeptides in
which one or more amino acids were inserted in the amino acid
sequence of SEQ ID NO:1 or 2 are also included as variants. Such
insertions can be made at any position of the polypeptide shown in
SEQ ID NO:1 or 2. Moreover, polypeptides in which one or more amino
acids are missing in comparison with SEQ ID NO:1 or 2 are also
considered to be variants of the polypeptides of SEQ ID NO:1 or 2.
Such deletions can apply to any amino acid position of the sequence
of SEQ ID NO:1 or 2.
[0060] Variants of TRPM4 will preferably have at least 80% sequence
identity, more preferably at least 85% sequence identity, and even
more preferably at least 90% sequence identity, e.g. 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% sequence identity, with the human
TRPM4 ion channel protein shown in SEQ ID NO:1 or 2 when these
sequences are optimally arranged, e.g. with the computer program
GAP or BESTFIT using the default gap method. Computer programs for
determination of the amino acid identity are known to the skilled
person.
[0061] Enzymatically active fragments of the sequence shown in SEQ
ID NO:1 or 2 or variants thereof are to be understood to refer to
peptides or polypeptides that differ from the amino acid sequence
shown in SEQ ID NO:1 or 2 or from the above-defined variants
thereof by the absence of one or more amino acids at the N-terminus
and/or at the C-terminus of the peptide or polypeptide.
[0062] Derivatives of the polypeptides shown in SEQ ID NO:1 or 2 or
of the variants thereof refer to polypeptides that possess amino
acids with structural modifications, for example, modified amino
acids, relative to a polypeptide shown in SEQ ID NO:1 or 2 or
variants thereof. These modified amino acids can be, e.g. amino
acids that have been altered by phosphorylation, glycosylation,
acetylation, thiolation, branching and/or cyclization. It is
preferred that the variants or derivatives of the TRPM4 polypeptide
shown in SEQ ID NO:1 or 2 or the active fragments of this
polypeptide or its variants retain at least 75%, and preferably up
to 80%, 85%, 90% or even up to 99% of the ion channel activity of
the TRPM4 polypeptide shown in SEQ ID NO:1 or 2.
[0063] In another aspect, the invention relates to the use of: (a)
a polypeptide comprising the amino acid sequence shown in SEQ ID
NO:1 or SEQ ID NO:2 or a variant thereof having at least 80%, more
preferably at least 90% sequence identity to the amino acid
sequence shown in SEQ ID NO:1 or SEQ ID NO:2; or (b) a
polynucleotide encoding a polypeptide of (a) or the complement
thereof for identifying pharmaceutically active compounds for
treating or preventing an inflammatory, neurodegenerative disease.
The neurodegenerative disease is preferably a disease that is
associated with glutamate excitotoxicity. More preferably, it is
selected from the group consisting of MS, Parkinson's disease,
Alzheimer's disease, and amyotrophic lateral sclerosis, wherein MS
is particularly preferred.
EXAMPLE 1
TRPM4 Deficiency Ameliorates Experimental Autoimmune
Encephalomyelitis (EAE)
[0064] In order to investigate whether TRPM4 modulates the
pathogenesis of EAE, knockout mice with a dysfunctional Trpm4 gene
(Trpm4.sup.-/-) and wild-type (WT) mice were immunized with the
myelin oligodendrocyte glycoprotein peptide 35-55 (MOG.sub.35-55)
in order to induce EAE in these animals. The sequence of the
MOG.sub.35-55 peptide used for immunization is shown in SEQ ID
NO:3. Briefly, C57BL/6 Trpm4.sup.-/- mice (Vennekens et al. (2007),
Nat Immunol. 8:312-320) and Trpm4.sup.+/+ littermates (referred to
as WT controls) were immunized subcutaneously with 200 .mu.g/mouse
MOG.sub.35-55 in complete Freund's adjuvant (Sigma-Aldrich)
containing 4 mg/ml Mycobacterium tuberculosis (H37Ra, Difco). In
addition, 200 ng pertussis toxin (Calbiochem) was injected
intravenously on the day of immunization and 48 h later. The mice
were sex and age (6-10 weeks) matched and were scored for clinical
signs every day over a period of 60 days by the following system:
0, no clinical deficits; 1, tail weakness; 2, hind limb paresis; 3,
partial hind limb paralysis; 3.5, full hind limb paralysis; 4, full
hind limb paralysis and fore limb paresis; 5, pre-morbid or dead.
Animals were sacrificed when their scores reached 4 or higher.
Clinical scores (FIG. 1a) and body weight changes (FIG. 1b) were
determined in triplicate for groups of WT EAE (n=15) and
Trpm4.sup.-/- EAE (n=10) mice after immunization.
[0065] Results: As can be taken from FIG. 1, TRPM4 deficiency
resulted in an overall reduced disease severity (P<0.01; FIG.
1a) and a significantly better recovery from weight loss
(P<0.01; FIG. 1b) compared with WT littermates during EAE. These
results indicate a decisive role of TRPM4 in mediating clinical
disability in EAE.
EXAMPLE 2
Analysis of Immune Cell Activation by Detecting Incorporation of
[methyl-.sup.3H]-thymidine
[0066] Since EAE is an autoimmune disease and TRPM4 was previously
shown to fulfil functions in immune cell activation and migration,
it was first examined whether the protective phenotype in
Trpm4.sup.-/- could be explained by altered immune cell activation
or infiltration. For this purpose, C57BL/6 Trpm4.sup.-/- mice and
WT controls were treated as described in Example 1. For assessing
T-cell proliferation, single cell suspensions from draining lymph
nodes and spleens were prepared 15 days after immunization from WT
EAE (n=6) and Trpm4.sup.-/- EAE (n=6) mice by homogenizing the
tissues through a 40 .mu.m cell strainer (BD Biosciences). Cells
were sedimented by centrifugation (300.times.g, 7 min, 4.degree.
C.) and splenic red blood cells were lysed in red blood cell lysis
buffer for 7 min at 4.degree. C. Cells were washed once in PBS and
resuspended in buffer. The lymph node cells obtained from the
immunized animals were cultured in 96-well plates (Sarstedt) at
2.times.10.sup.5 cells/well in mouse complete medium and
re-stimulated with different concentrations of the MOG.sub.35-55
peptide or anti-CD3 (145-2C11, eBioscience). T cell proliferation
was assessed by incorporation of [methyl-.sup.3H]-thymidine as
follows: After 2 days, cells were pulsed with 1 .mu.Ci
[methyl-.sup.3H]-thymidine (Amersham) per well for 16 h. Cells were
harvested and spotted on filtermats with a Harvester 96 MACH III M
(Tomtec) according to manufacturer's instructions. Incorporated
activity per 96-well was assessed in a beta counter (1450
Microbeta, Perkin-Elmer) in counts per minute (cpm). The
stimulation index of the applied peptides or antibodies was
calculated by dividing the mean incorporated activity of stimulated
wells by the mean of unstimulated control wells.
[0067] Results: The results of the [methyl-.sup.3H]-thymidine
incorporation studies are shown in FIG. 2a. It can be seen there
that Trpm4.sup.-/- mice show no EAE-relevant immune system
alterations. Evidently, the deletion of the gene Trpm4 did not
alter the proliferation of peripheral T cells being specific for
MOG.sub.35-55 as analyzed by .sup.3H-thymidine incorporation after
MOG.sub.35-55 immunization.
EXAMPLE 3
Analysis of CNS Infiltrates by Flow Cytometry
[0068] Since the protective phenotype observed in Example 1 could
not be explained by altered immune cell activation, CNS (brain and
spinal cord) infiltrates were analyzed by flow cytometry. For the
isolation of CNS-infiltrating immune cells, WT EAE (n=4) and
Trpm4.sup.-/- EAE (n=3) mice were sacrificed by CO.sub.2 inhalation
and immediately transcardially perfused with ice-cold PBS. Brain
and spinal cord were removed, minced with blades and digested in
collagenase/DNAseI solution (Roche) for 45 min at 37.degree. C.
Tissue was triturated through a 40 p.m cell strainer. The
homogenized tissue was washed in PBS (300.times.g, 10 min, and
4.degree. C.). Immune cells including microglia were separated from
myelin, other glia and neuronal cells by centrifugation over a
discontinuous percoll (GE Healthcare) gradient. The homogenized
tissue was resuspended in 30% isotonic percoll, transferred into a
15 ml Falcon tube and 78% isotonic percoll was layered underneath.
The gradient was centrifuged (1500.times.g, 30 min, 4.degree. C.)
and CNS-infiltrating immune cells were then recovered from the
gradient interphase. The isolated cell fraction was washed two
times in PBS and subsequently resuspended in buffer. The total
number of isolated CD45.sup.+ leukocytes was quantified using
TruCOUNT.RTM. beads (BD Bio-sciences). CNS-infiltrating leukocyte
subsets were identified as CD45.sup.intCD11b.sup.+ microglia,
CD45.sup.highNK1.1.sup.+CD3.sup.-NK cells,
CD45.sup.highNK1.1.sup.+CD3.sup.+NKT cells,
CD45.sup.highLy.sup.-6G.sup.+ neutrophils,
CD45.sup.highCD11b.sup.-c-Kit.sup.+ mast cells,
CD45.sup.highLy-6G-CD11b+CD11c-macrophages,
CD45.sup.highLy-6G-CD11b-CD11c.sup.+ DC,
CD45.sup.highLy-6G-CD11b-CD11c-CD3.sup.+ T cells and
CD45.sup.highLy-6G-CD11b-CD11c-CD45R.sup.+ B cells (all antibodies
are rat IgG except for CD3 and CD11c (hamster IgG) and NK1.1 (mouse
IgG), all from eBioscience except of c-Kit which was from
Biolegend).
[0069] Results: Equal total numbers of CNS-infiltrating CD45.sup.+
leukocytes (FIG. 2b) and the same subset composition of infiltrates
were found in Trpm4.sup.-/- and WT mice (FIG. 2c). Of note,
recruitment of dendritic cells and mast cells to the CNS was not
influenced by Trpm4 deletion (data not shown). Further, numbers of
FoxP3.sup.+ regulatory or effector T cells, expression of the
activation markers CD25 and CD69 as well as interferon-.gamma.
(IFN-.gamma.), interleukin (IL)-17A, IL-4 and IL-10 cytokine
production by CD4.sup.+ T cells were not significantly altered in
the periphery (spleen and lymph nodes) or CNS. Consequently, no
significant difference with regard to the numbers of inflammatory
lesions (HE) and extend of demyelination (LFB/PAS) (FIG. 2d) as
well as densities of infiltrates with T cells (CD3) and
macrophages/microglia (Mac3) were observed within spinal cords at
day 21 post immunization, a time point at which the earliest
significant differences in clinical disability between
Trpm4.sup.-/- and WT mice are observed. Also, no difference in
blood-brain-barrier integrity or serum glucose levels between
Trpm4.sup.-/- EAE and WT EAE mice could be detected (data not
shown).
EXAMPLE 4
Bone Marrow Chimeric Mice
[0070] Finally, bone marrow (BM) chimeric mice were established by
reconstituting lethally irradiated Trpm4.sup.-/- or WT mice with
bone marrow from either Trpm4.sup.-/- or WT mice. This was followed
by an active EAE induction in these chimeras. Bone marrow chimeric
mice were generated by lethal whole-body irradiation (9 Gy; 1
Gy/min) using a caesium-137 gamma irradiator (BIOBEAM 2000) of
5-6-week-old recipient WT and Trpm4.sup.-/- mice which were
reconstituted 24 h later with 5.times.10.sup.6 bone marrow cells
derived from tibiae and femurs from respective donors. The lethally
irradiated mice received bone marrow transplantations from either
genotype (WT into Trpm4.sup.-/-, n=5; Trpm4.sup.-/- into
Trpm4.sup.-/-, n=6; WT into WT, n=8; Trpm4/.sup.-/- into WT, n=6).
In addition to WT and Trpm4.sup.-/- cells, BM cells from CD45
congenic C57BL/6 Ly5.1 mice (CD45.1) were transferred into
irradiated C57BL/6 WT mice (CD45.2) and assessed reconstitution
(>95%) by FACS analysis of peripheral blood cells of mice 6
weeks after grafting. After recovery of the recipients for 6 weeks,
the mice were actively immunized with MOG.sub.35-55 to induce EAE.
The recipient mice were scored for their clinical deficits.
[0071] Results: The results of the reconstitution experiments are
depicted in FIG. 2e. The figure shows the results from the EAE
experiments as mean values and statistical analyses were performed
by two-way ANOVA; asterisks indicate statistical significance of
Trpm4.sup.-/- in WT vs. WT in Trpm4.sup.-/-; **P<0.01; other not
shown significances are: Trpm4.sup.-/- in WT vs. Trpm4.sup.-/- in
Trpm4.sup.-/-, P<0.05; WT in WT vs. WT in Trpm4.sup.-/-,
P<0.01; WT in WT vs. Trpm4.sup.-/- in Trpm4.sup.-/-, P<0.05;
Trpm4.sup.-/- in Trpm4.sup.-/- vs. WT in Trpm4.sup.-/- , n.s.; WT
in WT vs. Trpm4.sup.-/- in WT, n.s.; results from one
representative experiment out of two are shown. It can be seen that
Trpm4 deletion in donor BM did not affect the disease course.
Protection from disease was only observed in mice that received the
Trpm4.sup.-/- BM, but not in mice that received WT BM
(P<0.01).
[0072] Taking together the results from Examples 2-4, it is evident
that the absence of TRPM4 does not impair the activation or
recruitment of disease-relevant immune cells during autoimmune
inflammation of the CNS. It is therefore to be concluded that TRPM4
signalling within the CNS parenchyma is responsible for the altered
disease course in Trpm4.sup.-/- mice.
EXAMPLE 5
TRPM4 Expression in Neurons and Axons
[0073] In order to understand the contribution of TRPM4 to the
ameliorated EAE course, TRPM4 expression in the CNS of humans and
mice was analyzed.
[0074] a) Preparation and analysis of murine tissues
[0075] Mice were anesthetized with an intraperitoneal injection of
100 .mu.l per 10 g of body weight of a mixture of 10 mg/ml
esketamine hydrochloride (Pfizer), 1.6 mg/ml xylazine hydrochloride
(Bayer) and water. Afterwards the animals were perfused with 0.1 M
phosphate buffer and fixed with 4% paraformaldehyde (PFA) in 0.1 M
phosphate buffer. The spinal cords were resected and fixed for 30
min with 4% PFA and cryoprotected in 30% sucrose in PBS at
4.degree. C. Midcervical spinal cord sections were cut transversely
at 12 .mu.m with a freezing microtome (Leica Jung CM3000) and
stored in a cryoprotective medium (Jung) at -80 .degree. C. For
immunohistochemistry the sections were incubated in blocking
solution (5% normal donkey serum in PBS) containing 0.1% Triton
X-100 at room temperature for 45 min and subsequently stained
simultaneously or consecutively overnight at 4.degree. C. with
antibodies against the following structures: Phosphorylated
neurofilaments (SMI 31, mouse IgG, 1:1,000; Covance),
non-phosphorylated neurofilaments (SMI 32, mouse IgG, 1:1,000;
Covance), neuronal nuclei (NeuN, mouse IgG, 1:200, Millipore) or
TRPM4 (rabbit IgG, 1:100, Abcam). Secondary antibodies were Alexa
Fluor 488coupled donkey antibodies recognizing rabbit IgG,
Cy3-coupled goat antibodies recognizing mouse IgG and Alexa Fluor
488coupled goat antibodies recognizing mouse IgG (all 1:600,
Dianova). DNA was stained with Hoechst 33258 (Invitrogen). Control
experiments with no primary or secondary antibodies showed no
staining (data not shown). Analyses of the sections were done with
a Leica TCS SP2 confocal microscope. For quantification of axonal
loss multiple representative images were taken with a 63-fold lens
from the corticospinal tract and the dorsal column and axons were
counted with ImageJ software based on a minimum diameter of 6.15
.mu.m.sup.2. Threshold intensities were fixed across experimental
groups for each type of tissue examined. Accuracy of automated
counting technique was confirmed by manual counting of sample
images. Neuronal somata of the gray matter were counted
manually.
[0076] b) Preparation and analysis of human tissue
[0077] Histopathological analysis of MS tissue was performed on
PFA-fixed sections from human brain biopsies of patients with MS.
All tissue blocks were first classified with regard to lesional
activity. Avidin-biotin technique with 3,3-diaminobenzidine was
used for the visualization of bound primary antibodies. For
fluorescence immunohistochemistry, MS biopsies were consecutively
incubated with antibodies recognizing antigens in axons
(neurofilament, mouse IgG1, Clone 2F11, Dako). Bound antibodies
were visualized with species and immunoglobulin subtype specific
secondary antibodies (Cy2 anti-mouse IgG from Jackson
ImmunoResearch and Alexa555 anti-mouse IgG1 from Invitrogen).
Counterstaining of cells was performed with DAPI (Invitrogen).
[0078] c) Quantitative Real-Time PCR
[0079] Whole brain homogenates and cultured primary neurons after 4
weeks of culture (see below Example 7) of WT and Trpm4.sup.-/- mice
were analyzed by quantitative Real-Time PCR for TRMP4 expression.
RNA was purified by TRIzol Reagent (Invitrogen) and cDNA synthesis
was performed with RevertAid H Minus First Strand cDNA Synthesis
Kit (Fermentas) according to the manufacturers' protocols. For
quantitative Real-Time PCRTaqMan Gene Expression Assays
Mm00613159_ml (Trpm4) and Mm99999915_gl (Gapdh) were used with a
7900HT Fast Real-Time PCR System (all Applied Biosystems).
.DELTA.CT values were calibrated to whole brain cDNA of WT
mice.
[0080] Results: Trpm4 mRNA was detected by quantitative real-time
PCR of Trpm4 of transcripts from WT and Trpm4.sup.-/- whole brain
homogenates and compared with the Trpm4 mRNA in E16 hippocampal
neurons after 4 weeks of culture (FIG. 3c). An increased number of
transcripts was found in cultured hippocampal neurons (FIG. 3c),
while no signal was detectable in Trpm4-deficient neurons. In
addition, TRPM4 expression was recorded by immunohistochemistry in
spinal cord motor neurons in WT, but not in Trpm4.sup.-/- mice
(representative immunohistochemical stainings are depicted in FIG.
3a). Similarly, motor neurons of spinal cord autopsy from human
patients with MS were labelled by immunohistochemistry with an
antibody against human TRPM4 (FIG. 3a).
[0081] FIG. 3b shows co-localizations between TRPM4 and
phosphorylated and non-phosphorylated neurofilament H (SMI 31 and
SMI 32) in cervical spinal cord sections of healthy WT (upper
panels) and in acutely inflamed lesions of WT EAE mice (middle
panels; day 14 post immunization; scale bars: 50 .mu.m and inset 3
.mu.m) as well as TRPM4 and neurofilaments in periplaque white
matter lesions of MS patients (lower panels; scale bars: 50 .mu.m
and inset 12 .mu.m). TRPM4 immunoreactivity was observed within
axonal processes at the edge of EAE lesions in WT mice in contrast
to axons of healthy WT animals and Trpm4.sup.-/- mice
(representative immunohistochemical stainings in FIG. 3b, data for
Trpm4.sup.-/- not shown). Equally, TRPM4-positive axons were
identified in affected spinal cords of MS patients in the
periplaque white matter (representative immunohistochemical
stainings in FIG. 3b) and a substantial fraction of axons in the
periplaque white matter of MS brain lesions as well as cortical
neurons were labelled by TRPM4 antibodies (data not shown).
However, no change was detected in TRPM4 expression in neuronal
somata in the inflammatory vicinity in mice and humans (data not
shown). Together these data provide evidence that TRPM4 is
physiologically expressed within the neuronal soma and in a
fraction of axons of diseased EAE mice and MS patients, which
suggest a contribution of TRPM4 to inflammation-induced neuronal
and axonal degeneration.
EXAMPLE 6
Reduced Axonal/Neuronal Injury in Trpm4.sup.-/- Mice
[0082] Since TRPM4 expression patterns suggested an involvement in
axonal and neuronal injury mechanisms, it was tested whether the
observed ameliorated EAE phenotype of Trpm4.sup.-/- mice was
reflected in axonal immunohistochemical studies. For this purpose,
histopathological analysis of EAE tissue was performed on PFA-fixed
cross-sections (8 to 10 per animal) of lumbar and thoracic spinal
cords. Digital images of tissue sections were recorded at 200-fold
magnification using Zeiss MIRAX MIDI Slide Scanner (Carl Zeiss,
Microlmaging GmbH, Germany). Numbers of inflammatory foci per
spinal cord cross sections were quantified on hematoxylin and eosin
(HE) stained sections and the average of inflammatory foci per
analyzed cross-sections are expressed as inflammatory index. The
extent of demyelination was quantified by measuring the
demyelinated area of LFB/PAS-stained sections using a Mirax Viewer
(Carl Zeiss, MicroImaging GmbH, Germany). The area of demyelination
was calculated as percentage of total analyzed area of white matter
within a given section. Immunostaining for the amyloid precursor
protein (APP, clone 22C11, Chemicon) was used as a marker of acute
axonal damage. Mice were also stained for neurofilaments (SMI 31
and SMI 32) in the corticospinal tract and dorsal column and for
neuronal nuclei (NeuN) in the gray matter of cervical spinal cord
sections (FIG. 4b). Further immunostaining was performed to assess
infiltrates of activated macrophages/microglia (MAC3, Clone M3/84,
BP Pharmingen) and T cells (CD3, Clone CD3-12, AbDSerotec).
Avidin-biotin technique with 3,3-diaminobenzidine was used for the
visualization of bound primary antibodies. The average density of
activated macrophages/microglia and T cells was analyzed
automatically applying a custom-programmed script in Cognition
Network Language based on the Definiens Cognition Network
Technology.RTM. platform (Definiens Developer XD software).
Briefly, the programmed script first discriminates between tissue
and background (no tissue) by spectral difference detection.
Subsequently, the area of detected tissue (region of interest, ROI)
is calculated and immunostained cells (CD3 or Mac3, respectively)
within this ROI are detected based on their dark brown color. To
split cells that were localized in dense clusters into single
cells, nuclei were detected based on their blue color in Hemalaun
counterstaining. Only immunoreactive brown structures displaying a
blue nucleus in the center were further classified as "cells".
APP-positive axons within white matter spinal cord sections were
measured using an ocular counting grid and expressed as APP.sup.+
spheroids/mm.sup.2.
[0083] Results: Trpm4.sup.-/- mice showed reduced axonal and
neuronal loss during EAE (FIG. 4). Indeed, fewer axonal spheroids
were observed by immunostaining against amyloid precursor protein
(APP), an acute injury marker for axons, within EAE lesions in
Trpm4.sup.-/--deficient animals in comparison to WT animals at day
21 post immunization (dpi) (n=8 for WT and n=5 for Trpm4.sup.-/-;
P<0.05; FIG. 4a). Consequently, there was a marked preservation
of axons in the corticospinal tract (P<0.01) and dorsal column
(P<0.01) as well as of neuronal cell bodies in the gray matter
of the spinal cord (P<0.05) of Trpm4.sup.-/--EAE mice in
comparison to WTEAE mice in immunohistochemical analyses at day 60
post immunization (representative images in FIG. 4b; WT-control
(n=4), WT EAE (60 dpi; n=10), Trpm4.sup.-/--control (n=4) and
Trpm4.sup.-/- EAE (60 dpi; n=9); Scale bars: 10 .mu.m
(corticospinal tract and dorsal column) and 50 .mu.m (gray
matter)). These results indicate a profound resistance of axons and
neurons in Trpm4.sup.-/- mice towards hostile inflammatory
challenges leading to a neuro-axonal preservation and less clinical
disability.
EXAMPLE 7
Excitotoxic Cell Death is Driven by TRPM4-Dependent Inward
Currents
[0084] a) Resistance towards neurotoxic challenges in vitro
[0085] It was tested whether the increased neuro-axonal
preservation in Trpm4-deficient mice can be explained by a
resistance towards ATP depletion and glutamate-mediated Ca.sup.2+
influx in vitro. For this purpose, experiments were conducted with
cells obtained from neuronal cell culture. Briefly, on day 16 after
mating of Trpm4.sup.+/- mice, pregnant females were sacrificed by
CO.sub.2. The embryos were taken out and decapitated separately
followed by dissection and dissociation of the hippocampus. In
addition, a separate piece of tissue was taken for genotyping.
Subsequently, the cells were plated at a density of
1.times.10.sup.5 cells in 24-well plates with neurobasal medium
supplemented with L-glutamine, B-27 and penicillin/streptomycin
(all Invitrogen). Three days after culturing ARAC (Sigma-Aldrich)
was added to avoid proliferation of glial cells.
[0086] After 4 weeks of culture experimental challenges were added
(FIG. 5a). To examine the cell integrity, LDH concentrations were
measured in the supernatant according to the manufacturers'
protocol (Roche) from untreated and stressed neurons by ELISA
(VICTOR.sup.2, Wallac) 2, 4 and 6 h after addition of 50 nM
glutamate (Invitrogen), 300 .mu.M H.sub.2O.sub.2 (Sigma-Aldrich) or
0.5 .mu.M antimycin A (Sigma-Aldrich) (FIG. 5a). The percentages of
damaged cells were calculated by setting 2% Triton X-100 treated
cells as 100%. ATP levels of neuronal cells after treatment with
either glutamate or antimycin A were quantified from cell lysates
by using a luciferin based ATP determination kit (Molecular Probes)
after 4 h of antimycin A or glutamate administration.
[0087] Immunocytochemistry of treated and control neurons was
performed by fixing cells with 4% PFA for 30 min at room
temperature and permeabilizing them with ice cold 80% methanol for
6 min. The cells were stained simultaneously or consecutively
overnight at 4.degree. C. with the following antibodies: TRPM4
(rabbit IgG, 1:100, Abcam) and .beta.-tubulin III (mouse IgG,
1:500, Sigma-Aldrich). Secondary antibodies were Alexa Fluor
488coupled donkey antibodies recognizing rabbit IgG, Cy3-coupled
goat antibodies recognizing mouse IgG, Alexa Fluor 488-coupled
donkey antibodies recognizing mouse IgG and Alexa Fluor 488-coupled
goat antibodies recognizing mouse IgG (all 1:600, Dianova). Actin
filaments were stained with Alexa Fluor 555-coupled phalloidin
(Invitrogen) and DNA with Hoechst 33258 (Invitrogen). The cell
volumes of untreated neuronal cells (10 days in culture) and the
cell volumes of the cells which were treated with 50 nM glutamate
for 2 h were assessed by staining the cytoskeleton with
.beta.-tubulin III and Alexa Fluor 488-coupled donkey anti-mouse
antibodies. Z-stack images of whole neuronal cells were taken with
a confocal laser-scanning microscope (Leica TCS SP2) with a defined
step size. The cell bodies were rearranged with Imaris software
(bitplane) and the complete volumes were calculated by ImageJ
software.
[0088] Results: Treatment of cultured hippocampal neurons with
ATP-depleting (antimycin A) and calcium-dependent excitotoxic
(glutamate) stimuli resulted in a time-dependent loss of cell
integrity of WT neuronal cells, as measured by LDH release into the
supernatant, while cells from Trpm4.sup.-/- mice remained largely
unaffected by both stimuli (2 h, P<0.05; 4 and 6 h, P<0.01
for antimycin A; 2, 4 and 6 h, P<0.01 for glutamate; FIG. 5a).
Interestingly, glutamate treatment led to an even more profound
reduction of cytosolic ATP levels than antimycin A (all P<0.01;
FIG. 5b), while cytosolic ATP levels of WT and Trpm4.sup.-/- mice
were comparable in all experimental conditions. Consistently,
reduction of ATP by glucose deprivation resulted in a similar
protection of Trpm4.sup.-/- neurons (6 h, P<0.01; data not
shown).
[0089] b) Influence of Glutamate on TRPM4-Mediated Cation
Currents
[0090] Since a marked resistance of Trpm4.sup.-/- neurons against
glutamate-induced excitotoxicity was observed and additionally a
substantial reduction of neuronal ATP levels was detected under
these conditions, it was then examined whether neuronal
TRPM4-mediated cation currents are activated by glutamate
treatment. For this, whole-cell patch-clamp recordings were
performed with an EPC9 patch-clamp amplifier (HEKA Elektronik) at a
sampling rate of 20 kHz. Patch electrodes had a DC resistance
between 2 and 4 M.OMEGA. when filled with intracellular solution
for whole-cell patches. An Ag-AgCl wire was used as a reference
electrode. Capacitance and access resistance were monitored
continuously, and cell membrane capacitance values were used to
calculate current densities. Bath solution was composed of 156 mM
NaCl, 5 mM CaCl.sub.2, 10 mM glucose, and 10 mM HEPES at pH 7.4.
The pipette solution contained 156 mM CsCl, 1 mM MgCl.sub.2, 10 mM
EGTA and 10 mM HEPES at pH 7.2. Free Ca.sup.2+ in the pipette
solution was 7.4 .mu.M by addition of CaCl.sub.2. Holding potential
was 0 mV, and current traces were elicited by voltage ramps for 250
ms from -120 to +100 mV. During all recordings, 100 nM TTX
(Sigma-Aldrich) and 1 .mu.M nifedipine (Sigma-Aldrich) were added
to the bath solution.
[0091] Whole-cell patch-clamp recordings were performed on
hippocampal neurons of E16 embryos from WT and Trpm4.sup.-/- mice
after 10 days in culture under resting conditions (un-treated) and
after administration of 50 nM glutamate for 2 h. Normalized
current-voltage relationship for WT (untreated, n=11; glutamate
incubation, n=7) and Trpm4.sup.-/- (untreated, n=9; glutamate
incubation, n=7) cells obtained from 250 ms voltage ramps measured
in the wholecell patch-clamp configuration from -120 to +100 mV are
shown. Holding potential was 0 mV. In parallel, hippocampal neurons
of E16 embryos from WT (n=7) and Trpm4.sup.-/- (n=7) mice were
cultured for 10 days. Glutamate treated and control cells were
stained for their cytoskeleton by .beta.-tubulin III. Z-stacks of
whole neuronal cells were taken with a confocal laser-scanning
microscope and cell volumes were calculated. Representative
pictures of neuronal cells from WT and Trpm4.sup.-/- mice after
glutamate incubation for 2 h are shown (FIG. 5d: Scale bar 30
.mu.m).
[0092] Results: Under resting conditions no differences in current
density between hippocampal neurons from Trpm4.sup.-/- and WT mice
were detected by whole-cell patch-clamp recordings. Strikingly,
however, after incubation of the cells with glutamate, WT neurons
showed a strong TRPM4-dependent inward current at negative
potentials which was absent in Trpm4.sup.-/- neurons (FIG. 5c).
During electrophysiological measurements, an increased cell
capacity of glutamate-treated WT neurons compared to untreated
neurons was additionally detected, while this increase was absent
in Trpm4.sup.-/- neurons (P<0.05; FIG. 5d). Since a gain in
capacity can reflect an increase in cell volume, we analyzed the
cell size of neurons from Trpm4.sup.-/- and WT mice under resting
conditions and after glutamate treatment in immunofluorescent
stainings of the cytoskeleton. In agreement with the observed gain
in capacity, the cell volume of glutamate-treated WT neurons
increased substantially compared to untreated WT neurons
(P<0.01). By contrast, glutamate-treated Trpm4.sup.-/- neurons
showed similar cell volumes in treated and untreated conditions
(FIG. 5e).
[0093] Together, these results indicate that TRPM4 ion channels can
exert neuronal injury in the context of energy deficiency and
excitotoxic glutamate stimulation, and that TRPM4-mediated inward
currents are substantially increased after this stimulation with
subsequent oncotic cell swelling and neuronal cell death. As both
conditions, ATP depletion due to mitochondrial dysfunction and
substantially increased glutamate concentrations are abundant
phenomena in EAE and MS lesions (Campbell et al. (2011), Ann
Neurol. 69:481-492), it has to be concluded that a pathological
activation of TRPM4 occurs in EAE and MS. The results further imply
that a TRPM4-dependent excessive Na.sup.+ overload eventually
causes oncotic cell swelling and neuronal cell death.
EXAMPLE 8
Glibenclamide Reduces EAE Severity by TRPM4 Inhibition
[0094] Having established a decisive role of TRPM4 for neuro-axonal
degeneration under neuroinflammatory conditions, it was next
examined whether pharmacological inhibition of TRPM4 after EAE
induction is able to exert a neuroprotective effect in vivo.
Glibenclamide, a well-tolerated, FDA-approved oral antidiabetic
drug, has been reported to effectively inhibit TRPM4 (Damien et al.
(2007), Cardiovasc Res. 73:531-538; Becerra et al. (2011),
Cardiovasc Res. 91:677-684). The influence of this drug was tested
in groups of WT-EAE and Trpm4.sup.-/--EAE mice. WT EAE and
Trpm4.sup.-/- EAE mice were treated as described in Example 1 above
to determine mean clinical disability scores. Pharmacological
blockade of TRPM4 was achieved by daily administration of 10 .mu.g
glibenclamide or DMSO control by intraperitoneal injections after
onset of first clinical symptoms (n=8 for
Trpm4.sup.-/-+glibenclamide; for all other groups n=6). 25 mg
glibenclamide (Sigma-Aldrich) were solved in 5 ml dimethylsulfoxid
(DMSO, AppliChem) every day, and 200 .mu.l of this solution were
diluted in 9.8 ml phosphate buffered saline (PBS). Treatment was
started when first clinical symptoms occurred (day 8 after
immunization with MOG.sub.35-55). Mice received 100 .mu.l of this
solution or 2% DMSO in PBS as control. Due to the higher burden of
daily injections, mice that were treated with glibenclamide or
DMSO-control received only 2 mg/ml M. tuberculosis and 100 ng
pertussis toxin for EAE immunization. Healthy WT mice (WT control)
and WT EAE mice treated either with glibenclamide or vehicle (n=4
per group) were stained 30 days post immunization for
neurofilaments (SMI 31 and SMI 32) in the corticospinal tract and
dorsal column and for neuronal nuclei (NeuN) in the gray matter of
cervical spinal cord sections as described above. Numbers of axons
and somata were counted by ImageJ software.
[0095] Results: The results obtained from the glibenclamide study
are shown in FIG. 6. Glibenclamide treatment reduces clinical
disability and neurodegeneration in EAE mice. While glibenclamide
ameliorated disability in WT EAE mice in comparison to vehicle
control treated WT EAE animals (P<0.05), glibenclamide showed no
additional clinical improvement in Trpm4.sup.-/- EAE mice,
indicating that glibenclamide exerts its protective properties in
EAE via targeting TRPM4. This was substantiated by a preservation
of axons in the corticospinal tract (P<0.01) and dorsal column
(P<0.05) of glibenclamide treated WT EAE mice in comparison to
vehicle control treated WT EAE mice. Further, neuronal cell body
loss in the gray matter of the spinal cord in WT EAE mice
(P<0.05) was slightly diminished by glibenclamide treatment in
comparison to vehicle control treated mice, although this
preservation of neurons did not reach statistical significance
(FIG. 6b).
Sequence CWU 1
1
311214PRTHomo sapiens 1Met Val Val Pro Glu Lys Glu Gln Ser Trp Ile
Pro Lys Ile Phe Lys1 5 10 15Lys Lys Thr Cys Thr Thr Phe Ile Val Asp
Ser Thr Asp Pro Gly Gly 20 25 30Thr Leu Cys Gln Cys Gly Arg Pro Arg
Thr Ala His Pro Ala Val Ala 35 40 45Met Glu Asp Ala Phe Gly Ala Ala
Val Val Thr Val Trp Asp Ser Asp 50 55 60Ala His Thr Thr Glu Lys Pro
Thr Asp Ala Tyr Gly Glu Leu Asp Phe65 70 75 80Thr Gly Ala Gly Arg
Lys His Ser Asn Phe Leu Arg Leu Ser Asp Arg 85 90 95Thr Asp Pro Ala
Ala Val Tyr Ser Leu Val Thr Arg Thr Trp Gly Phe 100 105 110Arg Ala
Pro Asn Leu Val Val Ser Val Leu Gly Gly Ser Gly Gly Pro 115 120
125Val Leu Gln Thr Trp Leu Gln Asp Leu Leu Arg Arg Gly Leu Val Arg
130 135 140Ala Ala Gln Ser Thr Gly Ala Trp Ile Val Thr Gly Gly Leu
His Thr145 150 155 160Gly Ile Gly Arg His Val Gly Val Ala Val Arg
Asp His Gln Met Ala 165 170 175Ser Thr Gly Gly Thr Lys Val Val Ala
Met Gly Val Ala Pro Trp Gly 180 185 190Val Val Arg Asn Arg Asp Thr
Leu Ile Asn Pro Lys Gly Ser Phe Pro 195 200 205Ala Arg Tyr Arg Trp
Arg Gly Asp Pro Glu Asp Gly Val Gln Phe Pro 210 215 220Leu Asp Tyr
Asn Tyr Ser Ala Phe Phe Leu Val Asp Asp Gly Thr His225 230 235
240Gly Cys Leu Gly Gly Glu Asn Arg Phe Arg Leu Arg Leu Glu Ser Tyr
245 250 255Ile Ser Gln Gln Lys Thr Gly Val Gly Gly Thr Gly Ile Asp
Ile Pro 260 265 270Val Leu Leu Leu Leu Ile Asp Gly Asp Glu Lys Met
Leu Thr Arg Ile 275 280 285Glu Asn Ala Thr Gln Ala Gln Leu Pro Cys
Leu Leu Val Ala Gly Ser 290 295 300Gly Gly Ala Ala Asp Cys Leu Ala
Glu Thr Leu Glu Asp Thr Leu Ala305 310 315 320Pro Gly Ser Gly Gly
Ala Arg Gln Gly Glu Ala Arg Asp Arg Ile Arg 325 330 335Arg Phe Phe
Pro Lys Gly Asp Leu Glu Val Leu Gln Ala Gln Val Glu 340 345 350Arg
Ile Met Thr Arg Lys Glu Leu Leu Thr Val Tyr Ser Ser Glu Asp 355 360
365Gly Ser Glu Glu Phe Glu Thr Ile Val Leu Lys Ala Leu Val Lys Ala
370 375 380Cys Gly Ser Ser Glu Ala Ser Ala Tyr Leu Asp Glu Leu Arg
Leu Ala385 390 395 400Val Ala Trp Asn Arg Val Asp Ile Ala Gln Ser
Glu Leu Phe Arg Gly 405 410 415Asp Ile Gln Trp Arg Ser Phe His Leu
Glu Ala Ser Leu Met Asp Ala 420 425 430Leu Leu Asn Asp Arg Pro Glu
Phe Val Arg Leu Leu Ile Ser His Gly 435 440 445Leu Ser Leu Gly His
Phe Leu Thr Pro Met Arg Leu Ala Gln Leu Tyr 450 455 460Ser Ala Ala
Pro Ser Asn Ser Leu Ile Arg Asn Leu Leu Asp Gln Ala465 470 475
480Ser His Ser Ala Gly Thr Lys Ala Pro Ala Leu Lys Gly Gly Ala Ala
485 490 495Glu Leu Arg Pro Pro Asp Val Gly His Val Leu Arg Met Leu
Leu Gly 500 505 510Lys Met Cys Ala Pro Arg Tyr Pro Ser Gly Gly Ala
Trp Asp Pro His 515 520 525Pro Gly Gln Gly Phe Gly Glu Ser Met Tyr
Leu Leu Ser Asp Lys Ala 530 535 540Thr Ser Pro Leu Ser Leu Asp Ala
Gly Leu Gly Gln Ala Pro Trp Ser545 550 555 560Asp Leu Leu Leu Trp
Ala Leu Leu Leu Asn Arg Ala Gln Met Ala Met 565 570 575Tyr Phe Trp
Glu Met Gly Ser Asn Ala Val Ser Ser Ala Leu Gly Ala 580 585 590Cys
Leu Leu Leu Arg Val Met Ala Arg Leu Glu Pro Asp Ala Glu Glu 595 600
605Ala Ala Arg Arg Lys Asp Leu Ala Phe Lys Phe Glu Gly Met Gly Val
610 615 620Asp Leu Phe Gly Glu Cys Tyr Arg Ser Ser Glu Val Arg Ala
Ala Arg625 630 635 640Leu Leu Leu Arg Arg Cys Pro Leu Trp Gly Asp
Ala Thr Cys Leu Gln 645 650 655Leu Ala Met Gln Ala Asp Ala Arg Ala
Phe Phe Ala Gln Asp Gly Val 660 665 670Gln Ser Leu Leu Thr Gln Lys
Trp Trp Gly Asp Met Ala Ser Thr Thr 675 680 685Pro Ile Trp Ala Leu
Val Leu Ala Phe Phe Cys Pro Pro Leu Ile Tyr 690 695 700Thr Arg Leu
Ile Thr Phe Arg Lys Ser Glu Glu Glu Pro Thr Arg Glu705 710 715
720Glu Leu Glu Phe Asp Met Asp Ser Val Ile Asn Gly Glu Gly Pro Val
725 730 735Gly Thr Ala Asp Pro Ala Glu Lys Thr Pro Leu Gly Val Pro
Arg Gln 740 745 750Ser Gly Arg Pro Gly Cys Cys Gly Gly Arg Cys Gly
Gly Arg Arg Cys 755 760 765Leu Arg Arg Trp Phe His Phe Trp Gly Ala
Pro Val Thr Ile Phe Met 770 775 780Gly Asn Val Val Ser Tyr Leu Leu
Phe Leu Leu Leu Phe Ser Arg Val785 790 795 800Leu Leu Val Asp Phe
Gln Pro Ala Pro Pro Gly Ser Leu Glu Leu Leu 805 810 815Leu Tyr Phe
Trp Ala Phe Thr Leu Leu Cys Glu Glu Leu Arg Gln Gly 820 825 830Leu
Ser Gly Gly Gly Gly Ser Leu Ala Ser Gly Gly Pro Gly Pro Gly 835 840
845His Ala Ser Leu Ser Gln Arg Leu Arg Leu Tyr Leu Ala Asp Ser Trp
850 855 860Asn Gln Cys Asp Leu Val Ala Leu Thr Cys Phe Leu Leu Gly
Val Gly865 870 875 880Cys Arg Leu Thr Pro Gly Leu Tyr His Leu Gly
Arg Thr Val Leu Cys 885 890 895Ile Asp Phe Met Val Phe Thr Val Arg
Leu Leu His Ile Phe Thr Val 900 905 910Asn Lys Gln Leu Gly Pro Lys
Ile Val Ile Val Ser Lys Met Met Lys 915 920 925Asp Val Phe Phe Phe
Leu Phe Phe Leu Gly Val Trp Leu Val Ala Tyr 930 935 940Gly Val Ala
Thr Glu Gly Leu Leu Arg Pro Arg Asp Ser Asp Phe Pro945 950 955
960Ser Ile Leu Arg Arg Val Phe Tyr Arg Pro Tyr Leu Gln Ile Phe Gly
965 970 975Gln Ile Pro Gln Glu Asp Met Asp Val Ala Leu Met Glu His
Ser Asn 980 985 990Cys Ser Ser Glu Pro Gly Phe Trp Ala His Pro Pro
Gly Ala Gln Ala 995 1000 1005Gly Thr Cys Val Ser Gln Tyr Ala Asn
Trp Leu Val Val Leu Leu 1010 1015 1020Leu Val Ile Phe Leu Leu Val
Ala Asn Ile Leu Leu Val Asn Leu 1025 1030 1035Leu Ile Ala Met Phe
Ser Tyr Thr Phe Gly Lys Val Gln Gly Asn 1040 1045 1050Ser Asp Leu
Tyr Trp Lys Ala Gln Arg Tyr Arg Leu Ile Arg Glu 1055 1060 1065Phe
His Ser Arg Pro Ala Leu Ala Pro Pro Phe Ile Val Ile Ser 1070 1075
1080His Leu Arg Leu Leu Leu Arg Gln Leu Cys Arg Arg Pro Arg Ser
1085 1090 1095Pro Gln Pro Ser Ser Pro Ala Leu Glu His Phe Arg Val
Tyr Leu 1100 1105 1110Ser Lys Glu Ala Glu Arg Lys Leu Leu Thr Trp
Glu Ser Val His 1115 1120 1125Lys Glu Asn Phe Leu Leu Ala Arg Ala
Arg Asp Lys Arg Glu Ser 1130 1135 1140Asp Ser Glu Arg Leu Lys Arg
Thr Ser Gln Lys Val Asp Leu Ala 1145 1150 1155Leu Lys Gln Leu Gly
His Ile Arg Glu Tyr Glu Gln Arg Leu Lys 1160 1165 1170Val Leu Glu
Arg Glu Val Gln Gln Cys Ser Arg Val Leu Gly Trp 1175 1180 1185Val
Ala Glu Ala Leu Ser Arg Ser Ala Leu Leu Pro Pro Gly Gly 1190 1195
1200Pro Pro Pro Pro Asp Leu Pro Gly Ser Lys Asp 1205
121021069PRTHomo sapiens 2Met Val Val Pro Glu Lys Glu Gln Ser Trp
Ile Pro Lys Ile Phe Lys1 5 10 15Lys Lys Thr Cys Thr Thr Phe Ile Val
Asp Ser Thr Asp Pro Gly Gly 20 25 30Thr Leu Cys Gln Cys Gly Arg Pro
Arg Thr Ala His Pro Ala Val Ala 35 40 45Met Glu Asp Ala Phe Gly Ala
Ala Val Val Thr Val Trp Asp Ser Asp 50 55 60Ala His Thr Thr Glu Lys
Pro Thr Asp Ala Tyr Gly Glu Leu Asp Phe65 70 75 80Thr Gly Ala Gly
Arg Lys His Ser Asn Phe Leu Arg Leu Ser Asp Arg 85 90 95Thr Asp Pro
Ala Ala Val Tyr Ser Leu Val Thr Arg Thr Trp Gly Phe 100 105 110Arg
Ala Pro Asn Leu Val Val Ser Val Leu Gly Gly Ser Gly Gly Pro 115 120
125Val Leu Gln Thr Trp Leu Gln Asp Leu Leu Arg Arg Gly Leu Val Arg
130 135 140Ala Ala Gln Ser Thr Gly Ala Trp Ile Val Thr Gly Gly Leu
His Thr145 150 155 160Gly Ile Gly Arg His Val Gly Val Ala Val Arg
Asp His Gln Met Ala 165 170 175Ser Thr Gly Gly Thr Lys Val Val Ala
Met Gly Val Ala Pro Trp Gly 180 185 190Val Val Arg Asn Arg Asp Thr
Leu Ile Asn Pro Lys Gly Ser Phe Pro 195 200 205Ala Arg Tyr Arg Trp
Arg Gly Asp Pro Glu Asp Gly Val Gln Phe Pro 210 215 220Leu Asp Tyr
Asn Tyr Ser Ala Phe Phe Leu Val Asp Asp Gly Thr His225 230 235
240Gly Cys Leu Gly Gly Glu Asn Arg Phe Arg Leu Arg Leu Glu Ser Tyr
245 250 255Ile Ser Gln Gln Lys Thr Gly Val Gly Gly Thr Gly Ile Asp
Ile Pro 260 265 270Val Leu Leu Leu Leu Ile Asp Gly Asp Glu Lys Met
Leu Thr Arg Ile 275 280 285Glu Asn Ala Thr Gln Ala Gln Leu Pro Cys
Leu Leu Val Ala Gly Ser 290 295 300Gly Gly Ala Ala Asp Cys Leu Ala
Glu Thr Leu Glu Asp Thr Leu Ala305 310 315 320Pro Gly Ser Gly Gly
Ala Arg Gln Gly Glu Ala Arg Asp Arg Ile Arg 325 330 335Arg Phe Phe
Pro Lys Gly Asp Leu Glu Val Leu Gln Ala Gln Val Glu 340 345 350Arg
Ile Met Thr Arg Lys Glu Leu Leu Thr Val Tyr Ser Ser Glu Asp 355 360
365Gly Ser Glu Glu Phe Glu Thr Ile Val Leu Lys Ala Leu Val Lys Ala
370 375 380Cys Gly Ser Ser Glu Ala Ser Ala Tyr Leu Asp Glu Leu Arg
Leu Ala385 390 395 400Val Ala Trp Asn Arg Val Asp Ile Ala Gln Ser
Glu Leu Phe Arg Gly 405 410 415Asp Ile Gln Trp Arg Ser Phe His Leu
Glu Ala Ser Leu Met Asp Ala 420 425 430Leu Leu Asn Asp Arg Pro Glu
Phe Val Arg Leu Leu Ile Ser His Gly 435 440 445Leu Ser Leu Gly His
Phe Leu Thr Pro Met Arg Leu Ala Gln Leu Tyr 450 455 460Ser Ala Ala
Pro Ser Asn Ser Leu Ile Arg Asn Leu Leu Asp Gln Ala465 470 475
480Ser His Ser Ala Gly Thr Lys Ala Pro Ala Leu Lys Gly Gly Ala Ala
485 490 495Glu Leu Arg Pro Pro Asp Val Gly His Val Leu Arg Met Leu
Leu Gly 500 505 510Lys Met Cys Ala Pro Arg Tyr Pro Ser Gly Gly Ala
Trp Asp Pro His 515 520 525Pro Gly Gln Gly Phe Gly Glu Ser Met Tyr
Leu Leu Ser Asp Lys Ala 530 535 540Thr Ser Pro Leu Ser Leu Asp Ala
Gly Leu Gly Gln Ala Pro Trp Ser545 550 555 560Asp Leu Leu Leu Trp
Ala Leu Leu Leu Asn Arg Ala Gln Met Ala Met 565 570 575Tyr Phe Trp
Glu Met Gly Ser Asn Ala Val Ser Ser Ala Leu Gly Ala 580 585 590Cys
Leu Leu Leu Arg Val Met Ala Arg Leu Glu Pro Asp Ala Glu Glu 595 600
605Ala Ala Arg Arg Lys Asp Leu Ala Phe Lys Phe Glu Gly Met Gly Val
610 615 620Asp Leu Phe Gly Glu Cys Tyr Arg Ser Ser Glu Val Arg Ala
Ala Arg625 630 635 640Leu Leu Leu Arg Arg Cys Pro Leu Trp Gly Asp
Ala Thr Cys Leu Gln 645 650 655Leu Ala Met Gln Ala Asp Ala Arg Ala
Phe Phe Ala Gln Asp Gly Val 660 665 670Gln Ser Leu Leu Thr Gln Lys
Trp Trp Gly Asp Met Ala Ser Thr Thr 675 680 685Pro Ile Trp Ala Leu
Val Leu Ala Phe Phe Cys Pro Pro Leu Ile Tyr 690 695 700Thr Arg Leu
Ile Thr Phe Arg Lys Ser Glu Glu Glu Pro Thr Arg Glu705 710 715
720Glu Leu Glu Phe Asp Met Asp Ser Val Ile Asn Gly Glu Gly Pro Val
725 730 735Gly Leu Thr Pro Gly Leu Tyr His Leu Gly Arg Thr Val Leu
Cys Ile 740 745 750Asp Phe Met Val Phe Thr Val Arg Leu Leu His Ile
Phe Thr Val Asn 755 760 765Lys Gln Leu Gly Pro Lys Ile Val Ile Val
Ser Lys Met Met Lys Asp 770 775 780Val Phe Phe Phe Leu Phe Phe Leu
Gly Val Trp Leu Val Ala Tyr Gly785 790 795 800Val Ala Thr Glu Gly
Leu Leu Arg Pro Arg Asp Ser Asp Phe Pro Ser 805 810 815Ile Leu Arg
Arg Val Phe Tyr Arg Pro Tyr Leu Gln Ile Phe Gly Gln 820 825 830Ile
Pro Gln Glu Asp Met Asp Val Ala Leu Met Glu His Ser Asn Cys 835 840
845Ser Ser Glu Pro Gly Phe Trp Ala His Pro Pro Gly Ala Gln Ala Gly
850 855 860Thr Cys Val Ser Gln Tyr Ala Asn Trp Leu Val Val Leu Leu
Leu Val865 870 875 880Ile Phe Leu Leu Val Ala Asn Ile Leu Leu Val
Asn Leu Leu Ile Ala 885 890 895Met Phe Ser Tyr Thr Phe Gly Lys Val
Gln Gly Asn Ser Asp Leu Tyr 900 905 910Trp Lys Ala Gln Arg Tyr Arg
Leu Ile Arg Glu Phe His Ser Arg Pro 915 920 925Ala Leu Ala Pro Pro
Phe Ile Val Ile Ser His Leu Arg Leu Leu Leu 930 935 940Arg Gln Leu
Cys Arg Arg Pro Arg Ser Pro Gln Pro Ser Ser Pro Ala945 950 955
960Leu Glu His Phe Arg Val Tyr Leu Ser Lys Glu Ala Glu Arg Lys Leu
965 970 975Leu Thr Trp Glu Ser Val His Lys Glu Asn Phe Leu Leu Ala
Arg Ala 980 985 990Arg Asp Lys Arg Glu Ser Asp Ser Glu Arg Leu Lys
Arg Thr Ser Gln 995 1000 1005Lys Val Asp Leu Ala Leu Lys Gln Leu
Gly His Ile Arg Glu Tyr 1010 1015 1020Glu Gln Arg Leu Lys Val Leu
Glu Arg Glu Val Gln Gln Cys Ser 1025 1030 1035Arg Val Leu Gly Trp
Val Ala Glu Ala Leu Ser Arg Ser Ala Leu 1040 1045 1050Leu Pro Pro
Gly Gly Pro Pro Pro Pro Asp Leu Pro Gly Ser Lys 1055 1060
1065Asp321PRTartificialmyelin oligodendrocyte glycoprotein peptide
35-55 (MOG35-55) 3Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg
Val Val His Leu1 5 10 15Tyr Arg Asn Gly Lys 20
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