NZ614267B2 - New compositions for treating neurological disorders - Google Patents
New compositions for treating neurological disorders Download PDFInfo
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- NZ614267B2 NZ614267B2 NZ614267A NZ61426712A NZ614267B2 NZ 614267 B2 NZ614267 B2 NZ 614267B2 NZ 614267 A NZ614267 A NZ 614267A NZ 61426712 A NZ61426712 A NZ 61426712A NZ 614267 B2 NZ614267 B2 NZ 614267B2
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- torasemide
- baclofen
- drug
- trimetazidine
- intoxication
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Abstract
Provided are compositions comprising torasemide, trimetazidine, mexiletine, ifenprodil, moxifloxacin or bromocriptine for the treatment of neurological disorders. Examples of neurological disorders include Alzheimer's disease and related diseases, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, neuropathies, alcoholism, alcohol withdrawal, Huntington's disease and spinal cord injury. Further provided are compositions comprising one of the aforementioned drugs and one or more drugs selected from sulfisoxazole, methimazole, prilocaine, dyphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, ifenprodil, moxifloxacin, bromocriptine or torasemide. arkinson's disease, neuropathies, alcoholism, alcohol withdrawal, Huntington's disease and spinal cord injury. Further provided are compositions comprising one of the aforementioned drugs and one or more drugs selected from sulfisoxazole, methimazole, prilocaine, dyphylline, quinacrine, carbenoxolone, acamprosate, aminocaproic acid, baclofen, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, ifenprodil, moxifloxacin, bromocriptine or torasemide.
Description
NEW COMPOSITIONS FOR TREATING NEUROLOGICAL DISORDERS
FIELD OF THE INVENTION
The present invention relates to compositions and methods for the treatment of
neurological diseases and disorders. More particularly, this invention relates to novel
combinatorial therapies for such diseases, including Alzheimer's and related diseases,
Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Parkinson's disease, neuropathies,
alcoholism, alcohol withdrawal, Huntington's disease and spinal cord injury.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is the prototypic cortical dementia characterized by
memory deficit together with dysphasia (language disorder in which there is an
impairment of speech and of comprehension of speech), dyspraxia (disability to
coordinate and perform certain purposeful movements and gestures in the absence of
motor or sensory impairments) and agnosia (ability to recognize objects, persons,
sounds, shapes, or smells) attributable to involvement of the cortical association areas
(1-4).
AD is at present the most common cause of dementia. It is clinically
characterized by a global decline of cognitive function that progresses slowly and leaves
end-stage patients bound to bed, incontinent and dependent on custodial care. Death
occurs, on average, 9 years after diagnosis (5).
The incidence rate of AD increases dramatically with age. United Nation
population projections estimate that the number of people older than 80 years will
approach 370 million by the year 2050. Currently, it is estimated that 50% of people
older than age 85 years are afflicted with AD. Therefore, more than 100 million people
worldwide will suffer from dementia in 50 years. The vast number of people requiring
constant care and other services will severely affect medical, monetary and human
resources (6). Memory impairment is the early feature of the disease and involves
episodic memory (memory for day-today events). Semantic memory (memory for
verbal and visual meaning) is involved later in the disease. The pathological hallmark of
AD includes amyloid plaques containing beta-amyloid (Abeta), neurofibrillary tangles
(NFT) containing Tau and neuronal and synaptic dysfunction and loss (7-9). For the last
decade, two major hypotheses on the cause of AD have been proposed: the "amyloid
cascade hypothesis", which states that the neurodegenerative process is a series of
events triggered by the abnormal processing of the Amyloid Precursor Protein (APP)
(10), and the "neuronal cytoskeletal degeneration hypothesis" ( 11), which proposes that
cytoskeletal changes are the triggering events. The most widely accepted theory
explaining AD progression remains the amyloid cascade hypothesis (12-14) and AD
researchers have mainly focused on determining the mechanisms underlying the toxicity
associated with Abeta proteins. Microvascular permeability and remodeling, aberrant
angiogenesis and blood brain barrier breakdown have been identified as key events
contributing to the APP toxicity in the amyloid cascade (15). On contrary, Tau protein
has received much less attention from the pharmaceutical industry than amyloid,
because of both fundamental and practical concerns. Moreover, synaptic density change
is the pathological lesion that best correlates with cognitive impairment than the two
others.
Studies have revealed that the amyloid pathology appears to progress in a
neurotransmitter-specific manner where the cholinergic terminals appear most
vulnerable, followed by the glutamatergic terminals and finally by the GABAergic
terminals (9). Glutamate is the most abundant excitatory neurotransmitter in the
mammalian nervous system. Under pathological conditions, its abnormal accumulation
in the synaptic cleft leads to glutamate receptors overactivation (16). Abnormal
accumulation of glutamate in synaptic cleft leads to the overactivation of glutamate
receptors that results in pathological processes and finally in neuronal cell death. This
process, named excitotoxicity, is commonly observed in neuronal tissues during acute
and chronic neurological disorders.
It is becoming evident that excitotoxicity is involved in the pathogenesis of
multiple disorders of various etiology such as: spinal cord injury, stroke, traumatic brain
injury, hearing loss, alcoholism and alcohol withdrawal, alcoholic neuropathy, or
neuropathic pain as well as neurodegenerative diseases such as multiple sclerosis,
Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, and
Huntington's disease (17-19). The development of efficient treatment for these diseases
remains major public health issues due to their incidence as well as lack of curative
treatments.
NMDAR antagonists that target various sites of this receptor have been tested to
counteract excitotoxicity. Uncompetitive NMDAR antagonists target the ion channel pore
thus reducing the calcium entry into postsynaptic neurons. Some of them reached the
approval status. As an example, Memantine is currently approved in moderate to severe
Alzheimer’s disease. It is clinically tested in other indications that include a component of
excitotoxicity such as alcohol dependence (phase II), amyotrophic lateral sclerosis (phase
III), dementia associated with Parkinson (Phase II), epilepsy, Huntington’s disease (phase
IV), multiple sclerosis (phase IV), Parkinson’s disease (phase IV) and traumatic brain injury
(phase IV). This molecule is however of limited benefit to most Alzheimer’s disease
patients, because it has only modest symptomatic effects. Another approach in limiting
excitotoxicity consists in inhibiting the presynaptic release of glutamate. Riluzole, currently
approved in amyotrophic lateral sclerosis, showed encouraging results in ischemia and
traumatic brain injury models (20-23). It is at present tested in phase II trials in early
multiple sclerosis, Parkinson’s disease (does not show any better results than placebo) as
well as spinal cord injury. In 1995, the drug reached orphan drug status for the treatment of
amyotrophic lateral sclerosis and in 1996 for the treatment of Huntington’s disease.
WO2009/133128, WO2009/133141, WO2009/133142, and WO2011/054759, disclose
molecules which can be used in compositions for treating neurological disorders.
Despite active research in this area, there is still a need for alternative or improved
efficient therapies for neurological disorders, and, in particular, neurological disorders
which are related to glutamate and/or amyloid beta toxicity. The present invention provides
new treatments for such neurological diseases of the central nervous system (CNS) and the
peripheral nervous system (PNS).
SUMMARY OF INVENTION
In one or more aspects the present invention may advantageously provide new
therapeutic approaches for treating neurological disorders.
The invention stems, inter alia, from the unexpected discovery by the inventors that
Torasemide, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil and Moxifloxacin, alone
or in combinations, represent new and effective therapies for the treatment of neurological
disorders.
The invention therefore provides novel compositions and methods for treating
neurological disorders, particularly AD and related disorders, Multiple Sclerosis (MS),
Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease (PD), neuropathies (for instance
neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, Huntington's
disease (HD) and spinal cord injury.
More particularly, the invention relates to a composition, for use in the treatment of a
neurological disorder, comprising at least Torasemide, Trimetazidine, Mexiletine,
Ifenprodil, Moxifloxacin or Bromocriptine, or a salt, prodrug, derivative, or sustained
release formulation thereof.
A further aspect of the present invention relates to a composition comprising at least
one first compound selected from the group consisting of Torasemide, Trimetazidine,
Mexiletine, Ifenprodil, Moxifloxacin, and Bromocriptine, or a salt, prodrug, derivative of
any chemical purity, or sustained release formulation thereof, in combination with at least
one second compound distinct from said first compound, selected from Sulfisoxazole,
Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone, Acamprosate,
Aminocaproic acid, Baclofen, Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine,
Eplerenone, Fenoldopam, Leflunomide, Levosimendan, Sulodexide, Terbinafine,
Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil,
Torasemide, and Moxifloxacin salts, prodrugs, derivatives of any chemical purity, or
sustained release formulation thereof, for simultaneous, separate or sequential
administration.
A further aspect of the present invention relates to a composition, for use in the
treatment of a neurological disorder, comprising at least one first compound selected from
the group consisting of Torasemide, Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin,
and Bromocriptine, salts, prodrugs, derivatives of any chemical purity, or sustained release
formulation thereof, in combination with at least one second compound distinct from said
first compound, selected from Sulfisoxazole, Methimazole, Prilocaine, Dyphylline,
Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,
Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam, Leflunomide,
Levosimendan, Sulodexide, Terbinafine, Zonisamide, Etomidate, Phenformin,
Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil, Torasemide, and Moxifloxacin, salts,
prodrugs, derivatives of any chemical purity, or sustained release formulation thereof, for
simultaneous, separate or sequential administration.
The present invention also relates to a composition comprising at least one first
compound selected from the group consisting of Torasemide, Trimetazidine, Mexiletine,
Ifenprodil, Moxifloxacin and Bromocriptine, salt(s), prodrug(s), derivative(s) of any
chemical purity, or sustained release formulation(s) thereof, in combination with at least one
second compound distinct from said first compound, selected from Sulfisoxazole,
Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone, Acamprosate,
Aminocaproic acid, Baclofen, Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine,
Eplerenone, Fenoldopam, Leflunomide, Levosimendan, Sulodexide, Terbinafine,
Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil,
Torasemide, and Moxifloxacin salt(s), prodrug(s), derivative(s) of any chemical purity, or
sustained release formulation(s) thereof, and a pharmaceutically acceptable excipient, for
simultanous, separate or sequential administration.
Most preferred drug compositions comprise 1, 2, 3, 4 or 5 distinct drugs, even more
preferably 2, 3 or 4. Furthermore, the above drug compositions may also be used in further
combination with one or several additional drugs or treatments beneficial to subjects with a
neurological disorder.
The invention also relates to a method of treating a neurological disorder, the method
comprising administering to a subject in need thereof a drug or composition as disclosed
above.
A further aspect of this invention relates to a method of treating a neurological disorder,
the method comprising simultaneously, separately or sequentially administering to a subject
in need thereof a drug combination as disclosed above.
A further aspect of this invention relates to the use of at least one compound selected
from the group consisting of Torasemide, Trimetazidine, Mexiletine, Ifenprodil,
Bromocriptine and Moxifloxacin, or salt(s), prodrug(s), derivative(s) of any chemical purity,
or sustained release formulation(s) thereof, for the manufacture of a medicament for the
treatment of a neurological disorder.
A further aspect of this invention relates to the use of drug combinations disclosed
above, for the manufacture of a medicament for the treatment of a neurological disorder.
- 5A -
The invention may be used in any mammalian subject, particularly human subject, at
any stage of the disease.
In one aspect, the present invention relates to use of a composition comprising
Torasemide, or a salt, or sustained release formulation thereof, in the manufacture of a
medicament for the treatment of Alzheimer’s disease (AD) or an AD related disorder,
amyotrophic lateral sclerosis (ALS) or spinal cord injury (SCI) in a subject in need thereof.
In another aspect, the present invention relates to a composition comprising
Torasemide, or a salt or a sustained release formulation thereof, in combination with at least
one second compound, selected from Sulfisoxazole, Methimazole, Prilocaine, Dyphylline,
Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen, Cabergoline,
Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam, Leflunomide,
Levosimendan, Sulodexide, Terbinafine, Zonisamide, Etomidate, Phenformin,
Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil, and Moxifloxacin or salt(s), or
sustained release formulation(s) thereof, and a pharmaceutically acceptable excipient, for
simultaneous, separate or sequential administration.
BRIEF DESCRIPTION OF THE FIGURES
For all figures, * : p<0.05: significantly different from control (no intoxication);
"ns": no significant effect (ANOVA + Dunnett's Post-Hoc test)
Figure 1: Effect of selected drugs pre-treatment against human Abi injury in
HBMEC. A) Validation of the experimental model used for drug screening: lhr of
VEGF pre-treatment at lOnM significantly protected the capillary network from this
amyloid injury (+70% of capillary network compared to amyloid intoxication). The
intoxication is significantly prevented by Torasemide (B) and Bromocriptine (C) at
doses as low as of 400nM and 3.2pM respectively, whereas no or a weaker effect is
noticed for upper and lower doses. p<0.05: significantly different from Amyloid
intoxication.
Figure 2 : Effect of selected drugs pre-treatment on LDH release in human Abi
toxicity assays on rat primary cortical cells. A) Validation of the experimental model
used for drug screening: lhr of Estradiol (150ng/ml) pre-treatment significantly
protected the neurons from this amyloid injury (-70%), which is considered as a positive
control for neuroprotection. For all experiments, Abi produces a significant
intoxication compared to vehicle-treated neurons. The intoxication is significantly
prevented by Bromocriptine (40nM, -29%) (B), Trimetazidine (40nM, -94%) (C),
Ifenprodil (600nM, -94%) (D), Mexiletine (3.2nM, -73%) (E), Moxifloxacin (20nM, -
63%) (F). Note that for other drug concentrations, no or a weaker effect is noticed for
upper and lower doses. :p<0.05: significantly different from Abi intoxication.
Figure 3:Effect of Baclofen and Torasemide combination therapy on the total length of
capillary network in beta-amyloid intoxicated HBMEC cultures. The aggregated human
amyloid peptide (Abi 2.5 m M ) produces a significant intoxication, above 40%,
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Baclofen and Torasemide (A) whereas, at those concentrations,
Baclofen (B) and Torasemide (C) alone have no significant effect on intoxication. :
p<0.05, significantly different from Abi intoxication.
Figure 4: Effect of Sulfisoxazole and Torasemide combination therapy on the total
length of capillary network in beta-amyloid intoxicated HBMEC cultures. The
aggregated human amyloid peptide (Abi 2.5 M ) produces a significant intoxication,
above 40%, compared to vehicle-treated cells. This intoxication is significantly
prevented by the combination of Sulfisoxazole and Torasemide (A) whereas, at those
concentrations, Sulfisoxazole (B) and Torasemide (C) alone have no significant effect
on intoxication. p<0.05, significantly different from Abi intoxication.
Figure 5: Effect of Eplerenone and Torasemide combination therapy on the total length
of capillary network in beta-amyloid intoxicated HBMEC cultures. The aggregated
human amyloid peptide (Abi 2.5 m M ) produces a significant intoxication, above 40%,
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Eplerenone and Torasemide (A) whereas, at those concentrations,
Torasemide (B) and Eplerenone (C) alone have no significant effect on intoxication.
p<0.05, significantly different from Abi intoxication.
Figure 6: Effect of Bromocriptine and Sulfisoxazole combination therapy on the total
length of capillary network in beta-amyloid intoxicated HBMEC cultures. The
aggregated human amyloid peptide (Abi 2.5 M ) produces a significant intoxication,
above 40%, compared to vehicle-treated cells. This intoxication is significantly
prevented by the combination of Bromocriptine and Sulfisoxazole (A) whereas, at those
concentrations, Bromocriptine (B) and Sulfisoxazole (C) alone have no significant
effect on intoxication. p<0.05, significantly different from Abi intoxication.
Figure 7: Effect of Acamprosate and Ifenprodil combination therapy on LDH release in
human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Acamprosate
and Ifenprodil (A) whereas, at those concentrations, Acamprosate (B) and Ifenprodil
(C) alone have no significant effect on intoxication. p<0.05, significantly different
from Abi intoxication.
Figure 8: Effect of Baclofen and Mexiletine combination therapy on LDH release in
human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Baclofen
and Mexiletine (A) whereas, at those concentrations, Baclofen (B) and Mexiletine (C)
alone have no significant effect on intoxication. p=0.051, different from Abi
intoxication.
Figure 9: Effect of Baclofen and Trimetazidine combination therapy on LDH release in
human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Baclofen
and Trimetazidine (A) whereas, at those concentrations, Baclofen (B) and Trimetazidine
(C) alone have no significant effect on intoxication. p<0.05, significantly different
from Abi intoxication.
Figure 10: Effect of Cinacalcet and Mexiletine combination therapy on LDH release in
human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Cinacalcet
and Mexiletine (A) whereas, at those concentrations, Cinacalcet (B) and Mexiletine (C)
alone have no significant effect on intoxication. p<0.05, significantly different from
Abi intoxication.
Figure 11: Effect of Cinnarizine and Trimetazidine combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Cinnarizine and Trimetazidine (A) whereas, at those concentrations, Cinnarizine (B)
and Trimetazidine (C) alone have no significant effect on intoxication. p<0.05,
significantly different from Abi intoxication.
Figure 12: Effect of Trimetazidine and Zonisamide combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Trimetazidine and Zonisamide (A) whereas, at those concentrations, Trimetazidine (B)
and Zonisamide (C) alone have no significant effect on intoxication. p<0.05,
significantly different from Abi intoxication.
Figure 13: Effect of Terbinafine and Torasemide combination therapy on the total
length of capillary network in beta-amyloid intoxicated HBMEC cultures. The
aggregated human amyloid peptide (Abi 2.5 m M ) produces a significant intoxication,
above 40%, compared to vehicle-treated cells. This intoxication is significantly
prevented by the combination of Terbinafine and Torasemide (A) whereas, at those
concentrations, Terbinafine (B) and Torasemide (C) alone have no significant effect on
intoxication. p<0.05, significantly different from Abi intoxication.
Figure 14: Effect of Cinacalcet and Mexiletine combination therapy on the total length
of capillary network in beta-amyloid intoxicated HBMEC cultures. The aggregated
human amyloid peptide (Abi 2.5 m M ) produces a significant intoxication, above 40%,
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Cinacalcet and Mexiletine (A) whereas, at those concentrations,
Cinacalcet (B) and Mexiletine (C) alone have no significant effect on intoxication.
p<0.05, significantly different from Abi intoxication.
Figure 15: Effect of Baclofen and Torasemide combination therapy on LDH release in
human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Baclofen
and Torasemide whereas, at those concentrations, Baclofen and Torasemide alone have
no significant effect on intoxication. p<0.05, significantly different from Abi
intoxication.
Figure 16: Effect of Torasemide and Sulfisoxazole combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Sulfisoxazole and Torasemide (A) whereas, at those concentrations, Torasemide (B)
and Sulfisoxazole (C) alone have no significant effect on intoxication. p<0.05,
significantly different from Abi intoxication.
Figure 17: Effect of Moxifloxacin and Trimetazidine combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Moxifloxacin and Trimetazidine (A). Adjunction of Moxifloxacin allows an increase of
100% of the effect observed for Trimetazidine (C) alone, whereas, at the same
concentration, Moxifloxacin (B) alone has no significant effect on intoxication.
p<0.05, significantly different from Abi intoxication.
Figure 18: Effect of Moxifloxacin and Baclofen combination therapy on LDH release
in human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of
Moxifloxacin and Baclofen (A) whereas, at those concentrations, Moxifloxacin (B) and
Baclofen (C) alone have no significant effect on intoxication. p<0.05, significantly
different from Abi intoxication.
Figure 19: Effect of Moxifloxacin and Cinacalcet combination therapy on LDH release
in human Abi toxicity on rat primary cortical cells. The aggregated human amyloid
peptide (Abi IOmM ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of
Moxifloxacin and Cinacalcet (A) whereas, at those concentrations, Moxifloxacin (B)
and Cinacalcet (C) alone have no significant effect on intoxication. p<0.05,
significantly different from Abi intoxication.
Figure 20: Effect of Moxifloxacin and Zonisamide combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Moxifloxacin and Zonisamide (A). Adjunction of Moxifloxacin allows an increase of
81% of the effect observed for Zonisamide (C) alone, whereas, at the same
concentration, Moxifloxacin (B) alone has no significant effect on intoxication.
p<0.05, significantly different from Abi intoxication.
Figure 21: Effect of Moxifloxacin and Sulfisoxazole combination therapy on LDH
release in human Abi toxicity on rat primary cortical cells. The aggregated human
amyloid peptide (Abi 10m M ) produces a significant intoxication compared to vehicle-
treated neurons. This intoxication is significantly prevented by the combination of
Moxifloxacin and Sulfisoxazole (A) whereas, at those concentrations, Moxifloxacin (B)
and Sulfisoxazole (C) alone have no significant effect on intoxication. p<0.05,
significantly different from Abi intoxication.
Figure 22: Effect of Mexiletine (MEX) and Ifenprodil (IFN) combination therapy on
LDH release in human Abi toxicity on rat primary cortical cells. The aggregated
human amyloid peptide (Abi 10m M ) produces a significant intoxication compared to
vehicle-treated neurons. This intoxication is significantly prevented by the combination
of Mexiletine 25.6 pM and Ifenprodil 24 nM whereas, at those concentrations,
Mexiletine and Ifenprodil alone have no significant effect on intoxication. p<0.0572,
significantly different from Abi intoxication.
Figure 23: Effect of Baclofen (BCL) and Torasemide (TOR) combination therapy on
the total length of neurites network in beta-amyloid intoxicated cortical neurons. The
human amyloid peptide (Abi 2.5 m M ) produces a significant intoxication, above 15 %,
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Acamprosate and Baclofen; furthermore this combination allows an
enhancement of neurite growth. p<0.05, significantly different from Abi
intoxication.
Figure 24: Effect of Cinacalcet and Mexiletine combination therapy against glutamate
toxicity on neuronal cortical cells. The glutamate intoxication is significantly prevented
by the combination of Cinacalcet (64pM) and Mexiletine (25.6pM) whereas, at those
concentrations, Cinacalcet and Mexiletine alone have no significant effect on
intoxication. p<0.001, significantly different from glutamate intoxication; (ANOVA
+ Dunnett Post-Hoc test).
Figure 25: Effect of Sulfisoxazole and Torasemide combination therapy against
glutamate toxicity on neuronal cortical cells. The glutamate intoxication is significantly
prevented by the combination of Sulfisoxazole (6.8nM) and Torasemide (400nM)
whereas, at those concentrations, Sulfisoxazole and Torasemide alone have no
significant effect on intoxication. : p<0.001, significantly different from glutamate
intoxication; (ANOVA + Dunnett Post-Hoc test).
Figure 26: Effect of Torasemide (TOR) pre-treatment on LDH release in human Abi
toxicity assays on rat primary cortical cells. Abi produces a significant intoxication
compared to vehicle-treated neurons. The intoxication is significantly prevented by
Torasemide (200nM, -90%) 0 : pO.0001: significantly different from Abi
intoxication.
Figure 27: Comparison of Acamprosate and its derivative Homotaurine pre-treatment
on LDH release in human Abi toxicity assays on rat primary cortical cells. Abi
2
produces a significant intoxication compared to vehicle-treated neurons. The
intoxication is equally significantly prevented by Homotaurine and Acamprosate (99%,
8nM). : p<0.0001: significantly different from Abi intoxication.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides new compositions for treating neurological
disorders. The invention discloses novel use of drugs or novel drug combinations which
allow an effective correction of such diseases and may be used for patient treatment.
The invention is suited for treating any neurological disorder, whether central or
peripheral, particularly disorders wherein amyloid or glutamate excitotoxicity are
involved. Specific examples of such disorders include neurodegenerative diseases such
as Alzheimer's and related disorders, Multiple Sclerosis (MS), Amyotrophic Lateral
Sclerosis (ALS), Parkinson's Disease (PD), Huntington's Disease (HD), or other
neurological disorders like neuropathies (for instance alcoholic neuropathy or
neuropathic pain), alcoholism or alcohol withdrawal and spinal cord injury.
Neuropathies refer to conditions where nerves of the peripheral nervous system are
damaged, this include damages of the peripheral nervous system provoked by genetic
factors, inflammatory disease, or by chemical substance including drugs (vincristine,
oxaliplatin, ethyl alcohol). The treatment of neuropathies also includes the treatment of
neuropathic pain.
The invention is particularly suited for treating AD and related disorders. In the
context of this invention, the term "AD related disorder" includes senile dementia of
AD type (SDAT), Lewis body dementia, vascular dementia, mild cognitive impairment
(MCI) and age-associated memory impairment (AAMI).
As used herein, "treatment" includes the therapy, prevention, prophylaxis,
retardation or reduction of symptoms provoked by or of the causes of the above diseases
or disorders. The term treatment includes in particular the control of disease progression
and associated symptoms. The term treatment particularly includes i) a protection
against the toxicity caused by Amyloid Beta, or a reduction or retardation of said
toxicity, and/or ii) a protection against glutamate excitotoxicity, or a reduction or
retardation of said toxicity, in the treated subjects. The term treatment also designates an
improvement of cognitive symptom or a protection of neuronal cells.
Within the context of this invention, the designation of specific compounds is
meant to include not only the specifically named molecules, but also any
pharmaceutically acceptable salt, hydrate, derivatives (e.g., ester, ether), isomers,
racemate, conjugates, or prodrugs thereof, of any purity.
The term "prodrug" as used herein refers to any functional derivatives (or
precursors) of a compound of the present invention, which, when administered to a
biological system, generates said compound as a result of e.g., spontaneous chemical
reaction(s), enzyme catalyzed chemical reaction(s), and/or metabolic chemical
reaction(s). Prodrugs are usually inactive or less active than the resulting drug and can
be used, for example, to improve the physicochemical properties of the drug, to target
the drug to a specific tissue, to improve the pharmacokinetic and pharmacodynamic
properties of the drug and/or to reduce undesirable side effects. Some of the common
functional groups that are amenable to prodrug design include, but are not limited to,
carboxylic, hydroxyl, amine, phosphate/phosphonate and carbonyl groups. Prodrugs
typically produced via the modification of these groups include, but are not limited to,
esters, carbonates, carbamates, amides and phosphates. Specific technical guidance for
the selection of suitable prodrugs is general common knowledge (24-28). Furthermore,
the preparation of prodrugs may be performed by conventional methods known by those
skilled in the art. Methods which can be used to synthesize other prodrugs are described
in numerous reviews on the subject (25; 29-35). For example, Arbaclofen Placarbil is
listed in ChemID plus Advance database (http://chem.sis.nlm.nih.gov/chemidplus/) and
Arbaclofen Placarbil is a well known prodrug of Baclofen (36; 43).
The term "derivative" of a compound includes any molecule that is functionally and/or
structurally related to said compound, such as an acid, amide, ester, ether, acetylated
variant, hydroxylated variant, or an alkylated (C1-C6) variant of such a compound. The
term derivative also includes structurally related compound having lost one or more
substituent as listed above. For example, Homotaurine is a deacetylated derivative of
Acamprosate. Preferred derivatives of a compound are molecules having a substantial
degree of similarity to said compound, as determined by known methods. Similar
compounds along with their index of similarity to a parent molecule can be found in
numerous databases such as PubChem (http://pubchem.ncbi.nlm.nih.gov/search/) or
DrugBank (http://www.drugbank.ca/). In a more preferred embodiment, derivatives
should have a Tanimoto similarity index greater than 0.4, preferably greater than 0.5,
more preferably greater than 0.6, even more preferably greater than 0.7 with a parent
drug. The Tanimoto similarity index is widely used to measure the degree of structural
similarity between two molecules. Tanimoto similarity index can be computed by
software such as the Small Molecule Subgraph Detector (37-38) available online
(http://www.ebi.ac.uk/thornton-srv/software/SMSD/). Preferred derivatives should be
both structurally and functionally related to a parent compound, i.e., they should also
retain at least part of the activity of the parent drug, more preferably they should have a
protective activity against A b or glutamate toxicity.
The term derivatives also include metabolites of a drug, e.g., a molecule which results
from the (biochemical) modification(s) or processing of said drug after administration to
an organism, usually through specialized enzymatic systems, and which displays or
retains a biological activity of the drug. Metabolites have been disclosed as being
responsible for much of the therapeutic action of the parent drug. In a specific
embodiment, a "metabolite" as used herein designates a modified or processed drug that
retains at least part of the activity of the parent drug, preferably that has a protective
activity against A b toxicity or glutamate toxicity. Examples of metabolites include
hydroxylated forms of Torasemide resulting from the hepatic metabolism of the drug
(Drug bank database (39).
The term "salt" refers to a pharmaceutically acceptable and relatively non-toxic,
inorganic or organic acid addition salt of a compound of the present invention.
Pharmaceutical salt formation consists in pairing an acidic, basic or zwitterionic drug
molecule with a counterion to create a salt version of the drug. A wide variety of
chemical species can be used in neutralization reaction. Pharmaceutically acceptable
salts of the invention thus include those obtained by reacting the main compound,
functioning as a base, with an inorganic or organic acid to form a salt, for example, salts
of acetic acid, nitric acid, tartric acid, hydrochloric acid, sulfuric acid, phosphoric acid,
methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid or
citric acid. Pharmaceutically acceptable salts of the invention also include those in
which the main compound functions as an acid and is reacted with an appropriate base
to form, e.g., sodium, potassium, calcium, magnesium, ammonium, or choline salts.
Though most of salts of a given active principle are bioequivalents, some may have,
among others, increased solubility or bioavailability properties. Salt selection is now a
common standard operation in the process of drug development as teached by H . Stahl
and C.G Wermuth in their handbook (40).
The term "combination" or "combinatorial treatment/therapy" designates a
treatment wherein at least two or more drugs are co-administered to a subject to cause a
biological effect. In a combined therapy according to this invention, the at least two
drugs may be administered together or separately, at the same time or sequentially.
Also, the at least two drugs may be administered through different routes and protocols.
As a result, although they may be formulated together, the drugs of a combination may
also be formulated separately.
As disclosed in the examples, Torasemide, Trimetazidine, Mexiletine, Ifenprodil,
Bromocriptine and Moxifloxacin have a strong unexpected effect on biological
processes involved in neurological disorders. Furthermore, these compounds also
showed in vivo a very efficient ability to correct symptoms of such diseases. These
molecules, alone or in combination therapies, therefore represent novel approaches for
treating neurological disorders, such as Alzheimer's disease, Multiple Sclerosis,
Amyotrophic Lateral Sclerosis, Parkinson's Disease, Huntington's Disease,
neuropathies (for instance neuropathic pain or alcoholic neuropathy), alcoholism or
alcohol withdrawal, and spinal cord injury. Combinations of these drugs with other
selected compounds (see Table 2) are particularly advantageous because they produce a
surprising and unexpected synergistic effect at dosages where the drugs alone have
essentially no effect. Also, because of their efficacy, the herein disclosed drugs
combinations can be used at low dosages, which is a further very substantial advantage.
In this regard, in particular embodiment, the invention relates to a composition
for use in the treatment of AD, AD related disorders, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury, comprising at least Torasemide, Trimetazidine,
Mexiletine, Ifenprodil, Bromocriptine, or Moxifloxacin, or a salt, prodrug, derivative, or
sustained release formulation thereof.
The specific CAS number for each of these compounds is provided in Table 1
below. Table 1 cites also, in a non-limitative way, common salts, racemates, prodrugs,
metabolites or derivatives for these compounds used in the compositions of the
invention.
Table 1
The above molecules may be used alone or, preferably, in combination therapies
to provide the most efficient clinical benefit. In this regard, in a preferred embodiment,
the invention relates to a composition for use in the treatment of a neurological disorder,
preferably AD, AD related disorders, MS, PD, ALS, HD, neuropathies (for instance
neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal
cord injury, comprising any one of the above compounds in combination with at least
one distinct compound selected from Sulfisoxazole, Methimazole, Prilocaine,
Dyphylline, Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen,
Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam,
Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide, Etomidate,
Phenformin, Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin, Bromocriptine or
Torasemide, or a salt, prodrug, derivative, or sustained release formulation thereof.
The specific CAS number for each of these additional distinct compounds,
different from those of Table 1 is provided in Table 2 below:
Table 2
DRUG NAME CAS NUMBER
773379 ; 773376 ; 1077 ;
Acamprosate
36871
Aminocaproic Acid 602
11340; 665146; 693088;
Baclofen 702063; 637014; 637013 ;
8473534
814097
Cabergoline
Carbenoxolone 56973 or 74211
2987
Cinnarizine
Diethylcarbamazine 901 or 16422
4795
Dyphylline
Eplerenone 1077249
331252
Etomidate
Fenoldopam 672270 or 672279
Leflunomide 757066
Levosimendan 1415051
Methimazole 600
1510962 or 1868268 or 192927-
Moxifloxacin
63-2 or 3548122
Phenformin 1143 or 8346
Prilocaine 7216 or 142897 or 142898
836 or 696 or 61510
Quinacrine
Sulodexide 578211
Terbinafine 9 11616
Trimetazidine 501 17 or 131710
Zonisamide 682914
Specific examples of prodrugs of Baclofen are given in Hanafi et al, 201 1 (41),
particularly Baclofen esters and Baclofen ester carbamateswhich are of particular
interest for CNS targeting. Hence such prodrugs are particularly suitable for
compositions of this invention. Baclofen placarbil as mentioned before is also a well-
known prodrug and may thus be used instead of Baclofen in compositions of the
invention. Other prodrugs of Baclofen can be found in the following patent applications:
WO2010102071, US2009197958, WO2009096985, WO2009061934, WO2008086492,
US2009216037, WO2005066122, US201 1021571, WO2003077902, WO2010120370.
Useful prodrugs for acamprosate such as pantoic acid ester neopentyl sulfonyl
esters, neopentyl sulfonyl esters prodrugs or masked carboxylate neopentyl sulfonyl
ester prodrugs of acamprosate are notably listed in WO2009033069, WO2009033061,
WO2009033054 WO2009052191, WO2009033079, US 2009/0099253, US
2009/0069419, US 2009/0082464, US 2009/0082440, and US 2009/0076147.
In a preferred embodiment, the invention relates to a composition comprising:
- at least one first compound selected from Torasemide, Trimetazidine,
Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin salt(s), prodrug(s),
derivative(s) of any chemical purity, or sustained release formulation(s) thereof, in
combination with
- at least one second compound, distinct from said first compound, selected from
Sulfisoxazole, Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone,
Acamprosate, Aminocaproic acid, Baclofen, Cabergoline, Diethylcarbamazine,
Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam, Leflunomide, Levosimendan,
Sulodexide, Terbinafine, Zonisamide, Etomidate, Phenformin, Trimetazidine,
Mexiletine, Bromocriptine, Ifenprodil, Torasemide and Moxifloxacin, salt(s),
prodrug(s), derivative(s) of any chemical purity, or sustained release formulation(s)
thereof, for use in the treatment of a neurological disorder in a subject in need thereof.
In a particular embodiment, the invention relates to the use of these drugs or
compositions for treating AD or a related disorder in a subject in need thereof.
In a particular embodiment, the invention relates to the use of these drugs or
compositions for treating MS, PD, ALS, HD, neuropathies (for instance neuropathic
pain or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury,
in a subject in need thereof.
As disclosed in the examples, composition therapies using one or more of the
above-listed drugs lead to an efficient correction of Alzheimer's disease and other
neurological diseases. As illustrated in the experimental section, compositions
comprising at least Torasemide, Trimetazidine, Mexiletine, Ifenprodil, Bromocriptine,
and Moxifloxacin provide substantial therapeutic and biological effect to prevent the
toxic effects of amyloid b (A b ) protein or peptide on human cells. Moreover, in vivo,
these compositions lead to an improvement of cognitive symptoms as well as to an
inhibition of molecular pathways triggered by A b intoxication, within which glutamate
excitotoxicity. Hence they represent novel and potent methods for treating such disease.
The experimental section further shows that the above mentioned compositions are also
efficient i) in synergistically protecting in vitro neuronal cells from glutamate toxicity,
and ii) in conferring clinical benefit in in vivo models for diseases related to glutamate
excitotoxicity.
More preferably, drug compositions of the invention may comprise 1, 2, 3, 4 or 5
distinct drugs, even more preferably 2, 3 or 4 distinct drugs for combinatorial treatment
of Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury in a subject in need thereof. In a preferred
embodiment, the drugs of the invention are used in combination(s) for combined,
separate or sequential administration, in order to provide the most effective effect.
In a particular embodiment, the composition comprises (i) Torasemide and (ii) a
compound selected from Bromocriptine, Baclofen, Sulfisoxazole, Eplerenone or
Terbinafine, or a salt, prodrug, derivative, or sustained release formulation of said
compounds (i) and (ii).
In another particular embodiment, the composition comprises (i) Trimetazidine and
(ii) a compound selected from Baclofen, Cinnarizine, Zonisamide, or Moxifloxacin, or a
salt, prodrug, derivative, or sustained release formulation of said compounds (i) and (ii).
According to a further particular embodiment, the composition comprises (i)
Moxifloxacin and (ii) a compound selected from Baclofen, Cinacalcet, Zonisamide,
Sulfisoxazole, or Trimetazidine, or a salt, prodrug, derivative, or sustained release
formulation of said compounds (i) and (ii).
In another further particular embodiment, the composition comprises (i) Mexiletine
and (ii) a compound selected from Baclofen, Cinacalcet, Ifenprodil, or levosimendan or
a salt, prodrug, derivative, or sustained release formulation of said compounds (i) and
(ii).
A particular embodiment also relates to a composition comprising (i) Ifenprodil and
(ii) a compound selected from Acamprosate, Levosimendan, or Mexiletine or a salt,
prodrug, derivative, or sustained release formulation of said compounds (i) and (ii).
Preferred compositions of the invention, for use in the treatment of a neurological
disorder such as Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD,
neuropathies (for instance neuropathic pain or alcoholic neuropathy), alcoholism or
alcohol withdrawal, or spinal cord injury, comprise one of the following drug
combinations, for combined, separate or sequential administration:
- Baclofen and Torasemide,
- Eplerenone and Torasemide,
Acamprosate and Ifenprodil,
- Baclofen and Mexiletine,
- Baclofen and Trimetazidine,
- Bromocriptine and Sulfisoxazole,
Cinacalcet and Mexiletine,
Cinnarizine and Trimetazidine,
- Sulfisoxazole and Torasemide,
Trimetazidine and Zonisamide,
- Levosimendan and Mexiletine,
- Levosimendan and Ifenprodil,
- Levosimendan and Trimetazidine,
- Levosimendan and Moxifloxacin,
Terbinafine and Torasemide,
Moxifloxacin and Trimetazidine,
Moxifloxacin and Baclofen,
- Moxifloxacin and Cinacalcet,
Moxifloxacin and Zonisamide,
- Moxifloxacin and Sulfisoxazole, or
Mexiletine and Ifenprodil.
Examples of preferred compositions according to the invention comprising a
combination of at least three compounds, for combined, separate or sequential
administration, are provided below:
- Baclofen and Trimetazidine and Torasemide,
- Baclofen and Cinacalcet and Torasemide,
- Baclofen and Acamprosate and Torasemide,
- Levosimendan and Baclofen and Trimetazidine,
- Levosimendan and Aminocaproic acid and Trimetazidine,
- Levosimendan and Terbinafine and Trimetazidine, or
- Levosimendan and Sulfisoxazole and Trimetazidine.
Examples of preferred compositions according to the invention comprising a
combination of at least four compounds, for combined, separate or sequential
administration, are provided below:
Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide,
Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet,
- Baclofen and Acamprosate and Torasemide and Diethylcarbamazine, or
- Baclofen and Acamprosate and Torasemide and Ifenprodil.
As disclosed in the experimental section the above combination therapies of the
invention induce a strong neuroprotective effect against A b toxicity and give positive
results in behavioural performances and biochemical assays in vivo. The results show
that compositions of the invention i) efficiently correct molecular pathways triggered, in
vivo, by A b aggregates and ii) lead to an improvement of neurophysiological
impairments observed in diseased animals as neuron survival or synapse integrity.
Moreover, the results presented show also that the above combinations therapies have
an important synergistic neuroprotecting effect against glutamate excitotoxicity (figures
24 and 25, table 8), a pathway which is implicated in various neurological diseases as
AD, MS, PD, ALS, HD, neuropathies (for instance neuropathic pain or alcoholic
neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury. These therapies
give positive results in in vivo or in vitro models for these diseases.
In addition, in vivo results also show that compositions of the invention efficiently
restore Brain Blood Barrier integrity, which is known to be impaired in several
neurological diseases.
An object of this invention thus also resides in a composition as defined above
for treating a neurological disorder such as Alzheimer's disease (AD), AD related
disorders, MS, PD, ALS, HD, neuropathies (for instance alcoholic neuropathy or
neuropathic pain), alcoholism or alcohol withdrawal, or spinal cord injury.
A further object of this invention resides in the use of a composition as defined
above for the manufacture of a medicament for treating a neurological disorder such as
Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies (for
instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal,
or spinal cord injury.
The invention further provides a method for treating a neurological disorder such
as Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury, comprising administering to a subject in need thereof
an effective amount of a composition as disclosed above.
As indicated previously, the compounds in a combinatorial treatment or
composition of the present invention may be formulated together or separately, and
administered together, separately or sequentially and/or repeatedly.
In this regard, a particular object of this invention is a method for treating AD,
an AD related disorder, MS, PD, ALS, HD, neuropathies (for instance neuropathic pain
or alcoholic neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury in a
subject, comprising administering simultaneously, separately or sequentially to a subject
in need of such a treatment, an effective amount of a composition as disclosed above.
In a preferred embodiment, the invention relates to a method of treating
Alzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury in a subject in need thereof, comprising administering
to the subject an effective amount of Torasemide, Trimetazidine, Mexiletine, Ifenprodil,
Bromocriptine or Moxifloxacin, or salt(s) or prodrug(s) or derivative(s) or sustained
release formulation(s) thereof, preferably in a combination as disclosed above.
In another embodiment, this invention relates to a method of treating
Alzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury in a subject in need thereof, comprising
simultaneously, separately or sequentially administering to the subject at least one first
compound selected from the group consisting of Torasemide, Trimetazidine,
Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin salts, prodrugs, derivatives, or
any formulation thereof, in combination with at least one second compound distinct
from said first compound, selected from, Sulfisoxazole, Methimazole, Prilocaine,
Dyphylline, Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen,
Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam,
Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide, Etomidate,
Phenformin, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil, Torasemide, and
Moxifloxacin salts, prodrugs, derivatives, or any formulation thereof.
In a further embodiment, the invention relates to a method of treating
Alzheimer's disease (AD), an AD related disorder, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury comprising administering to a subject in need thereof,
at least one first compound selected from the group consisting of Torasemide,
Trimetazidine, Mexiletine, Ifenprodil, Bromocriptine and Moxifloxacin salts, prodrugs,
derivatives, or any formulation thereof, in combination with at least one second
compound distinct from said first compound, selected from, Sulfisoxazole,
Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone, Acamprosate,
Aminocaproic acid, Baclofen, Cabergoline, Diethylcarbamazine, Cinacalcet,
Cinnarizine, Eplerenone, Fenoldopam, Leflunomide, Levosimendan, Sulodexide,
Terbinafine, Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine,
Bromocriptine, Ifenprodil, Torasemide, and Moxifloxacin salts, prodrugs, derivatives,
or any formulation thereof.
Although very effective in vitro and in vivo, depending on the subject or specific
condition, the methods and compositions of the invention may be used in further
conjunction with additional drugs or treatments beneficial to the treated neurological
condition in the subjects. In this regard, in a particular embodiment, the drug(s) or
compositions according to the present invention may be further combined with Ginkgo
Biloba extracts. Suitable extracts include, without limitation, Ginkgo biloba extracts,
improved Ginkgo biloba extracts (for example enriched in active ingredients or lessened
in contaminant) or any drug containing Ginkgo biloba extracts.
Ginkgo Biloba extracts may be used in a composition comprising at least
Torasemide, Trimetazidine, Mexiletine, Bromocriptine, Ifenprodil and Moxifloxacin.
In preferred embodiments, Ginkgo Biloba extracts are used in combination with
anyone of the following drug combinations:
- Acamprosate and Ifenprodil,
Baclofen and Mexiletine,
- Baclofen and Torasemide,
- Baclofen and Trimetazidine,
- Bromocriptine and Sulfisoxazole,
- Cinacalcet and Mexiletine,
Cinnarizine and Trimetazidine,
- Eplerenone and Torasemide,
Sulfisoxazole and Torasemide,
Trimetazidine and Zonisamide,
- Levosimendan and Mexiletine,
- Levosimendan and Ifenprodil,
- Levosimendan and Trimetazidine,
- Levosimendan and Moxifloxacin,
Terbinafine and Torasemide,
- Moxifloxacin and Baclofen,
- Moxifloxacin and Cinacalcet,
Moxifloxacin and Zonisamide,
- Moxifloxacin and Sulfisoxazole,
Mexiletine and Ifenprodil,
- Baclofen and Trimetazidine and Torasemide,
- Baclofen and Cinacalcet and Torasemide,
- Baclofen and Acamprosate and Torasemide,
Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide,
Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet,
- Baclofen and Acamprosate and Torasemide and Diethylcarbamazine,
- Baclofen and Acamprosate and Torasemide and Ifenprodil,
- Levosimendan and Baclofen and Trimetazidine,
- Levosimendan and Aminocaproic acid and Trimetazidine,
- Levosimendan and Terbinafine and Trimetazidine, or
- Levosimendan and Sulfisoxazole and Trimetazidine.
Other therapies used in conjunction with drug(s) or drug(s) combination(s)
according to the present invention, may comprise one or more drug(s) that ameliorate
symptoms of Alzheimer's disease, an AD related disorder, MS, PD, ALS, HD,
neuropathies (for instance neuropathic pain or alcoholic neuropathy), alcoholism or
alcohol withdrawal, or spinal cord injury, or drug(s) that could be used for palliative
treatment of these disorders.
For instance, combinations of the invention can be used in conjunction with
Donepezil (CAS: 1200144), Gabapentine (CAS: 4782969; 601423),
Rivastigmine (1234412) or Memantine (CAS: 199822).
A further object of this invention relates to the use of a compound or
combination of compounds as disclosed above for the manufacture of a medicament for
the treatment of the above listed disorders, by combined, separate or sequential
administration to a subject in need thereof.
A further object of this invention is a method of preparing a pharmaceutical
composition, the method comprising mixing the above compounds in an appropriate
excipient or carrier.
The duration of the therapy depends on the stage of the disease or disorder being
treated, the combination used, the age and condition of the patient, and how the patient
responds to the treatment. The dosage, frequency and mode of administration of each
component of the combination can be controlled independently. For example, one drug
may be administered orally while the second drug may be administered intramuscularly.
Combination therapy may be given in on-and-off cycles that include rest periods so that
the patient's body has a chance to recover from any as yet unforeseen side-effects. The
drugs may also be formulated together such that one administration delivers all drugs.
The administration of each drug of the combination may be by any suitable
means that results in a concentration of the drug that, combined with the other
component, is able to ameliorate the patient condition or efficiently treat the disease or
disorder.
While it is possible for the active ingredients of the combination to be
administered as the pure chemical it is preferable to present them as a pharmaceutical
composition, also referred to in this context as pharmaceutical formulation. Possible
compositions include those suitable for oral, rectal, topical (including transdermal,
buccal and sublingual), or parenteral (including subcutaneous, intramuscular,
intravenous and intradermal) administration.
More commonly these pharmaceutical formulations are prescribed to the patient
in "patient packs" containing a number dosing units or other means for administration of
metered unit doses for use during a distinct treatment period in a single package, usually
a blister pack. Patient packs have an advantage over traditional prescriptions, where a
pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the
patient always has access to the package insert contained in the patient pack, normally
missing in traditional prescriptions. The inclusion of a package insert has been shown to
improve patient compliance with the physician's instructions. Thus, the invention
further includes a pharmaceutical formulation, as herein before described, in
combination with packaging material suitable for said formulations. In such a patient
pack the intended use of a formulation for the combination treatment can be inferred by
instructions, facilities, provisions, adaptations and/or other means to help using the
formulation most suitably for the treatment. Such measures make a patient pack
specifically suitable for and adapted for use for treatment with the combination of the
present invention.
The drug may be contained, in any appropriate amount, in any suitable carrier
substance (e.g., excipient, vehicle, support), which may represent 1-99% by weight of
the total weight of the composition. The composition may be provided in a dosage form
that is suitable for the oral, parenteral (e.g., intravenously, intramuscularly), rectal,
cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus,
the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates,
suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams,
plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables,
implants, sprays, or aerosols.
The pharmaceutical compositions may be formulated according to conventional
pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy
(20th ed.), ed. A . R . Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia
of Pharmaceutical Technology, eds. J . Swarbrick and J . C . Boylan, 1988-1999, Marcel
Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to
release the active drug substantially immediately upon administration or at any
predetermined time or time period after administration.
The controlled release formulations include (i) formulations that create a
substantially constant concentration of the drug within the body over an extended period
of time; (ii) formulations that after a predetermined lag time create a substantially
constant concentration of the drug within the body over an extended period of time; (iii)
formulations that sustain drug action during a predetermined time period by maintaining
a relatively, constant, effective drug level in the body with concomitant minimization of
undesirable side effects associated with fluctuations in the plasma level of the active
drug substance; (iv) formulations that localize drug action by, e.g., spatial placement of
a controlled release composition adjacent to or in the diseased tissue or organ; and (v)
formulations that target drug action by using carriers or chemical derivatives to deliver
the drug to a particular target cell type.
Administration of drugs in the form of a controlled release formulation is
especially preferred in cases in which the drug, either alone or in combination, has (i) a
narrow therapeutic index (i.e., the difference between the plasma concentration leading
to harmful side effects or toxic reactions and the plasma concentration leading to a
therapeutic effect is small; in general, the therapeutic index, TI, is defined as the ratio of
median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption
window in the gastro-intestinal tract; or (iii) a very short biological half-life so that
frequent dosing during a day is required in order to sustain the plasma level at a
therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled
release in which the rate of release outweighs the rate of metabolism of the drug in
question. Controlled release may be obtained by appropriate selection of various
formulation parameters and ingredients, including, e.g., various types of controlled
release compositions and coatings. Thus, the drug is formulated with appropriate
excipients into a pharmaceutical composition that, upon administration, releases the
drug in a controlled manner (single or multiple unit tablet or capsule compositions, oil
solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches,
and liposomes).
Solid Dosage Forms for Oral Use
Formulations for oral use include tablets containing the active ingredient(s) in a
mixture with non-toxic pharmaceutically acceptable excipients. These excipients may
be, for example, inert diluents or fillers (e.g., sucrose, microcrystalline cellulose,
starches including potato starch, calcium carbonate, sodium chloride, calcium
phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents
(e.g., cellulose derivatives including microcrystalline cellulose, starches including
potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g.,
acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch,
microcrystalline cellulose, carboxymethylcellulose sodium, methylcellulose,
hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene
glycol); and lubricating agents, glidants, and antiadhesives (e.g., stearic acid, silicas, or
talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents,
plasticizers, humectants, buffering agents, and the like.
The tablets may be uncoated or they may be coated by known techniques,
optionally to delay disintegration and absorption in the gastrointestinal tract and thereby
providing a sustained action over a longer period. The coating may be adapted to release
the active drug substance in a predetermined pattern (e.g., in order to achieve a
controlled release formulation) or it may be adapted not to release the active drug
substance until after passage of the stomach (enteric coating). The coating may be a
sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,
carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or
polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer,
cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl
methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or
ethylcellulose). A time delay material such as, e.g., glyceryl monostearate or glyceryl
distearate may be employed.
The solid tablet compositions may include a coating adapted to protect the
composition from unwanted chemical changes, (e.g., chemical degradation prior to the
release of the active drug substance). The coating may be applied on the solid dosage
form in a similar manner as that described in Encyclopedia of Pharmaceutical
Technology.
Several drugs may be mixed together in the tablet, or may be partitioned. For
example, the first drug is contained on the inside of the tablet, and the second drug is on
the outside, such that a substantial portion of the second drug is released prior to the
release of the first drug.
Formulations for oral use may also be presented as chewable tablets, or as hard
gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g.,
potato starch, microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or
an oil medium, for example, liquid paraffin, or olive oil. Powders and granulates may be
prepared using the ingredients mentioned above under tablets and capsules in a
conventional manner.
Controlled release compositions for oral use may, e.g., be constructed to release
the active drug by controlling the dissolution and/or the diffusion of the active drug
substance.
Dissolution or diffusion controlled release can be achieved by appropriate
coating of a tablet, capsule, pellet, or granulate formulation of drugs, or by
incorporating the drug into an appropriate matrix. A controlled release coating may
include one or more of the coating substances mentioned above and/or, e.g., shellac,
beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate,
glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,
polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate,
methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation, the matrix material
may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol,
carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate,
polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
A controlled release composition containing one or more of the drugs of the
claimed combinations may also be in the form of a buoyant tablet or capsule (i.e., a
tablet or capsule that, upon oral administration, floats on top of the gastric content for a
certain period of time). A buoyant tablet formulation of the drug(s) can be prepared by
granulating a mixture of the drug(s) with excipients and 20-75% w/w of hydrocolloids,
such as hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropylmethylcellulose. The obtained granules can then be compressed into
tablets. On contact with the gastric juice, the tablet forms a substantially water-
impermeable gel barrier around its surface. This gel barrier takes part in maintaining a
density of less than one, thereby allowing the tablet to remain buoyant in the gastric
juice.
Liquids for Oral Administration
Powders, dispersible powders, or granules suitable for preparation of an aqueous
suspension by addition of water are convenient dosage forms for oral administration.
Formulation as a suspension provides the active ingredient in a mixture with a
dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable
suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose,
sodium alginate, and the like.
Parenteral Compositions
The pharmaceutical composition may also be administered parenterally by
injection, infusion or implantation (intravenous, intramuscular, subcutaneous, or the
like) in dosage forms, formulations, or via suitable delivery devices or implants
containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
The formulation and preparation of such compositions are well known to those skilled
in the art of pharmaceutical formulation.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in
single-dose ampoules), or in vials containing several doses and in which a suitable
preservative may be added (see below). The composition may be in form of a solution, a
suspension, an emulsion, an infusion device, or a delivery device for implantation or it
may be presented as a dry powder to be reconstituted with water or another suitable
vehicle before use. Apart from the active drug(s), the composition may include suitable
parenterally acceptable carriers and/or excipients. The active drug(s) may be
incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for
controlled release. The composition may include suspending, solubilizing, stabilizing,
pH-adjusting agents, and/or dispersing agents.
The pharmaceutical compositions according to the invention may be in the form
suitable for sterile injection. To prepare such a composition, the suitable active drug(s)
are dissolved or suspended in a parenterally acceptable liquid vehicle. Among
acceptable vehicles and solvents that may be employed are water, water adjusted to a
suitable H by addition of an appropriate amount of hydrochloric acid, sodium
hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium
chloride solution. The aqueous formulation may also contain one or more preservatives
(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the drugs is
only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent
can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
Controlled release parenteral compositions may be in form of aqueous
suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil
suspensions, or emulsions. Alternatively, the active drug(s) may be incorporated in
biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.
Materials for use in the preparation of microspheres and/or microcapsules are, e.g.,
biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate),
poly(2-hydroxyethyl-L-glutamnine). Biocompatible carriers that may be used when
formulating a controlled release parenteral formulation are carbohydrates (e.g.,
dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in
implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g.,
poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).
Alternative routes
Although less preferred and less convenient, other administration routes, and
therefore other formulations, may be contemplated. In this regard, for rectal application,
suitable dosage forms for a composition include suppositories (emulsion or suspension
type), and rectal gelatin capsules (solutions or suspensions). In a typical suppository
formulation, the active drug(s) are combined with an appropriate pharmaceutically
acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated
gelatin, and various water-soluble or dispersible bases like polyethylene glycols.
Various additives, enhancers, or surfactants may be incorporated.
The pharmaceutical compositions may also be administered topically on the skin
for percutaneous absorption in dosage forms or formulations containing conventionally
non-toxic pharmaceutical acceptable carriers and excipients including microspheres and
liposomes. The formulations include creams, ointments, lotions, liniments, gels,
hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, and other kinds of
transdermal drug delivery systems. The pharmaceutically acceptable carriers or
excipients may include emulsifying agents, antioxidants, buffering agents,
preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents,
ointment bases, perfumes, and skin protective agents.
The preservatives, humectants, penetration enhancers may be parabens, such as
methyl or propyl p-hydroxybenzoate, and benzalkonium chloride, glycerin, propylene
glycol, urea, etc.
The pharmaceutical compositions described above for topical administration on
the skin may also be used in connection with topical administration onto or close to the
part of the body that is to be treated. The compositions may be adapted for direct
application or for application by means of special drug delivery devices such as
dressings or alternatively plasters, pads, sponges, strips, or other forms of suitable
flexible material.
Dosages and duration of the treatment
It will be appreciated that the drugs of the combination may be administered
concomitantly, either in the same or different pharmaceutical formulation or
sequentially. If there is sequential administration, the delay in administering the second
(or additional) active ingredient should not be such as to lose the benefit of the
efficacious effect of the combination of the active ingredients. A minimum requirement
for a combination according to this description is that the combination should be
intended for combined use with the benefit of the efficacious effect of the combination
of the active ingredients. The intended use of a combination can be inferred by
facilities, provisions, adaptations and/or other means to help using the combination
according to the invention.
Although the active drugs of the present invention may be administered in
divided doses, for example two or three times daily, a single daily dose of each drug in
the combination is preferred, with a single daily dose of all drugs in a single
pharmaceutical composition (unit dosage form) being most preferred.
The term "unit dosage form" refers to physically discrete units (such as capsules,
tablets, or loaded syringe cylinders) suitable as unitary dosages for human subjects,
each unit containing a predetermined quantity of active material or materials calculated
to produce the desired therapeutic effect, in association with the required
pharmaceutical carrier.
Administration is generally repeated. It can be one to several times daily for
several days to several years, and may even be for the life of the patient. Chronic or at
least periodically repeated long-term administration is indicated in most cases.
Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic,
pharmacodynamic or efficacy profile of a therapeutic) information about a particular
patient may affect the dosage used.
Except when responding to especially impairing cases when higher dosages may
be required, the preferred dosage of each drug in the combination usually lies within the
range of doses not above those usually prescribed for long-term maintenance treatment
or proven to be safe in phase 3 clinical studies.
One remarkable advantage of the invention is that each compound may be used
at low doses in a combination therapy, while producing, in combination, a substantial
clinical benefit to the patient. The combination therapy may indeed be effective at doses
where the compounds have individually no substantial effect. Accordingly, a particular
advantage of the invention lies in the ability to use sub-optimal doses of each
compound, i.e., doses which are lower than therapeutic doses usually prescribed,
preferably 1/2 of therapeutic doses, more preferably 1/3, 1/4, 1/5, or even more
preferably 1/10 of therapeutic doses. In particular examples, doses as low as 1/20, 1/30,
1/50, 1/100, or even lower, of therapeutic doses are used.
At such sub-optimal dosages, the compounds alone would be substantially
inactive, while the combination(s) according to the invention are fully effective.
A preferred dosage corresponds to amounts from 1% up to 50% of those usually
prescribed for long-term maintenance treatment.
The most preferred dosage may correspond to amounts from 1% up to 10% of
those usually prescribed for long-term maintenance treatment.
Specific examples of dosages of drugs for use in the invention are provided
below:
- Bromocriptine orally from about 0.01 to 10 mg per day, preferably less than 5
mg per day, more preferably less than 2.5 mg per day, even more preferably less
than 1 mg per day, such dosages being particularly suitable for oral
administration,
Ifenprodil orally from about 0.4 to 6 mg per day, preferably less than 3 mg per
day, more preferably less than 1.5 mg per day, even more preferably less than
0.75 mg per day, such dosages being particularly suitable for oral
administration,
- Mexiletine orally from about 6 to 120 mg per day, preferably less than 60 mg
per day, more preferably less than 30 mg per day, even more preferably less than
mg per day, such dosages being particularly suitable for oral administration,
- Moxifloxacin orally from about 4 to 40 mg per day, preferably less than 20 mg
per day, more preferably less than 10 mg per day, even more preferably less than
mg per day, such dosages being particularly suitable for oral administration,
Torasemide orally from about 0.05 to 4 mg per day, preferably less than 2 mg
per day, more preferably less than 1 mg per day, even more preferably less than
0.5 mg per day, such dosages being particularly suitable for oral administration,
- Trimetazidine orally from about 0.4 to 6 mg per day, preferably less than 3 mg
per day, more preferably less than 1.5 mg per day, even more preferably less
than 0.75 mg per day, such dosages being particularly suitable for oral
administration,
Acamprosate orally from about 1 to 400 mg per day,
- Aminocaproic Acid orally from about 0 .1 g to 2.4 g per day,
- Baclofen orally from about 0 .15 to 15 mg per day,
- Diethylcarbamazine orally from about 0.6 to 600 mg per day,
Cinacalcet orally from about 0.3 to 36 mg per day,
Cinnarizine orally from about 0.6 to 23 mg per day,
- Eplerenone orally from about 0.25 to 10 mg per day,
- Leflunomide orally from about 0 .1 to 10 mg per day,
- Levosimendan orally from about 0.04 to 0.8 mg per day,
Sulfisoxazole orally from about 20 to 800 mg per day,
Sulodexide orally from about 0.05 to 40 mg per day,
- Terbinafine orally from about 2.5 to 25 mg per day,
- Zonisamide orally from about 0.5 to 50 mg per day.
It will be understood that the amount of the drug actually administered will be
determined by a physician, in the light of the relevant circumstances including the
condition or conditions to be treated, the exact composition to be administered, the age,
weight, and response of the individual patient, the severity of the patient's symptoms,
and the chosen route of administration. Therefore, the above dosage ranges are intended
to provide general guidance and support for the teachings herein, but are not intended to
limit the scope of the invention.
The following examples are given for purposes of illustration and not by way of
limitation.
EXAMPLES
The care and husbandry of animals as well as the experimentations are
performed according to the guidelines of the Committeefor Research and Ethical Issue
ofthe l.A.S.P. (1983).
A) TREATMENT OF DISEASES RELATED TO A b TOXICITY
In this series of experiments, candidate compounds have been tested for their
ability to prevent or reduce the toxic effects of human Abi . Abi is the full length
-42 -42
peptide that constitutes aggregates found in biopsies from human patients afflicted with
AD. The drugs are first tested individually, followed by assays of their combinatorial
action. The effect is determined on various cell types, to further document the activity
of the compounds in in vitro models which illustrate different physiological features of
AD. In vivo studies are also performed in a mouse model for AD confirming this
protective effect by evaluating the effect of the compounds on i) the cognitive
performance of animals and ii) on molecular hallmarks (apoptosis induction, oxidative
stress induction, inflammation pathway induction) of AD.
I THE COMPOUNDS PREVENT TOXICITY OF HUMAN A
I. Protection against the toxicity of A 4 in human brain microvascular
Endothelial Cell model
Human brain microvascular endothelial cell cultures were used to study the protection
afforded by candidate compound(s) on Abi toxicity.
Human brain microvascular endothelial cerebral cells (HBMEC, ScienCell Ref: 1000,
frozen at passage 10) were rapidly thawed in a waterbath at +37°C. The supernatant was
immediately put in 9 ml Dulbecco's modified Eagle's medium (DMEM; Pan Biotech
ref: P04-03600) containing 10% of foetal calf serum (FCS; GIBCO ref 10270-106). Cell
suspension was centrifuged at 180 x g for 10 min at +4°C and the pellets were
suspended in CSC serum-free medium (CSC serum free, Cell System, Ref: SF-4Z0-
500-R, Batch 51407-4) with 1.6% of Serum free RocketFuel (Cell System, Ref: SF-
4Z0R, Batch 54102), 2% of Penicillin 10.000 U/ml and Streptomycin lOmg/ml
(PS ; Pan Biotech ref: P06-07100 batch 133080808) and were seeded at the density of
000 cells per well in 96 well-plates (matrigel layer biocoat angiogenesis system, BD,
Ref 354150, Batch A8662) in a final volume of IOOmI . On matrigel support, endothelial
cerebral cells spontaneously started the process of capillary network morphogenesis
(33).
Three separate cultures were performed per condition, 6 wells per condition.
Candidate compounds and Human treatment
Briefly, Abi peptide (Bachem, ref: H1368 batch 1010533) was reconstituted in define
culture medium at 20m M (mother solution) and was slowly shacked at +37 °C for 3
days in dark for aggregation. The control medium was prepared in the same conditions.
After 3 days, this aggregated human amyloid peptide was used on FIBMEC at 2.5 m M
diluted in control medium (optimal incubation time). The Abi peptide was added 2
hours after FIBMEC seeding on matrigel for 18 hours incubation.
One hour after FIBMEC seeding on matrigel, test compounds and VEGF- 165 were
solved in culture medium (+ 0.1 % DMSO) and then pre-incubated with FIBMEC for
lhour before the Abi application (in a final volume per culture well of IOOmI ) . One
hour after test compounds or VEGF incubation (two hours after cell seeding on
matrigel), IOOmI of Abi peptide was added to a final concentration of 2.5 m M diluted
in control medium in presence of test compounds or VEGF (in a 200 m ΐ total
volume/well), in order to avoid further drug dilutions.
Organization of cultures plates
VEGF- 165 known to be a pro-angiogenic isoform of VEGF-A, was used for all
experiment in this study as reference compound. VEGF-165 is one of the most abundant
VEGF isoforms involved in angiogenesis. VEGF was used as reference test compound
at IOhM .
The following conditions were assessed:
• Negative Control medium alone + 0.1% DMSO
• Intoxication: (2.5 m M ) for 18h
• Positive control. VEGF-165 ( IOhM ) ( 1 reference compound/culture) lhr
before the Abi (2.5 m M ) addition for a 18h incubation time.
· Test compounds: Test compound lhr before the Abi (2.5 m M )
addition for a 18h incubation time.
Capillary network quantification
Per well, 2 pictures with 4x lens were taken using InCell AnalyzerTM 1000 (GE
Healthcare) in light transmission. All images were taken in the same conditions.
Analysis of the angiogenesis networks was done using Developer software (GE
Healthcare). The total length of capillary network was assessed.
Data processing
All values are expressed as mean ± s.e. mean of the 3 cultures (n = 6 per condition).
Statistic analyses were done on the different conditions performing an ANOVA
followed by the Dunnett's test when it was allowed (Statview software version 5.0).
The values (as %) inserted on the graphs show the amyloid toxicity evolution. Indeed,
the amyloid toxicity was taken as the 100%> and the test compound effect was calculated
as a % of this amyloid toxicity.
Results
Results are shown in figure 1. They demonstrate that the drugs tested alone, induce a
substantial protective effect against the toxicity caused by A b peptide 1-42:
- Torasemide, at a low dosage of e.g., 400 nM, induces strong protective effect;
- Bromocriptine, at a low dosage of e.g., 3.2 nM, induces strong protective
effect.
The results also show that, unexpectedly, upper or lower drug concentrations in
comparison to the above mentioned drug concentrations, may worsen or rather have less
to no effect on A b toxicity in this model.
1- 2
1.2 Protection against the toxicity of A 4 on primary cortical neuron cells.
Test compound and Human amyloid-pi-42 treatment
Rat cortical neurons were cultured as described by Singer et al. (42). Briefly pregnant
female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar) and
the foetuses were removed from the uterus. The cortex was removed and placed in ice-
cold medium of Leibovitz (L15) containing 2% of Penicillin 10.000 U/ml and
Streptomycin lOmg/ml and 1% of bovine serum albumin (BSA). Cortices were
dissociated by trypsin for 20 min at 37°C (0.05%). The reaction was stopped by the
addition of Dulbecco's modified Eagle's medium (DMEM) containing DNasel grade II
and 10% of foetal calf serum (FCS). Cells were then mechanically dissociated by 3
serial passages through a 10 ml pipette and centrifuged at 515 x g for 10 min at +4°C.
The supernatant was discarded and the pellet of cells was re-suspended in a defined
culture medium consisting of Neurobasal supplemented with B27 (2%), L-glutamine
(0.2mM), 2% of PS solution and lOng/ml of BDNF. Viable cells were counted in a
Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a
density of 30 000 cells/well in 96 well-plates (wells were pre-coated with poly-L-lysine
(K^g/ml)) and were cultured at +37°C in a humidified air (95%)/C02 (5%)
atmosphere.
Briefly, Abi peptide was reconstituted in define culture medium at 40m M (mother
solution) and was slowly shook at +37 °C for 3 days in dark for aggregation. The
control medium was prepared in the same conditions.
After 3 days, the solution was used on primary cortical neurons as follows:
After 10 days of neuron culture, drug was solved in culture medium (+0.1 % DMSO)
and then pre-incubated with neurons for lhour before the Abi application (in a final
volume per culture well of 100 m ΐ) . One hour after drug(s) incubation, IOOmI of Abi
peptide was added to a final concentration of 10 m M diluted in presence of drug(s), in
order to avoid further drug(s) dilutions. Cortical neurons were intoxicated for 24 hours.
Three separate cultures were performed per condition, 6 wells per condition.
BDNF (50ng/ml) and Estradiol- b (150nM) were used as positive control and reference
compounds respectively.
Organization of cultures plates
Estradiol- b at 150nM was used as a positive control.
Estradiol- b was solved in culture medium and pre-incubated for 1 h before the
aggregated amyloid-Pi-42 application.
The following conditions were assessed:
- CONTROL PLAQUE: 12 wells/condition
• Negative Control: medium alone + 0.1% DMSO
• Intoxication: amyloid-Pi-42 (10 m M ) for 24h
• Reference compound: Estradiol (150nM) lhr.
- DRUG PLA TE: 6 wells/condition
• Negative Control: medium alone + 0.1% DMSO
• Intoxication: amyloid-Pi-42 (10 m M ) for 24h
• Drug :Drug - lhr followed by amyloid-Pi-42 (10 m M ) for 24h
Lactate dehydrogenase (LDH) activity assay
24 hours after intoxication, the supernatant was taken off and analyzed with
Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref: 11644793001, batch:
11800300). This colorimetric assay for the quantification of cell toxicity is based on the
measurement of lactate dehydrogenase (LDH) activity released from the cytosol of
dying cells into the supernatant.
Data processing
All values are expressed as mean ± s.e. mean of the 3 cultures (n = 6 per condition).
Statistic analyses were done on the different conditions (ANOVA followed by the
Dunnett's test when it was allowed, Statview software version 5.0).
Results
The results obtained for individual selected drugs in the toxicity assays on primary
cortical neuron cells are presented in figures 2 and 26. They demonstrate that the drugs
tested alone, induce a substantial protective effect against the toxicity caused by A b
peptide 1-42:
Trimetazidine, at a low dosage of e.g., 40 nM, induces strong protective
effect;
- Mexiletine, at a dose as low as 3.2 nM, induces a strong protective effect;
- Bromocriptine, at a dose as low as 40 nM, induces a strong protective effect;
Ifenprodil, at a dose as low as 600 nM, induces a strong protective effect;
- Moxifloxacin, at a dose as low as 20 nM, induces a strong protective effect.
- Torasemide, at a dose of 200 nM, induces a strong protective effect.
- Homotaurine, at a dose of 8 nM, induces a strong protective effect.
The obtained results also show that, unexpectedly, upper or lower drug concentrations
than those indicated above, may worsen or rather have less to no protective effect on A b
toxicity for neuronal cells.
1-42
II. COMBINED THERAPIES PREVENT TOXICITY OF HUMAN A 2
HBMEC cells.
The efficacy of drug combinations of the invention is assessed on human cells. The
protocol which is used in these assays is the same as described in section 1. 1 above.
Results
All of the tested drug combinations give protective effect against toxicity of human Abi .
peptide in HBMEC model, as shown in table 3 below and examplified in figures 3 to
6 and figures 13 and 14. The results clearly show that the intoxication by aggregated
human amyloid peptide (Abi 2.5 m M ) is significantly prevented by combinations of
the invention whereas, at those concentrations, drugs alone have no significant effect on
intoxication in the experimental conditions described above.
Table 3 :
Protective effect
DRUG COMBINATION in A intoxicated
HBMEC cells
Baclofen and Torasemide
Eplerenone and Torasemide
Bromocriptine and Sulfisoxazole
Sulfisoxazole and Torasemide
Terbinafine and Torasemide
Mexiletine and Cinacalcet
Baclofen and Trimetazidine and Torasemide
Baclofen and Cinacalcet and Torasemide
Baclofen and Acamprosate and Torasemide
Sulfisoxazole and Trimetazidine and Torasemide and
Zonisamide
Sulfisoxazole and Mexiletine and Torasemide and
Cinacalcet
Baclofen and Acamprosate and Torasemide and
Diethylcarbamazine
Baclofen and Acamprosate and Torasemide and Ifenprodil
Levosimendan and Baclofen and Trimetazidine
Levosimendan and Aminocaproic acid and Trimetazidine
Levosimendan and Terbinafine and Trimetazidine
Levosimendan and Sulfisoxazole and Trimetazidine
As exemplified in Figures 3 to 6, 13 and 14, the following drug combinations give
particularly interesting protective effects against toxicity of human Abi peptide in
intoxicated HBMEC cells:
- Baclofen and Torasemide,
Sulfisoxazole and Torasemide,
Torasemide and Eplerenone,
Sulfisoxazole and Bromocriptine,
Terbinafine and Torasemide, or
Cinacalcet and Mexiletine.
II.2 Effect of combined therapies on the toxicity of human A 4 peptide on primary
cortical neuron cells.
The efficacy of drug combinations of the invention is assessed on primary cortical
neuron cells. The protocol which is used in these assays is the same as described in
section 1.2 above.
Results
All of the tested drug combinations give protective effect against toxicity of human Abi .
peptide in primary cortical neuron cells, as shown in Table 4 below and exemplified
in figures 7 to 12 and 16 to 22. The results clearly show that the intoxication by
aggregated human amyloid peptide (Abi IOmM ) is significantly prevented by
combinations of the invention whereas, at those concentrations, drugs alone have no
significant effect on intoxication in the experimental conditions described above.
Table 4:
Protective effect
DRUG COMBINATIONS
in A intoxicated primary
cortical neuron cells
Acamprosate and Ifenprodil
Baclofen and Mexiletine
Baclofen and Trimetazidine
Baclofen and Torasemide
Cinacalcet and Mexiletine
Cinnarizine and Trimetazidine
Trimetazidine and Zonisamide
Levosimendan and Mexiletine +
Levosimendan and Ifenprodil
Levosimendan and Trimetazidine +
Levosimendan and Moxifloxacin
Mexiletine and Ifenprodil +
Moxifloxacin and Baclofen
Moxifloxacin and Cinacalcet
Moxifloxacin and Trimetazidine
Moxifloxacin and Sulfisoxazole
Moxifloxacin and Zonisamide
Torasemide and Sulfisoxazole
Baclofen and Trimetazidine and Torasemide
Baclofen and Cinacalcet and Torasemide
Baclofen and Acamprosate and Torasemide
Sulfisoxazole and Trimetazidine and Torasemide and
Zonisamide
Sulfisoxazole and Mexiletine and Torasemide and
Cinacalcet
Baclofen and Acamprosate and Torasemide and
Diethylcarbamazine
Baclofen and Acamprosate and Torasemide and
Ifenprodil
Levosimendan and Baclofen and Trimetazidine
Levosimendan and Aminocaproic acid and Trimetazidine
Levosimendan and Terbinafine and Trimetazidine
Levosimendan and Sulfisoxazole and Trimetazidine
As exemplified in Figures 7 to 12 and 15 to 22, the following drug combinations give
particularly interesting protective effects against toxicity of human Abi peptide in
intoxicated primary cortical neuron cells:
- Acamprosate and Ifenprodil,
Baclofen and Mexiletine,
- Baclofen and Torasemide,
- Baclofen and Trimetazidine,
Cinacalcet and Mexiletine,
- Cinnarizine and Trimetazidine,
Trimetazidine and Zonisamide.
Mexiletine and Ifenprodil,
Moxifloxacin and Baclofen,
- Moxifloxacin and Cinacalcet,
- Moxifloxacin and Trimetazidine,
- Moxifloxacin and Sulfisoxazole,
Moxifloxacin and Zonisamide, or
Torasemide and Sulfisoxazole.
II. 4. Protection of neurite growth against A 1 toxicity.
Test compounds and A b 1-42 treatment
Primary rat cortical neurons are cultured as described previously.
After 10 days of culture, cells are incubated with drugs. After 1 hour, cells are
intoxicated by 2.5 m M of beta-amyloid (1-42; Bachem) in defined medium without
BD but together with drugs. Cortical neurons are intoxicated for 24 hours. BDNF
(lOng/ml) is used as a positive (neuroprotective) control. Three independent cultures
were performed per condition, 6 wells per condition.
Neurites length
After 24 hours of intoxication, the supernatant is taken off and the cortical neurons are
fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min. After
permeabilization with 0.1% of saponin, cells are blocked for 2 h with PBS containing
1% foetal calf serum. Then, cells are incubated with monoclonal antibody anti
microtubule-assiociated-protein 2 (MAP-2; Sigma) This antibody is revealed with
Alexa Fluor 488 goat anti-mouse IgG (Molecular probe). Nuclei of neurons were
labeled by a fluorescent marker (Hoechst solution, SIGMA).
Per well, 10 pictures are taken using InCell AnalyzerTM 1000 (GE Healthcare) with
20x magnification. All pictures are taken in the same conditions. Analysis of the neurite
network is done using Developer software (GE Healthcare) in order to assess the total
length of neurite network.
Results
The combination of Baclofen and Torasemide induces a significant protective effect
against the toxicity of human Abi peptide (improvement of 531% of neurites
network) in primary cortical neuron cells as shown in figure 23. The results clearly
show that the intoxication by human amyloid peptide (Abi 2.5 m M ) is significantly
prevented by the combination and that, moreover, the combination enhances neurite
network in comparison with control.
Hence, this combination allows an effective protection of cortical neuron cells and of
cell neuronal networks against the toxicity of human Abi peptide. Moreover, such an
augmentation of neurites network confirms the efficacy of such drugs in neurological
disorders like spinal cord injury.
III THE COMPOUNDS PREVENT TOXICITYOF HUMAN A IN VIVO
-35
Animals
Male Swiss mice, are used throughout the study. Animals are housed in plastic cages,
with free access to laboratory chow and water, except during behavioural experiments,
and kept in a regulated environment, under a 12 h light/dark cycle (light on at 8:00
a.m.). Behavioral experiments are carried out in a soundproof and air-regulated
experimental room, to which mice have been habituated at least 30 min before each
experiment.
Amyloid peptide preparation and injection
The A peptide and scrambled A b _ peptide have been dissolved in sterile
-35
bidistilled water, and stored at -20°C until use. Light microscopic observation indicated
that incubating the A b _ peptide, but not the scrambled A b _ peptide, led the
2 3 2 3
presence of two types of insoluble precipitates, birefringent fibril-like structures and
amorphous globular aggregates. The b -amyloid peptides are then administered
intracerebroventricularly (i.c.v.). In brief, each mouse is anaesthetized lightly with
ether, and a gauge stainless-steel needle is inserted unilaterally 1 mm to the right of the
midline point equidistant from each eye, at an equal distance between the eyes and the
ears and perpendicular to the plane of the skull. Peptides or vehicle are delivered
gradually within approximately 3 s. Mice exhibit normal behaviour within 1 min after
injection. The administration site is checked by injecting Indian ink in preliminary
experiments. Neither insertion of the needle, nor injection of the vehicle have had a
significant influence on survival, behavioral responses or cognitive functions.
Drug(s) treatment
On day -1, i.e. 24 h before the A b _ peptide injection, drugs, drugs combination or the
vehicle solution are administered per os by gavage twice daily (at 8:00 am and 6:00
pm).
On day 0 (at 10:00 am), mice are injected i.c.v. with A b 25-35 peptide or scrambled A b
-35 peptide (control) in a final volume of 3 m ΐ (3 mM).
Between day 0 and day 7, drugs, drugs combination or the vehicle solution are
administered per os by gavage once or twice daily (at 8:00 am and 6:00 pm). One
animal group receives donepezil (reference compound - 1 mg/kg/day) per os by gavage
in a single injection (at 8:00 am). Drugs are solubilized in water and freshly prepared
just before each gavage administration.
On day 7, all animals are tested for the spontaneous alternation performance in the Y-
maze test, an index of spatial working memory.
On day 7 and 8, the contextual long-term memory of the animals is assessed using the
step-down type passive avoidance procedure.
On day 8, animals are sacrificed. Their brain is dissected and kept at -80°C for further
analysis.
Positive results are observed in behavioral performances and biochemical assays
performed 7 days after A b peptide icv injection, notably for the combinations listed
in table 5 .
Table 5:
Results in biochemical
DRUG COMBINATIONS and/or behavioral
assays
Baclofen and Torasemide
Mexiletine and Cinacalcet
Sulfisoxazole and Torasemide
Baclofen and Trimetazidine and Torasemide
Baclofen and Cinacalcet and Torasemide +
Baclofen and Acamprosate and Torasemide
Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide +
Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet
Baclofen and Acamprosate and Torasemide and Diethylcarbamazine +
Baclofen and Acamprosate and Torasemide and Ifenprodil
Levosimendan and Baclofen and Trimetazidine
Levosimendan and Aminocaproic acid and Trimetazidine
Levosimendan and Terbinafine and Trimetazidine +
Levosimendan and Sulfisoxazole and Trimetazidine
I V COMPOUNDS ENHANCED BEHA VIORAL AND COGNITIVE
PERFORMANCES OF INTOXICATED ANIMALS
Animals are intoxicated as in the above section.
Spontaneous alternation performance -YMaze Test
On day 7, all animals are tested for spontaneous alternation performance in the Y-maze,
an index of spatial working memory. The Y-maze is made of grey polyvinylchloride.
Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top,
and converging at an equal angle. Each mouse is placed at the end of one arm and
allowed to move freely through the maze during an 8 min session. The series of arm
entries, including possible returns into the same arm, are checked visually. An
alternation is defined as entries into all three arms on consecutive occasions. The
number of maximum alternations is therefore the total number of arm entries minus two
and the percentage of alternation is calculated as (actual alternations / maximum
alternations) x 100. Parameters include the percentage of alternation (memory index)
and total number of arm entries (exploration index). Animals that show an extreme
behavior (Alternation percentage < 25% or> 85% or number of arm entries < 10) are
discarded. Usually, it accounts for 0-5% of the animals. This test incidentally serves to
analyze at the behavioral level the impact and the amnesic effect induced in mice by the
A b 25-35 injection.
Passive avoidance test
The apparatus is a two-compartment (15 x 20x 15 cm high) box with one illuminated
with white polyvinylchloride walls and the other darkened with black polyvinylchloride
walls and a grid floor. A guillotine door separates each compartment. A 60 W lamp
positioned 40 cm above the apparatus lights up the white compartment during the
experiment. Scrambled footshocks (0.3 raA for 3 s) could be delivered to the grid floor
using a shock generator scrambler (Lafayette Instruments, Lafayette, USA). The
guillotine door is initially closed during the training session. Each mouse is placed into
the white compartment. After 5 s, the door raises. When the mouse enters the darkened
compartment and places all its paws on the grid floor, the door closes and the footshock
is delivered for 3 s. The step-through latency, that is, the latency spent to enter the
darkened compartment, and the number of vocalizations is recorded. The retention test
is carried out 24 h after training. Each mouse is placed again into the white
compartment. After 5 s the doors is raised, the step-through latency and the escape
latency, i.e. the time spent to return into the white compartment, are recorded up to
300 s.
Positive results are observed for each for the tested combinations listed in Table 6 .
Table 6
V COMPOUNDS OF THE INVENTION IMPROVE NEUROPHYSIOLOGICAL
CONCERN OF NEUROLOGICAL DISEASES
Combinations therapies are tested in the in vivo model of A b intoxication. Their effects
on several parameters which are affected in neurological diseases are assessed:
Caspases 3 and 9 expression level, considered as an indicator of apoptosis,
- Lipid peroxidation, considered as a marker for oxidative stress level,
GFAP expression assay, considered as a marker of the level of brain
inflammation,
Brain Blood Barrier integrity,
Overall synapse integrity (synaptophysin ELISA).
Brain Blood Barrier integrity
Experimental design about animal intoxication by A b is the same that in part III.
The potential protective effect of the combination therapies on the blood brain barrier
(BBB) integrity is analyzed in mice injected intracerebroventricularly (i.c.v.) with
oligomeric amyloid-P25-35 peptide (A b 25-35) or scrambled A b 25-35 control peptide
(Sc.AP), 7 days after injection.
On day 7 after the A b injection, animals are tested to determine the BBB integrity
by using the EB (Evans Blue) method. EB dye is known to bind to serum albumin after
peripheral injection and has been used as a tracer for serum albumin.
EB dye (2% in saline, 4 ml/kg) is injected intraperitoneal (i.p.) 3 h prior to the
transcardiac perfusion. Mice are then anesthetized with i.p. 200 m ΐ of pre-mix ketamine
80 mg/kg, xylazine 10 mg/kg, the chest is opened. Mice are perfused transcardially with
250 ml of saline for approximately 15 min until the fluid from the right atrium becomes
colourless. After decapitation, the brain is removed and dissected out into three regions:
cerebral cortex (left + right), hippocampus (left + right), diencephalon. Then, each brain
region is weighed for quantitative measurement of EB-albumin extravasation.
Samples are homogenized in phosphate-buffered saline solution and mixed by vortexing
after addition of 60% trichloroacetic acid to precipitate the protein. Samples are cooled
at 4°C, and then centrifuged 30 min at 10,000 g, 4°C. The supernatant is measured at
610 nm for absorbance of EB using a spectrophotometer.
EB is quantified both as
g/mg of brain tissue by using a standard curve, obtained by known
concentration of EB-albumin.
g/mg of protein.
As mentioned in table 7, combination therapies of the invention are efficient in
maintaining BBB integrity when compared with non-treated intoxicated animals.
Overall synapse integrity (synaptophysin ELISA)
Synaptophysin has been chosen as a marker of synapse integrity and is assayed using
a commercial ELISA kit (USCN , Ref. E90425Mu). Samples are prepared from
hippocampus tissues and homogenized in an extraction buffer specific to as described
by manufacturer and reference literature.
Tissues are rinsed in ice-cold PBS (0.02 mol/1, pH 7.0-7.2) to remove excess blood
thoroughly and weighed before nitrogen freezing and -80°C storage. Tissues are cut into
small pieces and homogenized in 1ml ice-cold phosphate buffer saline (PBS) solution
with a glass homogenizer. The resulting suspension is sonicated with an ultrasonic cell
disrupter or subjected to two freeze-thawing cycles to further break the cell membranes.
Then, homogenates are centrifugated for 5 min at 5,000 g and the supernatant is assayed
immediately.
All samples are assayed in triplicates.
Quantification of proteins is performed with the Pierce BCA (bicinchoninic acid)
protein assay kit (Pierce, Ref. #23221) to evaluate extraction performance and allow
normalization.
The total protein concentrations are then calculated from standard curve dilutions and
serve to normalize ELISA results.
Results (Table 7) show that combination therapies are efficient in maintaining an
overall Synaptophysin level in brain of treated animals when compared with the non-
treated intoxicated animals.
Oxidative stress assay
Mice are sacrificed by decapitation and both hippocampi are rapidly removed, weighted
and kept in liquid nitrogen until assayed. After thawing, hippocampus are homogenized
in cold methanol (1/10 w/v), centrifuged at 1,000 g during 5 min and the supernatant
placed in eppendorf tube. The reaction volume of each homogenate are added to FeS04
1 mM, H2S04 0.25 M, xylenol orange 1 mM and incubated for 30 min at room
temperature. After reading the absorbance at 580 nm (A580 1), 10 m ΐ of cumene
hydroperoxyde 1 mM (CHP) is added to the sample and incubated for 30 min at room
temperature, to determine the maximal oxidation level. The absorbance is measured at
580 nm (A580 2). The level of lipid peroxidation is determined as CHP equivalents
(CHPE) according to: CHPE = A580 1/A580 2 x [CHP] and expressed as CHP
equivalents per weight of tissue and as percentage of control group data.
Results (Table 7) show that combination therapies are efficient in reducing the overall
oxidative stress induced by A b in brain of treated animals when compared with the non-
treated intoxicated animals.
Caspase pathway induction assay and GFAP expression assay
Mice are sacrificed by decapitation and both hippocampi are rapidly removed, rinsed in
ice-cold PBS (0.02 mol/1, pH 7.0-7.2) to remove excess blood thoroughly weighted and
kept in liquid nitrogen until assayed. Tissues are cut into small pieces and homogenized
in 1ml ice-cold PBS with a glass homogenizer. The resulting suspension is sonicated
with ultrasonic cell disrupter or subjected to two freeze-thawing cycles to further break
the cell membranes. Then, homogenates are centrifugated at 5,000g during 5 min and
the supernatant is assayed immediately.
Experiments are conducted with commercial assay: Caspase-3 (USCN -
E90626Mu), Caspase-9 (USCN - E90627Mu), GFAP (USCN - E90068).
Quantification of proteins is performed with the Pierce BCA (bicinchoninic acid)
protein assay kit (Pierce, Ref. #23221) to evaluate extraction performance and allow
normalization.
Results (Table 7) show that combination therapies have a positive effect on
markers of apoptosis and inflammation in brain of treated animals when compared with
the non-treated intoxicated animals.
Table 7
B) PREVENTION OF GLUTAMATE TOXICITY ON NEURONAL CELLS
In this further set of experiment, candidate compounds have been tested for their
ability to prevent or reduce the toxic effects of glutamate toxicity on neuronal cells.
Glutamate toxicity is involved in the pathogenesis of neurological diseases or disorder
such as Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis,
Parkinson's Disease, Huntington's Disease, neuropathies, alcoholism or alcohol
withdrawal, or spinal cord injury.The drugs are first tested individually, followed by
assays of their combinatorial action.
Methods
The efficacy of drug combinations of the invention is assessed on primary cortical
neuron cells. The protocol which is used in these assays is the same as described in
section A .1.2 above.
Glutamate toxicity assays
The neuroprotective effect of compounds is assessed by quantification of the neurite
network (Neurofilament immunostaining (NF)) which specifically reveals the
glutamatergic neurons.
After 12 days of neuron culture, drugs of the candidate combinations are solved in
culture medium (+0.1% DMSO). Candidate combinations are then pre-incubated with
neurons for 1 hour before the Glutamate injury. One hour after incubation with,
Glutamate is added for 20 min, to a final concentration of 40m M , in presence of
candidate combinations, in order to avoid further drug dilutions. At the end of the
incubation, medium is changed with medium with candidate combination but without
glutamate. The culture is fixed 24 hours after glutamate injury. MK801
(Dizocilpinehydrogen maleate, 770867 - 20uM) is used as a positive control.
After permeabilization with saponin (Sigma), cells are blocked for 2h with PBS
containing 10% goat serum, then the cells are incubated with mouse monoclonal
primary antibody against Neurofilament antibody (NF, Sigma). This antibody is
revealed with Alexa Fluor 488 goat anti-mouse lgG.
Nuclei of cells are labeled by a fluorescent marker (Hoechst solution, SIGMA), and
neurite network quantified. Six wells per condition are used to assess neuronal survival
in 3 different cultures.
Results
All of the tested drug combinations give a protective effect against glutamate toxicity
for cortical neuronal cells. Results are shown in Table 8 below.
As exemplified in figures 24 and 25, combinations of the invention strongly protect
neurons from glutamate toxicity under experimental conditions described above. It is
noteworthy that an effective protection is noticed using drug concentrations at which
drugs used alone have no significant or lower protective effect.
Table 8
C) IMPROVEMENT OF OTHER DISORDERS RELATED TO
GLUTAMATE EXCITOXICITY USING COMBINATIONS OF THE
INVENTION
The above mentioned in vitro protective effect against glutamate toxicity of drugs and
drug combinations of the invention combined with the protective effects exemplified
herein in several AD models, prompted the inventors to test these drugs and
combinations in some models of other diseases in the pathogenesis of which glutamate
toxicity is also involved, as MS, ALS and neuropathic pain.
I) PROTECTIVE EFFECT OF COMBINATIONS IN AN IN VIVO MODEL OF
MULTIPLE SCLEROSIS.
A model in which myelin-oligodendrocyte glycoprotein-immunized (MOG-
immunized) mice develop chronic progressive EAE is used to demonstrate the
beneficial effect of compositions of the invention in multiple sclerosis treatment.
Animals and chemicals
C57L/6J female mice (8 weeks old) are purchased from Janvier (France); after two
weeks of habituation, female mice (10 weeks old) develop chronic paralysis after
immunization with MOG (Myelin Oligodendrocyte Glycoprotein) peptide. The
experimental encephalomyelitis is induced with the Hooke Kit MOG -55/CFA
Emulsion PTX (Pertussis toxin) for EAE Induction (EK-01 10, EK-01 15; Hooke
laboratories). The control kit is CK-01 15 (Hooke laboratories).
Experimentalprocedure
The experimental encephalomyelitis is induced by following procedure:
The day 0, two subcutaneous injections of 0.1 ml each are performed; one on upper
back of the mouse and one in lower back. Each injection contains 100 g of MOG -55
peptide (MEVGWYRSPF SRVVHLYRNGK), 200 g of inactivated Mycobacterium
tuberculosis H37Ra and is emulsified in Complete Freund's adjuvant (CFA) (Hooke
laboratories). The emulsion provides antigen needed to expand and differentiate MOG-
specific autoimmune T cells.
Two intraperitoneal injections of 500 ng of Pertussis toxin in PBS (Hooke kit) are
performed 2 hours (Day 0) and 24 hours (Day 1) after the MOG injection. Pertussis
toxin enhances EAE development by providing additional adjuvant.
Mice develop EAE 8 days after immunization and stay chronically paralyzed for the
duration of the experiment. After the immunization, mice are daily observed for clinical
symptoms in a blind procedure. Animals are kept in a conventional pathogen-free
facility and all experiments are carried out in accordance with guidelines prescribed by,
and are approved by, the standing local committee of bioethics.
Experimental groups and drug treatment:
Groups of female mice as disclosed are homogenized by weight before the
immunization:
Control group: vehicle injection in the same conditions of EAE mice (from Day
- 1 to Day 28, placebo is given daily)
- EAE group: MOG injection (day 0) + Pertussis toxin injections (Day 0 and 1) -
from Day - 1 to Day 28, placebo is given orally daily
- EAE + positive control: MOG injection (Day 0) + Pertussis toxin injections(Day
0 and 1) - from Day - 1 to Day 28, dexamethazone is given orally daily.
- EAE + treatment group: MOG injection (Day 0) + Pertussis toxin injections
(Day 0 and 1). The treatments start one Day before immunization and last until
Day 28.
The clinical scores are measured at Days 0812162126-28.
Statistica software (Statsoft Inc.) is utilized throughout for statistical analysis. ANOVA
analysis and Student's t test are employed to analyse clinical disease score. P < 0.05 is
considered significant.
Delays of disease occurrence, clinical score and delay of death, have been compared
between each group to the reference 'immu" group with Kaplan-Meier curves and a Cox
model (R package 'survival'). Resulting -values are unilateral and test the hypothesis
to be better than the reference 'immu' group.
The total clinical score is composed of the tail score, the hind limb score, the fore limb
score and the bladder score described as below:
Tail score:
Score=0 A normal mouse holds its tail erect when moving.
Score=l If the extremity of the tail is flaccid with a tendency to fall.
Score=2 If the tail is completely flaccid and drags on the table.
Hind limbs score:
Score=0 A normal mouse has an energetic walk and doesn't drag his paws
Score=l Either one of the following tests is positive:
A - Flip test: while holding the tail between thumb and index finger, flip the
animal on his back and observe the time it takes to right itself. A healthy
mouse will turn itself immediately. A delay suggests hind-limb weakness.
B - Place the mouse on the wire cage top and observe as it crosses from one
side to the other. If one or both limbs frequently slip between the bars we
consider that there is a partial paralysis.
Score=2 Both previous tests are positive.
Score=3
One or both hind limbs show signs of paralysis but some movements are
preserved; for example: the animal can grasp and hold on to the underside of
the wire cage top for a short moment before letting go.
Score=4 When both hind legs are paralyzed and the mouse drags them when moving.
Fore limbs score:
Score=0 A normal mouse uses its front paws actively for grasping and walking and
holds its head erect.
Score=l Walking is possible but difficult due to a weakness in one or both of the
paws, for example, the front paws are considered weak when the mouse has
difficulty grasping the underside of the wire top cage. Another sign of
weakness is head drooping.
Score=2 When one forelimb is paralyzed (impossibility to grasp and the mouse turns
around the paralyzed limb). At this time the head has also lost much of its
muscle tone.
Score=3 Mouse cannot move, and food and water are unattainable.
Bladder score:
Score=0 A normal mouse has full control of its bladder.
Score=l A mouse is considered incontinent when its lower body is soaked with urine.
The global score for each animal is determined by the addition of all the above
mentioned categories. The maximum score for live animals is 10.
Results-Combinations therapies are efficient in a MS model
A significant improvement of global clinical score is observed in "EAE+ treatment
group" mice, notably for the combinations listed in Table 9 .
Table 9
Improvement of the global clinical
Drug Combination
score in EAE anaimals
Baclofen-Torasemide
Baclofen-Acamprosate-Torasemide
Mexiletine and Cinacalcet
Sulfisoxazole and Torasemide
II PROTECTIVE EFFECT OF COMBINATIONS IN MODELS OFALS.
Combination therapies according to the present invention are tested in vitro, in a
coculture model, and in vivo, in a mouse model of ALS. Protocols and results are
presented in this section.
I l Protective effect against glutamate toxicity in primary cultures of nerve-muscle
co-culture
Primary cocultures of nerve-and muscle cells
Human muscle is prepared according to a previously described method from portions of
biopsie of healthy patient (44). Muscle cells are established from dissociated cells
(10000 cells per wells), plated in gelatin-coated 0.1% on 48 wells plate and grown in a
proliferating medium consisting of mix of MEM medium and M l 99 medium.
Immediately after satellite cells fusion, whole transverse slices of 13-day-old rat Wistar
embryos spinal cords with dorsal root ganglia (DRG) attached are placed on the muscle
monolayer 1 explant per well (in center area). DRG are necessary to achieve a good
ratio of innervations. Innervated cultures are maintained in mix medium. After 24h in
the usual co-culture neuritis are observed growing out of the spinal cord explants. They
make contacts with myotubes and induce the first contractions after 8 days. Quickly
thereafter, innervated muscle fibres located in proximity to the spinal cord explants, are
virtually continuously contracting. Innervated fibres are morphologically and spatially
distinct from the non-innervated ones and could easily be distinguished from them.
One co-culture is done (6 wells per conditions).
Glutamate injury
On day 27, co-cultures are incubated with candidate compounds or Riluzole one hour
before glutamate intoxication (60 m M ) for 20 min. Then, co-cultures are washed and
candidate compounds or Riluzole are added for an additional 48h. After this incubation
time, unfixed cocultures are incubated with a-bungarotoxin coupled with Alexa 488 at
concentration 500 nmol/L for 15 min at room temperature. Then, cocultures fixed by
PFA for 20 min at room temperature. After permeabilization with 0.1% of saponin, co-
cultures are incubated with anti-neurofilament antibody (NF).
These antibodies are detected with Alexa Fluor 568 goat anti-mouse IgG (Molecular
probe). Nuclei of neurons are labeled by a fluorescent marker (Hoechst solution).
Endpoints are (1) Total neurite length, (2) Number of motor units, (3) Total motor unit
area, which are indicative of motorneurone survival and functionality.
For each condition, 2 10 pictures per well are taken using InCell AnalyzerTM 1000
(GE Healthcare) with 20x magnification. All the images are taken in the same
conditions.
Results
Drugs of the invention effectively protect motorneurones and motor units in the
coculture model. Moreover an improvement of the protection is noticed when drugs are
used in combination for the drug combinations listed in table 10.
Table 10
II.2 —Combinations therapies are efficient in ALS Mouse model
Experiments are performed on male mice. Transgenic male mice B6SJL-
Tg(SODl)2Gur/J mice and their control (respectively SN2726 and SN2297 from
Jackson Laboratories, Ben Harbor, USA and distributed by Charles River in France) are
chosen in this set of experiments to mimic ALS.
Diseased mice express the SOD1-G93A transgene, designed with a mutant human
SODl gene (a single amino acid substitution of glycine to alanine at codon 93) driven
by its endogenous human SODl promoter. Control mice express the control human
SODl gene.
Drug administration
Mice are dosed with candidate drug treatment diluted in vehicle from 60th day after
birth till death. Diluted solutions of drug candidates are prepared with water at room
temperature just before the beginning of the administration.
· In drinking water :
Riluzole is added in drinking water at a final concentration of 6mg/ml (adjusted to each
group mean body weight) in 5% cyclodextrin. As a mouse drinks about 5 ml/day, the
estimated administrated dose is 30mg/kg/day which is a dose that was shown to increase
the survival of mice.
- Cyclodextrine is used as vehicle at the final concentration of 5 %, diluted in water at
room temperature from stock solution (cyclodextrin 20%).
• Oral administration (per os) :
- Drug combinations are administrated per os, daily.
- Cyclodextrine is used as vehicle at the final concentration of 5 %, diluted in water at
room temperature from stock solution (cyclodextrin 20%).
Clinical observation
The clinical observation of each mouse is performed daily, from the first day of
treatment (60 days of age) until the death (or sacrifice). Clinical observation consists in
studying behavioural tests: onset of paralysis, "loss of splay", "loss of righting reflex",
and general gait observation:
- Onset of paralysis: The observation consists of paralysis observation of each limb.
Onset of paralysis corresponds to the day of the first signs of paralysis.
- The loss of splay test consists of tremors or shaking notification and the position of
hind limb (hanging or splaying out) when the mouse is suspended by the tail.
- The loss of righting reflex test evaluates the ability of the mouse to right itself within
sec of being turned on either side. The righting reflex is lost when the mouse is
unable to right itself. The loss of righting reflex determines the end stage of disease: the
mouse unable to right itself is euthanized.
Results-Combinations therapies are efficient in ALS in vivo model
An improvement of the disease is observed for the diseased animals treated with the
drugs and drug combinations of the invention. Notably, drugs combinations listed in
Table 11 efficiently improve clinical score of these animals during the different stage of
the disease.
Table 11 :
III) PROTECTIVE EFFECT OF COMBINATIONS OF THE INVENTION IN
OXALIPLATINE INDUCED NEUROPATHY AS AN IN VIVO MODEL FOR
NEUROPATHIC PAIN
Combinatorial therapies of the present invention are tested in vivo, in suitable models of
peripheral neuropathy, i.e., acute model of oxaliplatin-induced neuropathy and chronic
model of oxaliplatin-induced neuropathy. The animals, protocols and results are
presented in this section.
Animal Husbandry
Sprague-Dawley rats (CERJ, France), weighing 150 - 175 g at the beginning of the
experimental of the Oxaliplatin treatment (D ) are used. Animals are housed in a limited
access animal facility in a temperature (19.5°C - 24.5°C) and relative humidity (45 % -
65 %) controlled room with a 12 h - light/dark cycle, with ad libitum access to standard
pelleted laboratory chow and water throughout the study. Animals are housed 4 or 5 per
cage and a one week-acclimation period is observed before any testing.
Experimental design
Four following groups of rats are used in all experiments:
Control groups :
Group 1 : Vehicle of Oxaliplatin (distilled water), i.p. / Vehicle of candidate
combination(s) (Distilled water), p.o. daily.
Group 2 : Oxaliplatin (distilled water), i.p. / Vehicle of candidate combination(s)
(Distilled water), p.o. daily.
Group 3 : Oxaliplatin 3 mg/kg i.p. / single drug in Distilled water, p.o. daily x 9 .
Tested composition groups :
Group 4 : Oxaliplatin 3 mg/kg i.p. / candidate combination(s) in Distilled water, p.o.
daily x 9 .
Group 5 : Oxaliplatin 3 mg/kg i.p. / Gabapentin (100 mg/kg) in Distilled water, p.o. on
testing days (i.e. Di & D );
Vehicle and test items are delivered daily from D-1 to D7 (the day before the last testing
day) whereas Gabapentin is administered on testing days (120 minutes before the test).
All treatments are administered in a coded and random order when it is possible. Doses
are expressed in terms of free active substance.
Neuropathy induction
Acute neuropathy is induced by a single intraperitoneal injection of oxaliplatin
(3 mg/kg).
Chronic peripheral neuropathy is induced by repeated intraperitoneal injections of
oxaliplatin (3 mg/kg, i.p.) on days 0, 2, 4 and 7 (CD= 12 mg/kg, i.p.). Chronic
neuropathy in humans is cumulative as well and is most commonly seen in patients who
have received total doses of oxaliplatin > or =540 mg/m which corresponds to -15
mg/kg as cumulative dose in rats (Cersosimo R.J. 2005).
The oxaliplatin-induced painful neuropathy in rat reproduces the pain symptoms in
oxaliplatin-treated patients:
The thermal hyperalgesia is the earliest symptom. It can be measured with the
acetone test or with the tail-immersion test;
- The mechanical hyperalgesia appears later. It can be quantified with the Von
Frey test or the paw pressure test.
Animal dosing and testing
All drug combinations are administered from the day before the first intraperitoneal
injection of oxaliplatin 3 mg/kg (D-l) and pursued daily orally until D7. During the
testing days (i.e. D l and D7), the drug combinations are administered after the test.
Animals from the reference-treated group (gabapentin) are dosed only during the testing
days.
Acetone test
Cold allodynia is assessed using the acetone test by measuring the responses to thermal
non-nociceptive stimulation on D l (around 24h after the first injection of oxaliplatin 3
mg/kg (acute effect of oxaliplatin), and D8 (chronic effect of oxaliplatin).
In the acetone test, latency of hindpaw withdrawal is measured after application of a
drop of acetone to the plantar surface of both hindpaws (reaction time) and the intensity
of the response is scored (cold score). Reaction time to the cooling effect of acetone is
measured within 20 sec (cut-off) after acetone application. Responses to acetone are
also graded to the following 4-point scale: 0 (no response); 1 (quick withdrawal, flick of
the paw); 2 (prolonged withdrawal or marked flicking of the paw); 3 (repeated flicking
of the paw with licking or biting).
For each experimental group, results are expressed as:
- The reaction time defined as the time expressed in sec required to elicit paw reaction
(mean of 6 measures for each rat together ± SEM).
- The cumulative cold score defined as the sum of the 6 scores for each rat together ±
SEM. The minimum score being 0 (no response to any of the 6 trials) and the maximum
possible score being 18 (repeated flicking and licking or biting of paws on each of the
six trials).
Statistical analyses
Student test, unilateral, type 3 is performed. The significance level is set as p < 0.05; all
the groups are compared to the diseased+vehicle group (oxaliplatin treated group).
Means and standard error mean are shown on the figures.
Results
Oxaliplatin induced a significant decrease in reaction time of paw withdrawal after
acetone application (diseased group + vehicle) during the time course. This decrease is
progressive and significant from day 1 (acute model of oxaliplatin-induced neuropathy)
to day 8 (chronic model) as compared to the vehicle group.
• Anti-allodynic effect in acute model of oxaliplatin-induced neuropathy
The drug combinations tested in acute model of oxaliplatin-induced neuropathy are
assessed with acetone test. Table 12 presents drug combinations (Group 4) which
induce a significant decrease in the cumulative cold score and a significant increase of
reaction time as compared to the oxaliplatin-vehicle treated group (Group 2). In
conclusion, these drug combinations protect animals from acute neuropathy induced by
oxaliplatin.
Table 12
+ = anti-allodynic effect obtained in Group 4 of rats, following analysis of the
cumulative cold scores and analysis of the reaction time in acetone tests, in acute
oxaliplatin-induced model.
• Anti-allodynic effect in chronic model of oxaliplatin-induced neuropathy
The drug combinations used in chronic model of oxaliplatin-induced neuropathy are
assessed with acetone test.
Table 13 presents drug combinations, for which, the reaction time and the cold score in
acetone test measured in the Group 4 (animals treated with drug combinations and
oxaliplatin) are respectively significantly increased and decreased after the treatment in
chronic model of neuropathy compared to the oxaliplatin-vehicle treated group (Group
2). In conclusion, these drug combinations protect animals from chronic neuropathy induced
by oxaliplatin.
Table 13
Drug combinations tested in Chronic Variation of the cold score Reaction time Anti-allodynic
model of neuropathy (Group 4) compared to Group 2 compared to Group 2 effect
decrease increase +
Baclofen-Torasemide
Baclofen-Acamprosate- decrease increase +
Torasemide
Mexiletine and Cinacalcet
decrease increase +
Sulfisoxazole and Torasemide
decrease increase +
+ = anti-allodynic effect obtained in Group 4 of rats, following analysis of the
cumulative cold scores and analysis of the reaction time in acetone tests, in chronic
oxaliplatin-induced model.
The reference in this specification to any prior publication (or information derived from
it), or to any matter which is known, is not, and should not be taken as an acknowledgment
or admission or any form of suggestion that that prior publication (or information derived
from it) or known matter forms part of the common general knowledge in the field of
endeavour to which this specification relates.
Throughout this specification and the claims which follow, unless the context requires
otherwise, the word "comprise", and variations such as "comprises" and "comprising", will
be understood to imply the inclusion of a stated integer or step or group of integers or steps
but not the exclusion of any other integer or step or group of integers or steps.
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Claims (4)
1. Use of a composition comprising Torasemide, or a salt, or sustained release formulation thereof, in the manufacture of a medicament for the treatment of Alzheimer’s disease (A D) or an AD related disorder, amyotrophic lateral sclerosis (A LS) or spinal cord injury ( S CI) in a subject in need thereof.
2. Use of claim 1, wherein said composition further comprises at least one distinct compound selected from Sulfisoxazole, Methimazole, Prilocaine, Dyphylline, Quinacrine, Carbenoxolone, Acamprosate, Aminocaproic acid, Baclofen, Cabergoline, Diethylcarbamazine, Cinacalcet, Cinnarizine, Eplerenone, Fenoldopam, Leflunomide, Levosimendan, Sulodexide, Terbinafine, Zonisamide, Etomidate, Phenformin, Trimetazidine, Mexiletine, Ifenprodil, Moxifloxacin or Bromocriptine, or a salt, or sustained release formulation thereof.
3. Use of claim 2, wherein said composition comprises at least one of the following combinations of compounds: Baclofen and Torasemide, Sulfisoxazole and Torasemide, Eplerenone and Torasemide, Terbinafine and Torasemide, Baclofen and Cinacalcet and Torasemide, Sulfisoxazole and Trimetazidine and Torasemide and Zonisamide, Sulfisoxazole and Mexiletine and Torasemide and Cinacalcet, Baclofen and Acamprosate and Torasemide and Diethylcarbamazine, or Baclofen and Acamprosate and Torasemide and Ifenprodil.
4. Use of any one of the preceding claims, wherein said composition further comprises a pharmaceutically acceptable carrier or excipient.
Applications Claiming Priority (5)
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EP11305217 | 2011-03-01 | ||
EP11305217.9 | 2011-03-01 | ||
US201161468658P | 2011-03-29 | 2011-03-29 | |
US61/468,658 | 2011-03-29 | ||
PCT/EP2012/053565 WO2012117073A2 (en) | 2011-03-01 | 2012-03-01 | New compositions for treating neurological disorders |
Publications (2)
Publication Number | Publication Date |
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NZ614267A NZ614267A (en) | 2015-07-31 |
NZ614267B2 true NZ614267B2 (en) | 2015-11-03 |
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