U.S. patent application number 16/694231 was filed with the patent office on 2020-03-19 for baclofen and acamprosate based therapy of neurological disorders.
The applicant listed for this patent is PHARNEXT. Invention is credited to ILYA CHUMAKOV, DANIEL COHEN, MICKAEL GUEDJ, SERGUEI NABIROCHKIN, EMMANUEL VIAL.
Application Number | 20200085790 16/694231 |
Document ID | / |
Family ID | 46758335 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200085790 |
Kind Code |
A1 |
COHEN; DANIEL ; et
al. |
March 19, 2020 |
BACLOFEN AND ACAMPROSATE BASED THERAPY OF NEUROLOGICAL
DISORDERS
Abstract
The present invention relates to combinations and methods for
the treatment of neurological disorders related to glutamate
excitotoxicity and Amyloid .beta. toxicity. More specifically, the
present invention relates to novel combinatorial therapies of
Alzheimer's disease, Alzheimer's disease related disorders,
amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's
disease, Huntington's disease, neuropathic pain, alcoholic
neuropathy, alcoholism or alcohol withdrawal, or spinal cord
injury, based on baclofen and acamprosate combination.
Inventors: |
COHEN; DANIEL; (SAINT CLOUD,
FR) ; CHUMAKOV; ILYA; (VAUX-LE-PENIL, FR) ;
NABIROCHKIN; SERGUEI; (CHATENAY-MALABRY, FR) ; VIAL;
EMMANUEL; (NICE, FR) ; GUEDJ; MICKAEL; (PARIS,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHARNEXT |
ISSY LES MOULINEAUX |
|
FR |
|
|
Family ID: |
46758335 |
Appl. No.: |
16/694231 |
Filed: |
November 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15494732 |
Apr 24, 2017 |
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16694231 |
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14861169 |
Sep 22, 2015 |
9636316 |
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15494732 |
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14479614 |
Sep 8, 2014 |
9144558 |
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14861169 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/10 20180101; A61K
31/13 20130101; A61K 31/27 20130101; A61P 43/00 20180101; A61K
9/0053 20130101; A61K 31/137 20130101; A61K 31/185 20130101; A61K
31/164 20130101; A61K 31/195 20130101; A61K 31/185 20130101; A61K
31/42 20130101; A61K 31/64 20130101; A61K 31/145 20130101; A61K
31/195 20130101; A61K 31/42 20130101; A61K 31/64 20130101; A61P
25/16 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/4045 20130101; A61K 31/137 20130101; A61K 31/197
20130101; A61K 31/138 20130101; A61K 31/44 20130101; A61K 2300/00
20130101; A61K 31/138 20130101; A61P 21/02 20180101; A61K 31/27
20130101; A61K 31/445 20130101; A61K 31/325 20130101; A61K 31/13
20130101; A61K 31/445 20130101; A61K 31/55 20130101; A61P 29/00
20180101; A61P 21/00 20180101; A61P 25/28 20180101; A61K 9/2004
20130101; A61K 45/06 20130101; A61K 31/197 20130101; A61K 31/428
20130101; A61P 25/00 20180101; A61K 31/44 20130101; A61K 31/55
20130101; A61P 25/14 20180101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/4045 20060101
A61K031/4045; A61K 31/197 20060101 A61K031/197; A61K 31/445
20060101 A61K031/445; A61K 45/06 20060101 A61K045/06; A61K 31/13
20060101 A61K031/13; A61K 31/27 20060101 A61K031/27; A61K 31/55
20060101 A61K031/55; A61K 31/185 20060101 A61K031/185; A61K 31/195
20060101 A61K031/195; A61K 31/325 20060101 A61K031/325; A61K 9/00
20060101 A61K009/00 |
Claims
1. A composition comprising (i) baclofen, (ii) acamprosate, and
(iii) idalopirdine, or pharmaceutically acceptable salts,
derivatives or prodrugs thereof.
2. The composition of claim 1, wherein the compounds are in
admixture with a pharmaceutically acceptable carrier or
excipient.
3. The composition of claim 1, which comprises a dose of baclofen
of less than 150 mg.
4. The composition of claim 1, which comprises a dose of
acamprosate of less than 1000 mg.
5. The composition of claim 1, which comprises a dose between 0.4
mg and 50 mg of acamprosate and between 6 and 15 mg of
baclofen.
6. The composition of claim 1, which comprises baclofen,
acamprosate and idalopirdine as the only active agents.
7. The composition of claim 1, which is a solid formulation
suitable for oral administration.
8. A method for treating Alzheimer in a human subject in need
thereof, comprising administering to said subject and effective
amount of baclofen, acamprosate and idalopirdine, or pharmaceutical
acceptable salt(s) or derivative(s) thereof.
9. The method of claim 8, wherein the compounds are in admixture
with a pharmaceutically acceptable carrier or excipient.
10. The method of claim 8, wherein baclofen, or the
pharmaceutically acceptable salt or derivative thereof, is
administered at a dose of less than 150 mg.
11. The method of claim 8, wherein acamprosate, or the
pharmaceutically acceptable salt or derivative thereof, is
administered at a dose of less than 1000 mg.
12. The method of claim 8, wherein acamprosate, or the
pharmaceutically acceptable salt or derivative thereof, is
administered at a dose between 0.4 mg and 50 mg, and baclofen, or
the pharmaceutically acceptable salt or derivative thereof, is
administered at a dose between 6 mg and 15 mg, twice daily.
13. The method of claim 8, wherein baclofen, acamprosate and
idalopirdine, or the pharmaceutically acceptable salt(s) or
derivative(s) thereof, are the only agents administered for
treating Alzheimer disease.
14. The method of claim 8, wherein baclofen, acamprosate and
idalopirdine, or the pharmaceutically acceptable salt(s) or
derivative(s) thereof, are formulated or administered together,
separately or sequentially.
15. The method of claim 8, wherein baclofen, acamprosate and
idalopirdine, or the pharmaceutically acceptable salt(s) or
derivative(s) thereof, are administered orally.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to combinations and methods
for the treatment of neurological diseases and disorders. More
specifically, the present invention relates to novel combinatorial
therapy of neurological disorders, based on baclofen and
acamprosate combination.
BACKGROUND OF THE INVENTION
[0002] 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. Special symptoms
such as spastic paraparesis (weakness affecting the lower
extremities) can also be involved [1-4].
[0003] Incidence of Alzheimer's disease increases dramatically with
the age. 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]
[0004] United Nations 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].
[0005] 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. By contrast, working memory (short-term memory
involving structures and processes used for temporarily storing and
manipulating information) and procedural memory (unconscious memory
that is long-term memory of skills and procedure) are preserved
until late. As the disease progresses, the additional features of
language impairment, visual perceptual and spatial deficits,
agnosias and apraxias emerge.
[0006] The classic picture of Alzheimer's disease is sufficiently
characteristic to allow identification in approximately 80% of
cases [7]. Nevertheless, clinical heterogeneity does occur and this
is not only important for clinical management but provides further
implication of specific medication treatments for functionally
different forms [8].
[0007] The pathological hallmark of AD includes amyloid plaques
containing beta-amyloid (Abeta), neurofibrillary tangles (NFT)
containing Tau and neuronal and synaptic dysfunction and loss
[9-11]. 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) [12], and the "neuronal cytoskeletal degeneration
hypothesis" [13], which proposes that cytoskeletal changes are the
triggering events. The most widely accepted theory explaining AD
progression remains the amyloid cascade hypothesis [14-16] 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 (BBB) breakdown have been identified as key
events contributing to the APP toxicity in the amyloid cascade
[17]. On the 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 better 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 [11]. 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 receptor overactivation [18].
Abnormal accumulation of glutamate in synaptic clefts 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.
[0008] 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
frontotemporal dementia, multiple sclerosis, Alzheimer's disease,
amyotrophic lateral sclerosis, Parkinson's disease, and
Huntington's disease [19-21]. The development of efficient
treatment for these diseases remains a major public health issue
due to their incidence as well as lack of curative treatments.
[0009] Two kinds of medication are used for improving or slowing
down symptoms of AD which lay on some acetylcholinesterase
modulators and a blocker of NMDA glutamate receptors, memantine
[22, 23].
[0010] 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 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's disease (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 of inhibiting the presynaptic release of glutamate.
Riluzole, currently approved in amyotrophic lateral sclerosis,
showed encouraging results in ischemia and traumatic brain injury
models [24-26]. 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. The use of NMDA receptor antagonists such as memantine,
felbamate, acamprosate and MRZ 2/579 for treating depression has
also been suggested in US2010076075.
[0011] WO2009133128, WO2009133141, WO2009133142 and WO2011054759
disclose drug combinations for use in the treatment of AD.
[0012] 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
[0013] It is an object of the present invention to provide new
therapeutic methods and compositions for treating neurological
disorders. More particularly, the invention relates to compositions
and methods for treating neurological disorders related to
glutamate and/or amyloid beta (A.beta.) toxicity, based on a
combination of baclofen and acamprosate.
[0014] The invention stems, inter alia, from the unexpected
discovery, by the inventors, that the combination of baclofen and
acamprosate provides substantial and unexpected benefit to patients
with Alzheimer's disease. Moreover, the inventors have surprisingly
discovered that this combination provides substantial and
unexpected protection of neuronal cells against various injuries
encountered in neurological disorders including glutamate toxicity.
Thus, this combination of baclofen and acamprosate constitutes an
efficient treatment for patients suffering from, predisposed to, or
suspected to suffer from neurological disorders. The inventors have
found that combinations of the invention are efficient in
counteracting the toxic cellular effects of A.beta. peptides and in
correcting the cognitive impairments in relation with such
toxicity. The invention further demonstrates that baclofen and
acamprosate administration does improve memory functions in aged
subjects as well as cognitive functions, in particular by
correcting cognition-related electro-physiological features in mild
AD patients.
[0015] An object of this invention therefore relates to
compositions comprising a combination of baclofen and acamprosate
for use in the treatment of a neurological disorder, particularly
AD and related disorders, multiple sclerosis (MS), amyotrophic
lateral sclerosis (ALS), Parkinson's disease (PD), neuropathies
(for instance neuropathic pain or alcoholic neuropathy),
frontotemporal dementia (FTD), alcoholism or alcohol withdrawal,
Huntington's disease (HD) and spinal cord injury.
[0016] The compositions of the invention may contain baclofen and
acamprosate as the only active ingredients. Alternatively, the
compositions may comprise additional active ingredient(s). In this
regard, a further object of this invention relates to a composition
comprising a combination of baclofen, acamprosate, and at least one
third compound selected from sulfisoxazole, methimazole,
prilocaine, dyphylline, quinacrine, carbenoxolone, aminocaproic
acid, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine,
eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide,
terbinafine, zonisamide, etomidate, phenformin, trimetazidine,
mexiletine, ifenprodil, moxifloxacin, bromocriptine or torasemide,
for use in the treatment of neurological disorders in a subject in
need thereof.
[0017] As it will be further disclosed in the present application,
the compounds in a combinatorial therapy of the invention may be
administered simultaneously, separately, sequentially and/or
repeatedly to the subject.
[0018] The invention also relates to any pharmaceutical composition
per se comprising a combination of at least two compounds as
defined above.
[0019] The compositions of the invention typically further comprise
one or several pharmaceutically acceptable excipients or carriers.
Also, the compounds as used in the present invention may be in the
form of a salt, hydrate, ester, ether, acid, amide, racemate, or
isomer. They may also be in the form of sustained-release
formulations. Prodrugs or derivatives of the compounds may be used
as well.
[0020] In a preferred embodiment, a compound is used as such or in
the form of a salt, hydrate, ester, ether or sustained release form
thereof. A particularly preferred salt for use in the present
invention is acamprosate calcium.
[0021] In another preferred embodiment, a prodrug or derivative is
used.
[0022] A further object of this invention is a method of preparing
a pharmaceutical composition, the method comprising mixing baclofen
and acamprosate, in a pharmaceutically acceptable excipient or
carrier.
[0023] Another object of this invention relates to a method for
treating a neurological disorder in a mammalian subject in need
thereof, preferably a human subject in need thereof, the method
comprising administering to said subject an effective amount of a
combination of the invention.
[0024] A further object of this invention relates to a method for
treating Alzheimer's disease (AD) or a related disorder in a
mammalian subject in need thereof, preferably a human subject in
need thereof, the method comprising administering to said subject
an effective amount of a combination of the invention.
[0025] A preferred object of this invention relates to a method for
treating a neurological disorder in a mammalian subject in need
thereof, preferably a human subject in need thereof, the method
comprising simultaneously, separately or sequentially administering
to said subject an effective amount of baclofen and
acamprosate.
[0026] A particular object of this invention relates to a method of
treating Frontotemporal Dementia (FTD) in a mammalian subject in
need thereof, preferably a human subject in need thereof, the
method comprising administering to said subject an effective amount
of a combination of the invention.
[0027] A more preferred object of this invention relates to a
method for treating AD or a related disorder in a mammalian subject
in need thereof, preferably a human subject in need thereof, the
method comprising simultaneously, separately or sequentially
administering to said subject an effective amount of baclofen and
acamprosate.
[0028] Another particular object of this invention relates to a
method of treating Age Associated Memory Impairment (AAMI) in a
mammalian subject in need thereof, preferably a human subject in
need thereof, the method comprising administering to said subject
an effective amount of a combination of the invention.
[0029] The invention may be used for treating a neurological
disorder in any mammalian subject, preferably in any human subject,
at any stage of the disease. As it will be disclosed in the
examples, the compositions of the invention are able to ameliorate
the pathological condition of said subjects.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1: Validation of the experimental model of human
beta-amyloid toxicity on endothelial cells used for drug screening.
One hour of VEGF pre-treatment at 10 nM significantly protected the
capillary network from this amyloid injury (+70% of capillary
network compared to amyloid intoxication).
[0031] FIGS. 2A-2C: Effect of baclofen (BCL) and acamprosate (ACP)
combination therapy on the total length of capillary network in
beta-amyloid intoxicated HBMEC cultures. The human amyloid peptide
(A.beta..sub.1-42 2.5 .mu.M) produces a significant intoxication,
above 40%, compared to vehicle-treated cells. This intoxication is
significantly prevented by the combination of acamprosate and
baclofen (FIG. 2A) whereas, at those concentrations, acamprosate
(FIG. 2B) and baclofen (FIG. 2C) alone have no significant effect
on intoxication. : p<0.05, significantly different from
A.beta..sub.1-42 intoxication; *: p<0.05, significantly
different from vehicle; "ns" no significant effect (ANOVA+Dunnett
Post-Hoc test).
[0032] FIG. 3: Effect of baclofen (BCL) and terbinafine (TBN)
combination therapy on the total length of capillary network in
beta-amyloid intoxicated HBMEC cultures. The human amyloid peptide
(A.beta..sub.1-42 2.5 .mu.M) produces a significant intoxication,
above 40%, compared to vehicle-treated cells. This intoxication is
prevented by the combination of terbinafine and baclofen. *:
p<0.05: significantly different from control (no
intoxication).
[0033] FIG. 4: Validation of the experimental model of human beta
amyloid toxicity on neuronal cells used for drug screening. One
hour of Estradiol (150 nM) or BDNF (50 ng/mL) pre-treatment
significantly protected the neurons from this amyloid injury
(-94%), which is considered as a positive control for
neuroprotection. *: p<0.05, significantly different from control
(no intoxication); : p<0.05, significantly different from
A.beta..sub.1-42 intoxication.
[0034] FIGS. 5A-5C: Effect of acamprosate (ACP) and baclofen (BCL)
combination therapy on LDH release in human A.beta..sub.1-42
toxicity on rat primary cortical cells. The human amyloid peptide
(A.beta..sub.1-42 10 .mu.M) produces a significant intoxication
compared to vehicle-treated neurons. This intoxication is
significantly prevented by the combination of acamprosate and
baclofen (FIG. 5A) whereas, at those concentrations, acamprosate
(FIG. 5B) and baclofen (FIG. 5C) alone have no significant effect
on intoxication. : p<:0.05, significantly different from
A.beta..sub.1-42 intoxication; *: p<:0.05, significantly
different from vehicle; "ns" no significant effect. (ANOVA+Dunnett
Post-Hoc test).
[0035] FIG. 6: Effect of cinacalcet (CNC) and sulfisoxazole (SFX)
combination therapy on LDH release in human A.beta..sub.1-42
toxicity on rat primary cortical cells. The human amyloid peptide
(A.beta..sub.1-42 10 .mu.M) produces a significant intoxication
compared to vehicle-treated neurons. This intoxication is prevented
by the combination of cinacalcet and sulfisoxazole. *: p<0.05,
significantly different from vehicle (ANOVA+Dunnett Post-Hoc
test).
[0036] FIG. 7: Effect of acamprosate (ACP) and baclofen (BCL)
combination therapy on the total length of neurite network in
beta-amyloid intoxicated cortical neurons. The human amyloid
peptide (A.beta..sub.1-42 2.5 .mu.M) produces a significant
intoxication, above 15%, compared to vehicle-treated cells. This
intoxication is significantly prevented by the combination of
acamprosate and baclofen whereas, at those concentrations,
acamprosate and baclofen alone have no significant effect on
intoxication. *: p<0.05, significantly different from
A.beta..sub.1-42 intoxication; *: p<0.05, significantly
different from vehicle (ANOVA+Dunnett Post-Hoc test).
[0037] FIG. 8: Effect of acamprosate and baclofen combination
therapy on behaviour as defined by Y-maze test. The amyloid peptide
produces a significant decrease in cognition as measured by
percentage of alternation (53.8% versus 73.5%). This deleterious
effect is significantly prevented (48.2% protection) by the
combination of acamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg
bid). .diamond.: p<0.05, significantly different from
A.beta..sub.25-35 intoxication; *: p<0.05, significantly
different from control (ANOVA+Dunnett Post-Hoc test).
[0038] FIG. 9: Effect of acamprosate and baclofen combination
therapy on memory as defined by passive avoidance (escape latency).
The amyloid peptide produces a significant decrease in memory
performances as measured by escape latency compared to control.
This deleterious effect is significantly prevented (complete
protection) by the combination of acamprosate (0.2 mg/kg bid) and
baclofen (3 mg/kg bid). .diamond.: p<:0.05, significantly
different from A.beta..sub.25-35 intoxication; *: p<0.05,
significantly different from control (ANOVA+Dunnett Post-Hoc
test).
[0039] FIG. 10: Effect of acamprosate and baclofen combination
therapy on memory as defined by passive avoidance (step-through
latency). The amyloid peptide produces a significant decrease in
memory performance as measured by step-through latency, above 44%,
compared to control. This deleterious effect is significantly
prevented (78.8% protection) by the combination of acamprosate (0.2
mg/kg bid) and baclofen (3 mg/kg bid) whereas, at those
concentrations, acamprosate and baclofen alone have a lower effect
on intoxication. .diamond.: p<0.05, significantly different from
A.beta..sub.25-35 intoxication; *: p<0.05, significantly
different from control (ANOVA+Dunnett Post-Hoc test).
[0040] FIG. 11: Effect of acamprosate and baclofen combination
therapy on neuron's density in the hippocampus. The amyloid peptide
produces a significant decrease in neuronal density as measured by
the number of neurons per millimeter in the hippocampus, above 21%,
compared to control. This neuronal injury is significantly
prevented (63.2% of injured neurons are protected) by the
combination of acamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg
bid). .diamond.: p<0.05, significantly different from
A.beta..sub.25-35 intoxication; *: p<0.05, significantly
different from control (ANOVA+Dunnett Post-Hoc test).
[0041] FIG. 12: Effect of acamprosate and baclofen combination
therapy on the blood-brain barrier (BBB) integrity. The amyloid
peptide affects the blood-brain barrier, inducing a significant
increase of its permeability, above 51%, compared to control. Those
damages on the blood-brain barrier are significantly prevented
(66.6% of the integrity restored) by the combination of acamprosate
(0.2 mg/kg bid) and baclofen (3 mg/kg bid). .diamond.: p<0.05,
significantly different from A.beta..sub.25-35 intoxication, *.
p<0.05, significantly different from control (ANOVA+Dunnett
Post-Hoc test).
[0042] FIG. 13: Effect of acamprosate and baclofen combination
therapy on synaptic density as reflected by the synaptophysin
concentration. The amyloid peptide affects synapse function,
inducing a significant decrease in the synaptophysin concentration
in the brain, above 34%, compared to control. Those damages on the
synaptic density are significantly prevented (76%) by the
combination of acamprosate (0.2 mg/kg bid) and baclofen (3 mg/kg
bid). .diamond.: p<0.05, significantly different from
A.beta..sub.25-35 intoxication, *: p<0.05, significantly
different from control (ANOVA+Dunnett Post-Hoc test).
[0043] FIG. 14: Protective effect of acamprosate and baclofen
combination therapy on oxidative stress in the hippocampus. The
amyloid peptide induces a significant increase in oxidative stress
in the hippocampus as measured by lipid peroxydation, above 59%,
compared to control. This oxidative stress is significantly
prevented (65.9%) by the combination of acamprosate (0.2 mg/kg bid)
and baclofen (3 mg/kg bid). .diamond.: p<0.05, significantly
different from A.beta..sub.25-35 intoxication; *: p<0.05,
significantly different from control (ANOVA+Dunnett Post-Hoc
test).
[0044] FIG. 15: Effect of baclofen and acamprosate combination
therapy against glutamate toxicity on neuronal cortical cells.
Glutamate intoxication is significantly prevented by the
combination of baclofen (400 nM) and acamprosate (1.6 nlM) whereas,
at those concentrations, baclofen and acamprosate alone have no
significant effect on intoxication. .diamond.: p<0.001,
significantly different from glutamate intoxication; (ANOVA+Dunnett
Post-Hoc test).
[0045] FIG. 16: Effect of donepezil, acamprosate and baclofen
combination therapy on behaviour and cognitive performance as
defined by the Y-maze test. The amyloid peptide produces a
significant decrease in cognition as measured by percentage of
alternation (51.5% versus 71.8%). This deleterious effect is
significantly prevented (98% protection) by the combination of
donepezil (0.25 mg/kg/day), acamprosate (32 .mu.g/kg bid) and
baclofen (480 .mu.g/kg bid), whereas at those concentrations the
drugs alone have no significant effect. .diamond.: p<0.01,
significantly different from A.beta..sub.25-35 intoxication; *:
p<0.01, significantly different from control (ANOVA+Dunnett
Post-Hoc test).
[0046] FIG. 17: Comparison of protective effect of acamprosate and
its derivative homotaurine pre-treatment in human A.beta..sub.1-42
toxicity assays on rat primary cortical cells. A.beta..sub.1-42
produces a significant intoxication compared to vehicle-treated
neurons. The intoxication is equally significantly prevented by
homotaurine and acamprosate (99%, 8 nM). .diamond.: p<0.0001:
significantly different from A.beta..sub.1-42 intoxication
(ANOVA+Dunnett Post-Hoc test).
[0047] FIG. 18: Effect of acamprosate and baclofen combination
therapy on the development of chronic progressive experimental
autoimmune encephalomyelitis (EAE) as defined by clinical score.
Immunization induces a significant decrease in physical features as
measured by clinical score. This deleterious effect is
significantly prevented (p-value<0.01) by the combination of
acamprosate (2 mg/kg/day) and baclofen (30 mg/kg/day).
[0048] FIG. 19: Effect of memantine, acamprosate and baclofen
combination therapy on behaviour and cognitive performances as
defined by Y-maze test. The amyloid peptide produces a significant
decrease in cognition as measured by percentage of alternation
(57.7% versus 69.7%). This deleterious effect is significantly
prevented (79.2% protection) by the combination of memantine (0.5
mg/kg/day), acamprosate (32 .mu.g/kg bid) and baclofen (480
.mu.g/kg bid), whereas at those concentrations the drugs alone have
no significant effect. .diamond..diamond.: p<0.01, .diamond.:
p<0.05, significantly different from A.beta..sub.25-35
intoxication; *: p<0.05, significantly different from control
(ANOVA+Dunnett Post-Hoc test).
[0049] FIG. 20: Baclofen (BCL)-acamprosate (ACP) combination
protects synapse integrity from A.beta..sub.1-42 toxicity.
A.beta..sub.1-42 (0.3 .mu.M) intoxication induces a loss in
synaptic junctions between hippocampal neurons (almost 34%), as
determined by quantification of colocalization of PSD93 and
synaptophysin proteins (black bar). This effect is significantly
reversed upon treatment with BCL and ACP combination (80 nM and
0.32 nM respectively) by up to 62% (light grey bar). ***:
p<0.001, significantly different from A.beta. intoxicated
cells.
[0050] FIGS. 21A-21D: Baclofen-acamprosate combination reduces
A.beta..sub.1-42-induced changes within neurones in vitro. (FIG.
21A) A.beta..sub.1-42 intoxication induces oxidative stress as
shown by the increase of 18% of MetO residues within the cells
cultured in the presence of A.beta..sub.1-42 compared to control.
Baclofen (0.32 nM) and acamprosate (80 nM) treatment significantly
lowers A.beta..sub.1-42 induced oxidative stress as reported by a
drop of 60% of MetO residues in treated, intoxicated cells. (FIG.
21B) A.beta..sub.1-42 intoxication results in the triggering of
apoptotic events as the increase of cytoplasmic cytochrome C (Cyto
C) released from mitochondria (above 150% control). Baclofen and
acamprosate combination significantly reduces by more than 30% the
release of mitochondrial Cyto C in intoxicated cells. (FIG. 21C)
A.beta..sub.1-42 intoxication results in an increase of 46% of
phosphorylated Tau protein (pTau.sup.Ser212/Thr214) within the
cells, compared to control, non-intoxicated cells. Quantity of
phosphorylated Tau protein in A.beta..sub.1-42 intoxicated neurons
is significantly reduced by more than 90% upon treatment with
baclofen and acamprosate. (FIG. 21D) Baclofen-acamprosate
combination diminishes by more than 90% the release of glutamate in
the culture medium of A.beta..sub.1-42-intoxicated cells. Values
are mean.+-.s.e.m. ***: p<0.001 versus A.beta. (ANOVA+Dunnett
Post-Hoc test).
[0051] FIGS. 22A-22D: Acamprosate (ACP) acts through GABA.sub.A,
glycine and metabotropic glutamatergic receptor signaling.
A.beta..sub.1-42 (10 .mu.M) produces a significant intoxication of
cells (-, black bars, up to 40% of death) compared to
vehicle-treated neurons (control, white bars). This intoxication is
efficiently prevented by acamprosate 8 nM (grey bars, up to 71%
improvement). Muscimol, a GABA.sub.A receptor agonist (FIG. 22A),
strychnine, a glycine receptor antagonist (FIG. 22B), DHPG, a
mGluR1/5 agonist (FIG. 22C) and (2R,4R)-APDC, a mGluR2/3 agonist
(FIG. 22D) all block ACP protection of rat primary neuronal cells.
Values are mean.+-.s.e.m. ***: p<0.001 versus A.beta., or ACP
pretreated and A.beta. intoxicated cells (ANOVA+Dunnett Post-Hoc
test). "Control": non-A.beta. intoxicated non treated cells; "-":
A.beta. intoxicated cells. mGluR1/5 and mGluR2/3: metabotropic
glutamate group I and II receptors respectively.
[0052] FIG. 23: Simultaneous activity on three main features of AD
within given concentration ranges, i.e., baclofen: from 80 nM to 1
.mu.M and acamprosate: from 320 pM to 4 nM. Baclofen-acamprosate
combination has been found to be neuroprotective and to improve, at
the same time, synapse function (neuron plasticity) and endothelial
cell function (angiogenesis). S: drug concentrations at which the
combination exerts a synergistic effect in the corresponding in
vitro models.
[0053] FIGS. 24A-24B: A one-month treatment with
baclofen-acamprosate combination (acamprosate 0.2 mg/kg bid and
baclofen 3 mg/kg bid) is effective in improving cognition in
hAPPS.sub.L mice of 8 months, when compared to non-treated
transgenic mice both during the acquisition phase or the test
phase. (FIG. 24A) On day 4 the latency observed for treated
transgenic mice is significantly different from that exhibited by
non-treated transgenic mice. (FIG. 24B) Escape latency for swim 1
and swim 2 in treated transgenic mice is significantly different
from non-treated transgenic mice. Globally performances of all
treated transgenic mice are better than those of non-treated
transgenic mice and similar to those of non-transgenic animals.
Values are mean.+-.s.e.m. *: p<0.05, treated transgenic animals
are different from non-treated transgenic animals (ANOVA+Dunnett
Post-Hoc test); .sup..largecircle..largecircle..largecircle.:
p<0.001. Performances of baclofen-acamprosate-treated animals
are globally significantly different from performances of
non-treated transgenic animals (ANOVA+Dunnett Post-Hoc test).
[0054] FIG. 25: Baclofen-acamprosate combination efficiently
reduces scopolamine-induced cognitive impairments in humans. Raw
data scores in Groton Maze Learning Test (GMLT) are plotted along
the vertical scale. An increase in the score corresponds to an
impairment of performance in GMLT. Scopolamine (administered at H3)
induces a rapid decrease in cognitive performance in
placebo-treated subjects (circles, dotted line) which lasts for
approximately 6 hours (H9) after scopolamine injection.
Baclofen-acamprosate mix (squares, grey/solid line) is efficient
over this period in reducing the deleterious effects of scopolamine
on cognitive performance. A significant improvement in cognitive
scores is observed in the time window corresponding to the higher
plasmatic concentrations of baclofen and acamprosate (shaded bar
below the horizontal scale, dark: higher plasmatic concentrations,
light: lower plasmatic concentrations).
[0055] FIG. 26: ADAS-cog (Alzheimer's Disease Assessment Scale
Cognitive Subscale) score evolution (change from baseline) during
single blind Challenge-De-challenge-Re-challenge (CDR) clinical
trial on 24 mild AD subjects. ADAS-cog score is evaluated at the
beginning and the end of each period during visits to the
neurophysiologist (V). The efficiency of baclofen-acamprosate mix
is supported by the clear succession of improvement, worsening and
improvement phases in correlation with the challenge (plain line),
de-challenge (dotted line) and re-challenge phases (plain line).
Data are obtained from the gathering of the whole data from dose 1
and dose 2 administered patients. Cognition is significantly
improved considering the whole duration of the study (V1 versus V4
score comparison, p<0.05); a significant improvement is also
noticed at the end of the challenge phase (V1 versus V2 score
comparison, p<0.01).
[0056] FIG. 27: Records of P300 wave all along the
Challenge-De-challenge-Re-challenge (CDR) study, in a subject
suffering from mild AD treated with 0.4 mg acamprosate and 6 mg
baclofen, each bid. Baclofen-acamprosate combination is efficient
in correcting the electrophysiological mechanisms underlying
cognitive processes in AD patients. ERPs were recorded at each
visit (V1-V4) to the neurophysiologist. Each curve is a modelling
of the whole recordings from the different electrodes. It clearly
appears that, from V2 to V4, the P300 subcomponent waves
consistently shift to the left during all the duration of the
study, thereby showing a decrease in latency. An improvement of
amplitude is also observed (Student's T-test on paired data).
[0057] FIG. 28: A 17-week treatment with baclofen-acamprosate
combination efficiently improves working memory performances of
aged mice (more than 28 months). Percentage of alternation in
T-maze test is significantly improved (by more than 50%) in treated
aged mice when compared to non-treated mice. Values are
mean.+-.s.e.m. ***p<0.001, treated aged animals are
significantly different from non-treated aged animals
(ANOVA+Dunnett Post-Hoc test).
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention provides new methods and compositions
for treating neurological disorders. The invention discloses novel
drug combinations which allow an effective correction of such
diseases and may be used in any mammalian subject.
[0059] The invention is suited for treating any neurological
disorder, whether central or peripheral, particularly disorders
wherein nerve or neuron injuries, 3 amyloid, BBB breakdown or
glutamate excitotoxicity are involved. Specific examples of such
disorders include neurodegenerative diseases, neuropathies, spinal
cord injury, and substance abuse such as alcoholism.
Definitions
[0060] "Neurodegenerative disorders" refers to diseases, such as
Alzheimer's disease (AD) and related disorders, amyotrophic lateral
sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease (PD),
and Huntington's disease (HD), encompassing a progressive loss of
function and death of neurons.
[0061] "Neuropathies" refers to conditions where nerves of the
peripheral nervous system are damaged; this includes damage of the
peripheral nervous system provoked by genetic factors, inflammatory
disease, or chemical substance including drugs (vincristine,
oxaliplatin, ethyl alcohol). The treatment of neuropathies also
includes the treatment of neuropathic pain.
[0062] The invention is particularly suited for treating AD and
related disorders. In the context of this invention, the term
"related disorder" includes senile dementia of AD type (SDAT),
frontotemporal dementia (FTD), vascular dementia, mild cognitive
impairment (MCI) and age-associated memory impairment (AAMI).
[0063] 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
symptoms or a protection of neuronal cells.
[0064] Within the context of this invention, the designation of a
specific drug or compound is meant to include not only the
specifically named molecule, but also any pharmaceutically
acceptable salt, hydrate, derivative, isomer, racemate, conjugate,
prodrug or derivative thereof of any chemical purity.
[0065] The terms "combination" or "combinatorial treating/therapy"
designate a treatment wherein at least baclofen and acamprosate 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 baclofen and acamprosate 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.
[0066] 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, generate said
compound as a result of, e.g., spontaneous chemical reaction(s),
enzyme catalysed 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 [27-31]. 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 [28, 32-38]. For example, arbaclofen placarbil is
listed in the ChemID plus Advance database (website:
chem.sis.nlm.nih.gov/chemidplus/) and arbaclofen placarbil is a
well-known prodrug of baclofen [39, 40].
[0067] 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 alkylated (C1-C6) variant of such a
compound. The term derivative also includes structurally related
compounds 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 (see Worldwide Website:
pubchem.ncbi.nlm.nih.gov/search/) or DrugBank (see Worldwide
Website: 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
[41, 42] available online (see Worldwide Website:
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.beta. or glutamate toxicity.
[0068] The term "derivative" also includes metabolites of a drug,
e.g., molecules which result from the (biochemical) modification(s)
or processing of said drug after administration to an organism,
usually through specialized enzymatic systems, and which display or
retain 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.beta. toxicity or glutamate
toxicity.
[0069] The term "salt" refers to a pharmaceutically acceptable and
relatively non-toxic, inorganic or organic salt of a compound of
the present invention. Pharmaceutical salt formation consists of
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 reactions.
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, tartaric 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 taught by
H. Stahl and C. G. Wermuth in their handbook [43].
[0070] In a preferred embodiment, the designation of a compound is
meant to designate the compound per se, as well as any
pharmaceutically acceptable salt, hydrate, isomer, racemate, ester
or ether thereof.
[0071] In a more preferred embodiment, the designation of a
compound is meant to designate the compound as specifically
designated per se, as well as any pharmaceutically acceptable salt
thereof.
[0072] In a particular embodiment, a sustained-release formulation
of the compound is used.
Compositions and Methods of the Invention
[0073] As discussed above, the invention relates to particular drug
combinations which have a strong unexpected effect on several
biological processes involved in neurological disorders. These drug
combinations therefore represent novel approaches for treating
neurological disorders, such as Alzheimer's disease and related
disorders, 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. More specifically, the
invention discloses compositions, comprising baclofen in
combination with acamprosate, which provide a significant effect in
vivo on neurological disorders.
[0074] Indeed, the invention shows, in the experimental part, that
combination therapies comprising baclofen and acamprosate can
substantially improve the condition of patients afflicted with
neurological disorders. In particular, the inventors have
surprisingly discovered that baclofen and acamprosate combinations
have a strong, unexpected effect on the length of the capillary
network, on LDH release in beta-amyloid intoxicated nervous cells
as well as on the length of the neurite network, and represent new
therapeutic approaches for AD. They have also discovered that
baclofen-acamprosate combinations are efficient in lowering the
A.beta. induced oxidative stress, apoptosis, glutamate release and
phosphorylated Tau accumulation in neuronal cells.
[0075] Also, the examples show that, in a combination therapy of
the invention, baclofen may be effective at a dose of 80 nM or
less, and acamprosate may be effective at a dose of 1 nM or less.
These results are remarkable and particularly advantageous since,
at such low doses, any possible side effects are avoided. Moreover
the inventors have been able to determine plasmatic and/or brain
concentration ranges at which such a combination therapy exerts a
simultaneous protective activity against the three aspects of
A.beta. toxicity in relation with AD pathogenesis, i.e.,
angiogenesis, neuronal protection and neuronal plasticity.
Furthermore, these combinations effectively protect neuronal cells
from various afflictions such as glutamate toxicity and oxidative
stress, and prevent BBB permeabilization and neuronal cell induced
apoptosis, which are involved in several neurological
disorders.
[0076] The present invention therefore proposes a novel therapy for
neurological disorders, based on baclofen and acamprosate
compositions. More particularly, the present invention therefore
proposes a novel therapy for Alzheimer's disease and related
disorders, 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, based on baclofen and
acamprosate combinations.
[0077] In this regard, in a particular embodiment, the invention
relates to a composition comprising baclofen and acamprosate.
[0078] In a further embodiment, the invention relates to a
composition comprising baclofen and acamprosate 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.
[0079] In a further embodiment, the invention relates to the use of
baclofen and acamprosate for the manufacture of a medicament for
the treatment of AD, AD-relateddisorders, MS, PD, ALS, HD,
neuropathies (for instance neuropathic pain or alcoholic
neuropathy), alcoholism or alcohol withdrawal, or spinal cord
injury.
[0080] Illustrative CAS numbers for baclofen and acamprosate are
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-US-00001 TABLE 1 Class or Tanimoto Drug CAS Numbers
similarity index Acamprosate and related compounds Acamprosate
77337-76-9; 77337-73-6 NA Homotaurine 3687-18-1 0.73 Ethyl Dimethyl
Ammonio / 0.77 Propane Sulfonate Taurine 107-35-7 0.5 Baclofen and
related compounds Baclofen 1134-47-0; 66514-99-6; NA 69308-37-8;
70206-22-3; 63701-56-4; 63701-55-3 3-(p-chlorophenyl)-4- /
Metabolite hydroxybutyric acid Arbaclofen placarbil 847353-30-4
Prodrug
[0081] Specific examples of prodrugs of baclofen are given in
Hanafi et al. [44], particularly baclofen esters and baclofen ester
carbamates, which are of particular interest for CNS targeting.
Hence such prodrugs are particularly suitable for compositions of
this invention. Arbaclofen 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, US2011021571, WO2003077902, and
WO2010120370.
[0082] Useful prodrugs for acamprosate such as pantoic acid ester
neopentyl sulfonyl esters, neopentyl sulfonyl ester prodrugs of
acamprosate 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.
[0083] Baclofen and acamprosate combination therapy may be used
alone or may be further combined with additional compounds. In this
regard, in a particular embodiment, the compositions of the
invention may further comprise at least one compound selected from
sulfisoxazole, methimazole, prilocaine, dyphylline, quinacrine,
carbenoxolone, aminocaproic acid, cabergoline, diethylcarbamazine,
cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide,
levosimendan, sulodexide, terbinafine, zonisamide, etomidate,
phenformin, trimetazidine, mexiletine, ifenprodil, moxifloxacin,
bromocriptine or torasemide. Illustrative CAS numbers for each of
these compounds are provided in Table 2 below:
TABLE-US-00002 TABLE 2 DRUG NAME CAS NUMBER Aminocaproic Acid
60-32-2 Bromocriptine 25614-03-3 Cabergoline 81409-90-7
Carbenoxolone 5697-56-3 Cinacalcet 226256-56-0 Cinnarizine 298-57-7
Diethylcarbamazine 90-89-1 Dyphylline 479-18-5 Eplerenone
107724-20-9 Etomidate 33125-97-2 Fenoldopam 67227-57-0 Ifenprodil
23210-56-2 or 23210-58-4 Leflunomide 75706-12-6 Levosimendan
141505-33-1 Methimazole 60-56-0 Mexiletine 5370-01-4 or 31828-71-4
Moxifloxacin 354812-41-2 Phenformin 114-86-3 Prilocaine 721-50-6 or
14289-31-7 or 14289-32-8 Quinacrine 83-89-6 Sulfisoxazole 127-69-5
Sulodexide 57821-29-1 Terbinafine 91161-71-6 Torasemide 56211-40-6
or 72810-59-4 Trimetazidine 5011-34-7 or 13171-25-0 Zonisamide
68291-97-4
[0084] In a particular embodiment, the invention relates to the use
of this combination for treating AD or a related disorder in a
subject in need thereof.
[0085] In another particular embodiment, the invention relates to
the use of this combination for treating an AD-related disorder
selected from senile dementia of AD type (SDAT), frontotemporal
dementia (FTD), vascular dementia, mild cognitive impairment (MCI)
and age-associated memory impairment (AAMI).
[0086] A particular object of this invention relates to a method of
treating frontotemporal dementia (FTD) in a mammalian subject in
need thereof, preferably a human subject in need thereof, the
method comprising administering to said subject an effective amount
of a combination of the invention.
[0087] In a particular embodiment, the invention relates to the use
of this combination 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.
[0088] As disclosed in the examples, composition therapies using at
least baclofen and acamprosate have a strong unexpected effect on
biological processes leading to neuronal injuries. Furthermore,
these combinations also showed in vivo a very efficient ability to
correct symptoms of neurological diseases. These combinations
therefore represent novel approaches for treating neurological
disorders, such as AD, MS, ALS, PD, HD, neuropathies (for instance
neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, and spinal cord injury. These compositions efficiently
prevent toxicity of amyloid .beta. (A.beta.) peptide or glutamate
excitotoxicity in neuronal cells. More particularly, as shown in
the experimental section, these compositions are efficient at
counteracting, simultaneously, the detrimental effects of
intoxication by A.beta. oligomers at the synaptic, neuronal and
endothelial levels. Such combination of effects is particularly
advantageous and leads to a significant improvement of the disease
both in several in vivo models for AD and in clinical trials.
Indeed, in vivo, these compositions lead to an improvement of
several cognitive symptoms as well as to protection of neuronal
cells.
[0089] Furthermore the experimental section shows that the
above-mentioned compositions are also efficient at i)
synergistically protecting in vitro neuronal cells from glutamate
excitotoxicity, and ii) conferring clinical benefit in in vivo
models for diseases related to glutamate excitotoxicity.
[0090] Hence they represent novel and potent methods for treating
such disorders.
[0091] The compositions of the invention may comprise 2, 3, 4 or 5
distinct drugs, 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.
[0092] 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: [0093] baclofen and acamprosate, [0094] baclofen
and acamprosate and diethylcarbamazine, [0095] baclofen and
acamprosate and cinacalcet, [0096] baclofen and acamprosate and
sulfisoxazole, [0097] baclofen and acamprosate and torasemide,
[0098] baclofen and acamprosate and ifenprodil, [0099] baclofen and
acamprosate and mexiletine. [0100] baclofen and acamprosate and
eplerenone, [0101] baclofen and acamprosate and levosimendan,
[0102] baclofen and acamprosate and terbinafine, or [0103] baclofen
and acamprosate and leflunomide.
[0104] As disclosed in the experimental section, combinatorial
therapies of the invention provide substantial therapeutic and
biological effects to improve Alzheimer's disease or related
disorders in both animal model and human subjects. They induce a
strong neuroprotective effect against AB toxicity, notably through
the inhibition of A.beta.-induced apoptosis and oxidative stress.
They also give positive results in behavioural performances and
biochemical assays in rodents. Results show that compositions of
the invention in vivo: (i) efficiently correct molecular pathways
triggered by AD oligomers, and (ii) lead to an improvement of
neurophysiological impairments observed in diseased animals as
neuron survival or synapse integrity. Results also show that
compositions of the invention efficiently restore the blood-brain
barrier (BBB) and prevent, retard, or lessen apoptosis triggering,
which are known to be impaired in several neurological diseases.
Combinatorial therapies are also efficient at correcting memory
impairment in aged animals. Results from clinical trials in human
subjects also show an activity on cognitive performances of AD
patients.
[0105] Moreover, the results presented also show that the above
combinations therapies have an important synergistic
neuroprotective effect against glutamate excitotoxicity (FIG. 15),
a pathway which is implicated in various neurological diseases such
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.
[0106] Furthermore, the particularly high synergistic interaction
observed for these two drugs through the combinatorial treatment of
the invention allows the use of drug concentrations showing no
effect when used in single-drug treatment. Moreover, as shown in
the experimental section, the baclofen and acamprosate combination
causes an enhanced therapeutic benefit in treating Alzheimer's
disease compared to other therapeutic combinations. These
compositions efficiently prevent the toxic effects of amyloid
.beta. protein or peptide on human cells and in an in vivo model
and represent novel and potent methods for treating such
disorder.
[0107] An object of this invention thus also resides in a
composition as defined above for treating a neurological disorder
such as AD, AD-related disorders, MS, PD, ALS, HD, neuropathies
(for instance alcoholic neuropathy or neuropathic pain), alcoholism
or alcohol withdrawal, or spinal cord injury.
[0108] As indicated previously, in a combination therapy of this
invention, the compounds or drugs may be formulated together or
separately, and administered together, separately or
sequentially.
[0109] 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.
[0110] 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.
[0111] A further object of the invention is a method of 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 method comprising simultaneously,
separately or sequentially administering to a subject in need
thereof an effective amount of a composition as disclosed
above.
[0112] In a preferred embodiment, the invention relates to a method
of 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 in a subject in need
thereof, comprising administering simultaneously, separately or
sequentially to the subject an effective amount of baclofen and
acamprosate.
[0113] The compositions of the invention typically comprise one or
several pharmaceutically acceptable carriers or excipients. Also,
for use in the present invention, the drugs or compounds are
usually mixed with pharmaceutically acceptable excipients or
carriers.
[0114] In this regard, 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.
[0115] In a particular embodiment, the method comprises mixing
baclofen and acamprosate in an appropriate excipient or
carrier.
[0116] According to preferred embodiments of the invention, as
indicated above, the compounds are used as such or in the form of a
pharmaceutically acceptable salt, prodrug, derivative, or sustained
release formulation thereof.
[0117] Although very effective in vitro and in vivo, depending on
the subject or specific condition, the combination therapy of the
invention may further be used in conjunction, association or
combination with additional drugs or treatments beneficial to the
treated neurological condition in the subject.
[0118] 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, results
also show that the above combination therapies have an important
synergistic neuroprotective effect when combined with donepezil
(FIG. 16) or memantine (FIG. 19), thereby allowing the use of low
doses of said compounds and avoiding or lessening side effects.
Thereby, illustrative therapies which can be used with combinations
of the invention are donepezil (CAS: 120014-06-4), galantamine
(CAS: 357-70-0), gabapentine (CAS: 478296-72-9; 60142-96-3),
rivastigmine (CAS: 123441-03-2) or memantine (CAS: 19982-08-2). The
above CAS numbers are only given in an illustrative way, and common
salts, enantiomeric forms, racemates, prodrugs, metabolites or
derivatives of the above compounds should be also considered.
[0119] 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
contaminants) or any drug containing Ginkgo biloba extracts.
[0120] In another particular embodiment, the drug(s) or
compositions according to the present invention may be further
combined with drugs or compounds which are currently under phase
III clinical trial for AD. These drugs or compounds include
Epigallocatechin-3-gallate, Human neutral insulin, Idalopirdine,
Vanutide cridificar, Durin-Leuprolide acetate, Gantenerumab,
Latrepirdine hydrochloride, Solanezumab, Masitinib mesylate,
Encenicline hydrochloride, Leuco methylthioninium salt, IGIV,
Lu-AE-58-054, VP4896, INM-176, R04909832, SK-PCB70M, AC-1204, and
MK8931.
[0121] Furthermore, the inventors have been able to decipher the
molecular mechanisms underlying the unexpected efficiency of
combinations of the invention. The results show that (i) baclofen
exerts a Gaba B agonistic activity which is essential for
neuroprotective activity and (ii) acamprosate exerts a particular
pattern of biological interactions (Table 3) that is essential for
a neuroprotective effect in the context of AD or AD-related
disorders
TABLE-US-00003 TABLE 3 Group I Group II metabotropic metabotropic
Gaba A Glycine glutamatergic glutamatergic receptors receptors
receptors receptors Acamprosate antagonist agonist antagonist
antagonist activity* *pattern of activity that has been shown
necessary to afford a protective effect against toxicity of
A.beta.
[0122] As shown in the experimental section, counteracting only one
of these mechanisms of action results in an almost total abolition
of the neuroprotective effect of acamprosate against A.beta.
toxicity (FIG. 22). Notably, the inventors show for the first time
that, in the context of AD pathogenesis, acamprosate exerts an
antagonistic effect on GABA A receptors which is essential to
afford neuroprotection against A.beta.. Hence, together with the
agonistic action of baclofen on GABA B receptors, the concerted
action of acamprosate on the four identified receptor families
leads to a particularly efficient therapeutic effect as described
in this whole disclosure. This concerted action on several targets
when using only one drug thus makes acamprosate of particular
interest.
[0123] Furthermore, based on the knowledge of the pattern of action
provided in the present application, it is now possible to design
and use alternative combinations of drugs in replacement of, e.g.,
acamprosate. Such alternative combinations should exhibit the same
pattern of biological interaction as shown for acamprosate in Table
3 and, in particular, they should (i) antagonize GABA A receptors
and Group I and II metabotropic receptors, and (ii) lead to
increased activity of glycinic receptors.
[0124] Consequently, in a particular embodiment, this invention
relates to a combination of baclofen with a drug having an
antagonist activity on GABA A receptors, as well as on Group I and
II metabotropic receptors, and an agonist activity on glycinic
receptors.
[0125] In a particular embodiment, the invention relates to a
combination of baclofen with a combination of drugs, said
combination of drugs having an antagonistic activity toward GABA A
receptors and Group I and II metabotropic receptors, as well as an
agonistic activity toward glycinic channels. Said combination of
drugs used as an alternative to acamprosate can comprise 2, 3, 4, 5
or even 6 drugs in order to mimic the pleiotropic activity of
acamprosate. Ideally, some of the drugs used in said composition
act on several of the targets identified by the inventors. More
preferably some of the drugs used in said composition act on 2, or
3 or 4 of the targets identified by the inventors. Hence, more
preferably, the combination of drugs used as an alternative to
acamprosate comprises 2, 3 or 4 drugs.
[0126] An agonistic or antagonistic activity toward a
receptor/channel refers to a direct action on said receptor/channel
or to an indirect action leading to the activation or inhibition of
said receptor/channel.
[0127] An agonistic or antagonist activity also refers to a
positive or negative allosteric modulation, respectively.
[0128] Drugs suitable to elaborate such a combination of drugs are
listed in Table 4 below with their corresponding CAS number, IUPAC
name or related articles, for illustrative purposes only.
TABLE-US-00004 TABLE 4 antagonists of group I mGluR GRM1 GRM5 AIDA
168560-79-0 ADX 10059 1166398-32-8; 757949-98-7 A-794278
869802-57-3 AZD-2066 1403991-95-6; 934282-55-0; A-794282
869802-44-8 934338-70-2 A-841720 869802-58-4 AZD6538 Raboisson et
al 2012 [45] A-850002 869802-73-3 AZD9272 1166398-50-0 Bay-36-7620
232605-26-4 Basimglurant 802906-73-6 FTIDC Suzuki et al 2007 [46]
Dipraglurant 872363-17-2 JNJ-16259685 409345-29-5 Fenobam
57653-26-6; 63540-28-3 LY-367385 198419-91-9 GRN529 1253291-12-1
RO0711401 714971-87-6 LY-344545 201851-20-9 YM-202074 299900-83-7
Mavoglurant 543906-09-8 YM-230888 Kohara et al 2007 [47] MPEP
219911-35-0 YM-298198 748758-45-4 MRZ-8676 Dekundy et al 2011 [48]
MTEP 329205-68-7; 1186195-60-7 Rufinamide 106308-44-5 SIB-1757
31993-01-8 SIB-1893 7370-21-0 Antagonists of group II m GluR (GRM2
and 3) BCI-632 569686-87-9 BCI-1038 (N/A) Prodrug of B632 BCI-1206
(N/A) Prodrug of B632 BCI-1283 (N/A) Prodrug of B632 BCI-838
(1R,2R,3R,5R,6R)-2-Amino-3-[(3,4-dichlorobenzyl)oxy]-
6-fluoro-6-[(heptyloxy)carbonyl]bicyclo[3.1.0]hexane- 2-carboxylic
acid RG-1578 911115-16-7 RO4491533 579482-31-8 LY-341495
201943-63-7 APICA 170847-18-4 EGLU 170984-72-2 Antagonists of Gaba
A receptors .alpha..sub.5IA 215874-86-5 beta-Cce 74214-62-3
beta-Ccm 69954-48-9 beta-Cct 93835-05-3 bicuculline 485-49-4;
56083-00-2 BTS-72-664
(R)-7-[1-(4-chlorophenoxy)]ethyl]-1,2,4-triazolo(1,5-alpha)pyri-
midine cicutoxin 505-75-9 clarithromycin 81103-11-9 DMCM 82499-00-1
FG71-42 78538-74-6 FGIN-1-27 142720-24-9 FGIN-1-44
2-hexyl-indole-3-acetamide-N-benzene-tricarboxylic acid flumazenil
78755-81-4 gabazine 104104-50-9 L-655708 Ethyl
(S)-11,12,13,13a-Tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-
a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate lorediplon
917393-39-6 MK0777 252977-51-8 oenanthotoxin 20311-78-8 pentetrazol
54-95-5 picrotoxin 124-87-8 pitrazepin 90685-01-1 PWZ-029
8-chloro-3-(methoxymethyl)-5-methyl-4H-imidazo[1,5-a] [1,4]
benzodiazepin-6-one R04882224
3,10-dichloro-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-
d][1,4]diazepine RO15-3505 ethyl
7-chloro-5-methyl-6-oxo-5,6-dihydro-4H-imidazo[1,5-
a][1,4]benzodiazepine-3-carboxylate Ro15-4513
ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-
benzodiazepine-3-carboxylate RO-4938581
3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-
a][1,2,4]triazolo[1,5-d][1,4]diazepine RU-5135 78774-26-2 RY-023
(tert-butyl-8-(trimethylsilyl)
acetylene-5,6-dihydro-5-methyl-6-oxo-4H- imidazo [1,5a] [1,4]
benzodiazepine-3-carboxylate RY-024
t-butyl-8-ehtynyl-(5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-
a)[1,4]benzodiazepine-3-carboxylate RY-024
t-butyl-8-ehtynyl-(5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-
a)[1,4]benzodiazepine-3-carboxylate RY-80 ethyl
8-ethynyl-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-
3-carboxylate S8510 [2-(3-isoxazolyl)-3,6,7,9-tetrahydroimidazo
[4,5-d] pyrano [4,3-b] pyridine monophosphate monohydrate]
sarmazenil 78771-13-8 SR-42641 105537-78-8 SR-95103 96440-63-0
thiocolchicoside 602-41-5 thujone 1125-12-8; 546-80-5; 471-15-8;
33766-30-2 TP003 628690-75-5 US-1010 516-55-2 ZK-93423 89592-45-0
Agonists of Glycinic channels anandamide 94421-68-8 cycloserine
68-41-7 dimethylglycine 1118-68-9 dronabinol 1972-08-3 enflurane
13838-16-9 glycine 56-40-6 Halothane 151-67-7 HU 210 112830-95-2
hypotaurine 300-84-5 isoflurane 26675-46-7 milacemide 76990-56-2
sarcosine 203-538-6 serine 302-84-1; 56-45-1; 312-84-5 sevoflurane
28523-86-6 taurine 107-35-7 trimethylglycine 107-43-7; 17146-86-0
alanine 302-72-7; 56-41-7; 338-69-2 .beta. alanine 107-95-9 WIN
55212-2 131543-23-2 arachidonyl- 53847-30-6 glycerol
[0129] In a particular embodiment, said combination of drugs used
as an alternative to acamprosate comprises at least one compound
selected from .alpha..sub.5TA, beta-Cce, beta-Ccm, beta-Cct,
bicuculline, BTS-72-664, cicutoxin, clarithromycin, DMCM, FG71-42,
FGIN-1-27, FGIN-1-44, flumazenil, gabazine, L-655708, lorediplon,
MK0777, oenanthotoxin, pentetrazol, picrotoxin, pitrazepin,
PWZ-029, R04882224, RO15-3505, Ro15-4513, RO-4938581, RU-5135,
RY-023, RY-024, RY-024, RY-80, S8510, sarmazenil, SR-42641,
SR-95103, thiocolchicoside, thujone, TP003, US-1010, or ZK-93423,
which are known as (direct or indirect) antagonists or inverse
agonists of the GABA A receptors.
[0130] In a preferred embodiment, said combination of drugs used as
an alternative to acamprosate comprises thiocolchicoside.
[0131] In another particular embodiment, antagonists or inverse
agonists of the GABA A receptors of use can be, but are not limited
to, one of those described in WO2011/153377, WO2012/059482,
EP2457569, and WO2011/024115, which are herein incorporated by
reference.
[0132] In an embodiment, antagonists of the Group I metabotropic
receptors that can be used in a combination of drugs used as an
alternative to acamprosate may be, but are not limited to, one of
those disclosed in WO2004014370A2, WO2004014881A2, WO2004014902A2,
WO2005080356A1, WO2005080363A1, WO2005080386A1, WO2006014185A1,
WO2007021574A1, WO2007021575A2, WO2007130825A2, WO2009051556A1,
WO2009054785A1, WO2009054786A1, WO2009054789A1, WO2009054790A1,
WO2009054791A1, WO2009054792A1, WO2009054793 A1, WO2009054794A1,
WO2010123451A1, WO02068417A2, WO2005066155A1, WO2004000316A1,
WO2005080379A1, WO2005080397A2, WO2007040982A1, WO2010019100A1,
WO2007130820A2, WO2007130821 A2, WO2007130822A2, WO2007130823 A2,
WO2007130824A2, WO2008041075A1, WO2009054787A1, WO9926927A2,
WO2004069813A1, WO2012127393 A1, WO2003047581A1, and WO2008128968,
which are incorporated herein by reference.
[0133] Further antagonists of the Group I metabotropic receptors
that can be used are disclosed in WO2012108831 A1 and are
incorporated herein by reference.
[0134] Yet further antagonists of the Group I metabotropic
receptors that can be used are disclosed in WO2012127393A1 and are
incorporated herein by reference.
[0135] Other antagonists of the Group I metabotropic receptors that
can be used are disclosed in WO2010048095A2 and are incorporated
herein by reference.
[0136] Antagonists of the Group I metabotropic receptors have been
the focus of numerous research programs in drug discovery which are
summarized by Jaeschke et al (2007) [50], Carroll (2008) [51] and
Emmitte (2013) [52]. All the antagonists of the Group I
metabotropic receptors disclosed in these reviews are incorporated
herein by reference and can be considered to be used in a
combination of drugs used as an alternative to acamprosate.
[0137] In a particular embodiment, said combination of drugs used
as an alternative to acamprosate comprises at least one compound
having an antagonistic activity for mGluR1 metabotropic receptors
and/or at least one compound having an antagonistic activity for
mGluR5 metabotropic receptors.
[0138] In a particular embodiment, said combination of drugs used
as an alternative to acamprosate comprises at least one compound
selected from AIDA, A-794278, A-794282, A-841720, A-850002,
Bay-36-7620, FTIDC, JNJ-16259685, LY-367385, RO0711401, YM-202074,
YM-230888, YM-298198, ADX 10059, AZD-2066, AZD6538, AZD9272,
basimglurant, dipraglurant, fenobam, GRN529, LY-344545,
mavoglurant, MPEP, MRZ-8676, MTEP, rufinamide, SIB-1757, and
SIB-1893, which are known as (direct or indirect) antagonists of
the Group I metabotropic receptors.
[0139] In a preferred embodiment, said combination of drugs used as
an alternative to acamprosate comprises rufinamide.
[0140] In another preferred embodiment, said combination of drugs
used as an alternative to acamprosate comprises a compound selected
from AIDA, A-794278, A-794282, A-841720, A-850002, Bay-36-7620,
FTIDC, JNJ-16259685, LY-367385, RO0711401, YM-202074, YM-230888,
YM-298198 and a compound selected from ADX 10059, AZD-2066,
AZD6538, AZD9272, basimglurant, dipraglurant, fenobam, GRN529,
LY-344545, mavoglurant, MPEP, MRZ-8676, MTEP, rufinamide, SIB-1757,
and SIB-1893.
[0141] In a particular embodiment, said combination of drugs used
as an alternative to acamprosate comprises a compound acting as an
antagonist of both mGluR1 and mGluR5 receptors.
[0142] In an embodiment, combination of drugs used as an
alternative to acamprosate comprises a drug that acts as an
antagonist or negative allosteric modulator of mGluR2 or mGluR3
(group II) metabotropic glutamate receptors.
[0143] In another embodiment, combination of drugs used as an
alternative to acamprosate comprises a drug that acts as an
antagonist or negative allosteric modulator of mGluR2 and mGluR3
(group II) metabotropic glutamate receptors.
[0144] Antagonists of group 11 metabotropic glutamate receptors
comprise, but are not limited to, EGLU, APTCA, LY-341495, BCI-632
or its prodrugs BCI-1038, BCI-1206, BCI-1283, BCI-838, or one of
the 2-amino-bicyclo [3.1.0] hexane-2,6-dicarboxylic ester
derivatives disclosed in WO2005000791, US2012028982, US2012004232,
US2010298561, US2009306408. US2007021394, or 5H-thiazolo (3,2-a)
pyrimidine derivatives disclosed in U.S. Pat. No. 5,958,931, which
are incorporated herein by reference.
[0145] Negative allosteric modulators of the Group II metabotropic
receptors that can be used in a combination of drugs used as an
alternative to acamprosate comprise, but are not limited to,
DT-2228 as disclosed in Froestl et al (2012) [53] and incorporated
herein by reference, RG-1578, R04432717, RO4491533, dihydrobenzo
[1,4] diazepin-2-one derivatives disclosed in Hempstapat et al 2007
[54] and incorporated herein by reference, and compounds described
in WO 2014/064028, WO 01/29011, WO 01/29012, WO 02/083652, WO
02/083665, WO 02/098864, WO 03/066623, WO 2005/014002, WO
2005/040171, WO2005/123738, WO 2006/084634, WO 2006/099972, WO
2007/039439, WO 2007/110337 and WO 2008/119689 and incorporated
herein by reference.
[0146] In another embodiment, said combination of drugs used as an
alternative to acamprosate comprises a drug that acts as an agonist
or positive allosteric modulator of the glycinic channels.
[0147] In a particular embodiment, said combination of drugs
comprises anandamide, cycloserine, dimethylglycine, dronabinol,
enflurane, glycine, halothane, HU 210, hypotaurine, isoflurane,
milacemide, sarcosine, serine, sevoflurane, taurine,
trimethylglycine, D and/or L alanine, or .beta. alanine, which are
known as agonists or positive allosteric modulators of the glycinic
channels.
[0148] Other agonists or positive allosteric modulators of the
glycinic channels that can be used in said combination of drugs
comprise propofol derivatives described in WO 2010/067069, which
are incorporated herein by reference. Agonists or positive
allosteric modulators of the glycinic channels also comprise, but
are not limited to, tropines and nortropines which are described in
Maksay el al. (2007) [55] and are also herein incorporated by
reference, as well as those listed in Yevenes & Zeilhofer
[56].
[0149] Therapy according to the invention may be provided at home,
the doctor's office, a clinic, a hospital's outpatient department,
or a hospital, so that the doctor can observe the therapy's effects
closely and make any adjustments that are needed.
[0150] The duration of the therapy depends on the stage of the
disease being treated, 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.
[0151] 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's condition or efficiently treat the disease or
disorder.
[0152] While it is possible for the drugs of the combination to be
administered as the pure chemicals, 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.
[0153] More commonly these pharmaceutical formulations are
prescribed to the patient in "patient packs" containing a number of
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
formulation. 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 use 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.
[0154] The drug may be contained, in any appropriate amount, in any
suitable carrier substance. The drug may be present in an amount of
up to 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., intravenous, intramuscular), 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.
[0155] 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).
[0156] 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.
[0157] 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.
[0158] Administration of drugs in the form of a controlled release
formulation is especially preferred in cases in which the drug 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 gastrointestinal
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.
[0159] 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).
[0160] Solid Dosage Forms for Oral Use Formulations for oral use
include tablets containing the composition of the invention 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); or lubricating agents, glidants, or
antiadhesives (e.g., stearic acid, silicas, or talc). Other
pharmaceutically acceptable excipients can be colorants, flavoring
agents, plasticizers, humectants, buffering agents, and the
like.
[0161] 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 provide 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.
[0162] 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 to that described in Encyclopedia of
Pharmaceutical Technology.
[0163] Drugs may be mixed together in the tablet, or may be
partitioned. For example, a first drug is contained on the inside
of the tablet, and a 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.
[0164] 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.
[0165] 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.
[0166] Dissolution or diffusion-controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulated
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 hydrogel,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycol. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0167] 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
[0168] 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
[0169] 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.
[0170] 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, and/or dispersing agents.
[0171] The pharmaceutical compositions according to the invention
may be in a 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 pH 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.
[0172] 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 polygalactin,
poly-(isobutyl cyanoacrylate), and
poly(2-hydroxyethyl-L-glutamine). 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
[0173] 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 glycol. Various additives,
enhancers, or surfactants may be incorporated.
[0174] 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.
[0175] The preservatives, humectants, and penetration enhancers may
be parabens, such as methyl or propyl p-hydroxybenzoate,
benzalkonium chloride, glycerin, propylene glycol, urea, etc.
[0176] 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
[0177] 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 use the combination according to the
invention.
[0178] Therapeutically effective amounts of the drugs in a
combination of this invention include, e.g., amounts that are
effective for reducing Alzheimer's disease symptoms, halting or
slowing the progression of the disease once it has become
clinically manifest, or preventing or reducing the risk of
developing the disease.
[0179] 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.
[0180] Administration 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.
[0181] 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.
[0182] The amount of each drug in a preferred unit dosage
composition depends upon several factors including the
administration method, the body weight and the age of the patient,
the stage of the disease, and the risk of potential side effects
considering the general health status of the person to be treated.
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.
[0183] Except when responding to especially impairing cases, where
higher dosages may be required, the preferred dosage of each drug
in the combination will usually lie within the range of doses not
above the dosage usually prescribed for long-term maintenance
treatment or proven to be safe in phase 3 clinical studies.
[0184] 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 individually have little or no 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.
[0185] At such sub-therapeutic dosages, the compounds would exhibit
no side effects, while the combination(s) according to the
invention are fully effective in treating Alzheimer's disease or AD
related diseases.
[0186] A preferred dosage corresponds to amounts from 1% up to 50%
of those usually prescribed for long-term maintenance
treatment.
[0187] The most preferred dosage may correspond to amounts from 1%
up to 10% of those usually prescribed for long-term maintenance
treatment.
[0188] Specific examples of dosages of drugs for use in the
invention are provided below: [0189] Acamprosate between 0.1 and
1000 mg per day, preferably less than 400 mg per day, more
preferably less than 200 mg per day, even more preferably 100 mg
per day or less, furthermore preferably between 0.5 mg and 100 mg
per day, typically 0.8 mg per day, 2 mg per day, 20 mg per day, 40
mg per day, or 80 mg per day, such dosages being particularly
suitable for oral administration, [0190] Baclofen between 0.01 to
150 mg per day, preferably less than 100 mg per day, more
preferably less than 50 mg per day, most preferably between 5 and
40 mg per day, even more preferably less than 35 mg per day,
typically 12 mg per day, 24 mg per day, or 30 mg per day, such
dosages being particularly suitable for oral administration, [0191]
Aminocaproic acid orally from about 0.1 g to 2.4 g per day, [0192]
bromocriptine orally from about 0.01 to 10 mg per day, [0193]
diethylcarbamazine orally from about 0.03 to 400 mg per day, [0194]
cabergoline orally from about 1 to 10 .mu.g per day, [0195]
cinacalcet orally from about 0.3 to 36 mg per day, [0196]
cinnarizine orally from about 0.6 to 23 mg per day, [0197]
dyphylline orally from about 9 to 320 mg per day, [0198] eplerenone
orally from about 0.25 to 10 mg per day, [0199] ifenprodil orally
from about 0.4 to 6 mg per day, [0200] leflunomide orally from
about 0.1 to 10 mg per day, [0201] levosimendan orally from about
0.04 to 0.8 mg per day, [0202] mexiletine orally from about 6 to
120 mg per day, [0203] moxifloxacin orally from about 4 to 40 mg
per day, [0204] phenformin orally from about 0.25 to 15 mg per day,
[0205] quinacrine orally from about 1 to 30 mg per day, [0206]
sulfisoxazole orally from about 20 to 800 mg per day, [0207]
sulodexide orally from about 0.05 to 40 mg per day, [0208]
terbinafine orally from about 2.5 to 25 mg per day, [0209]
torasemide orally from about 0.05 to 4 mg per day, [0210]
trimetazidine orally from about 0.4 to 6 mg per day, and [0211]
zonisamide orally from about 0.5 to 50 mg per day, administered in
one, two, or three doses daily.
[0212] When the composition comprises, as active ingredients, only
baclofen and acamprosate, these two compounds may be used in
different ratios, e.g., at a weight ratio of acamprosate/baclofen
comprised from between 0.05 to 1000 (W:W), preferably between 0.05
to 100 (W:W), more preferably between 0.05 to 50 (W:W).
[0213] As already mentioned, the above compounds in the therapies
of the invention can be used as specifically designated per se, as
well as any pharmaceutically acceptable salts, enantiomers,
racemates, prodrugs, metabolites or derivatives thereof.
[0214] In a particularly preferred embodiment, combinatorial
therapies of the invention comprise administering between 0.4 mg
and 50 mg acamprosate and 6 mg to 15 mg baclofen, twice daily.
[0215] In an embodiment, combinatorial therapies of the invention
comprise administering 0.4 mg acamprosate and 6 mg baclofen, twice
daily.
[0216] In a preferred embodiment, combinatorial therapies of the
invention comprise administering 1 mg acamprosate and 15 mg
baclofen, twice daily.
[0217] In yet another preferred embodiment, combinatorial therapies
of the invention comprise administering 10 mg acamprosate and 6 mg
baclofen, twice daily.
[0218] In another preferred embodiment, combinatorial therapies of
the invention comprise administering 20 mg acamprosate and 12 mg
baclofen, twice daily.
[0219] In still another embodiment, combinatorial therapies of the
invention comprise administering 40 mg acamprosate and 12 mg
baclofen, twice daily.
[0220] In another particular embodiment, besides comprising
administering one of the above baclofen-acamprosate regimens,
therapies of the invention also comprise administering donepezil or
memantine either at their usual dose and regimen (i.e., as an
add-on therapy) or even at a lower dose, from 1% up to 50% of those
usually prescribed for the treatment of AD.
[0221] In an even more particular embodiment, combinatorial
therapies of the invention further comprise administering 0.6 mg
donepezil twice daily.
[0222] Consequently, in a particularly preferred embodiment,
combinatorial therapies of the invention comprise administering
between 0.4 mg and 50 mg of acamprosate, 6 mg to 15 mg baclofen,
and 0.6 mg donepezil, twice daily.
[0223] In an embodiment, combinatorial therapies the invention
comprise administering 0.4 mg acamprosate, 6 mg baclofen, and 0.6
mg donepezil, twice daily.
[0224] In a preferred embodiment, combinatorial therapies of the
invention comprise administering 1 mg acamprosate, 15 mg baclofen,
and 0.6 mg donepezil, twice daily.
[0225] In yet another preferred embodiment, combinatorial therapies
of the invention comprise administering 20 mg acamprosate, 12 mg
baclofen, and 0.6 mg donepezil, twice daily.
[0226] In a particularly preferred embodiment, combinatorial
therapies of the invention comprise administering 40 mg
acamprosate, 12 mg baclofen, and 0.6 mg donepezil, twice daily.
[0227] As previously stated, the compounds in a combinatorial
therapy of the invention may be administered simultaneously,
separately, sequentially and/or repeatedly to the subject.
Particularly, the above dosing regimens can be simultaneously
orally administered using suitable tablets. Also, a particular
object of this invention relates to a tablet comprising acamprosate
and baclofen, suitable for the dosing regimen of a dose of
acamprosate between 0.4 and 50 mg and a dose of baclofen between 6
and 15 mg, to be administered twice daily.
[0228] A further particular object of this invention relates to a
tablet comprising acamprosate and baclofen, suitable for the dosing
regimen of 0.4 mg acamprosate and 6 mg baclofen, twice daily.
[0229] Another particular object of this invention relates to a
tablet comprising acamprosate and baclofen, suitable for the dosing
regimen of 1 mg acamprosate and 15 mg baclofen, twice daily.
[0230] Another particular object of this invention relates to a
tablet comprising acamprosate and baclofen, suitable for the dosing
regimen of 20 mg acamprosate and 12 mg baclofen, to be administered
twice daily.
[0231] A particular object of this invention relates to a tablet
comprising acamprosate and baclofen, suitable for the dosing
regimen of 40 mg acamprosate and 12 mg baclofen, to be administered
twice daily.
[0232] An even more particular object of this invention relates to
a tablet comprising baclofen, acamprosate and donepezil, said
tablet being suitable for any of the above mentioned dosages of
baclofen and acamprosate and also for the dosing regimen of 0.6 mg
donepezil, twice daily.
[0233] A more particular object of the invention is a scored tablet
suitable for the administration of any of the above dosing
regimens, said tablet being cleavable in 2, 3 and/or 4 part as a
function of the dose to be administered at each taking.
[0234] As mentioned above, a single unit dosage form containing the
combinations of the invention is most preferred. Alternatively,
where a separate administration would be considered more proper,
combinations of the invention can be provided in the form of a unit
dosage package, such unit dosage package being configured to hold a
first unit dosage comprising acamprosate and a second unit dosage
comprising baclofen. In a particular embodiment unit dosages are
tablets. In another particular embodiment the first unit dosage
comprises between 0.4 and 50 mg of acamprosate and the second unit
dosage comprises between 6 and 15 mg baclofen.
[0235] In another embodiment the first unit dosage is suitable for
the dosing regimen of 0.4 mg acamprosate, twice daily.
[0236] In another embodiment the first unit dosage is suitable for
the dosing regimen of 1 mg acamprosate, twice daily.
[0237] In another embodiment the first unit dosage is suitable for
the dosing regimen of 20 mg acamprosate, twice daily.
[0238] In another embodiment the first unit dosage is suitable for
the dosing regimen of 40 mg acamprosate, twice daily.
[0239] In an embodiment the first unit dosage is suitable for the
dosing regimen of 1 mg acamprosate, twice daily.
[0240] In another embodiment the second unit dosage is suitable for
the dosing regimen of 6 mg baclofen, twice daily.
[0241] In another embodiment the second unit dosage is suitable for
the dosing regimen of 12 mg baclofen, twice daily.
[0242] In another embodiment the second unit dosage is suitable for
the dosing regimen of 15 mg baclofen, twice daily.
[0243] In another particular embodiment, the above mentioned unit
dosage package comprises a third unit dosage comprising donepezil.
In a more particular embodiment this third unit dosage is suitable
for the dosing regimen of 0.6 mg donepezil, twice daily. In a
particular embodiment said unit dosage package contains a number of
unit dosages suitable for 1, 2, 3, 4, 5, 6, 7 days of treatment or
even more, preferably several weeks of treatment.
[0244] It will be understood that the amount of the drug actually
administered will be determined by a physician, in light of the
relevant circumstances including the condition 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.
[0245] The following examples are given for purposes of
illustration and not by way of limitation.
Examples
[0246] The care and husbandry of animals as well as the experiments
are performed according to the guidelines of the Committee for
Research and Ethical Issue of the I.A.S.P. (1983).
[0247] I. Treatment of Diseases Related to A.beta. Toxicity
[0248] In this series of experiments, candidate combinations have
been tested for their ability to prevent or reduce the toxic
effects of human A.beta..sub.1-42. A.beta..sub.1-42 is the
full-length peptide that constitutes aggregates found in biopsies
from human patients afflicted with AD. The effect is determined on
various cell types, to further document the activity of the
combinations 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 combinations on i) the cognitive
performance of the animals and ii) molecular hallmarks (apoptosis
induction, oxidative stress induction, inflammation pathway
induction) of AD. Clinical results show that baclofen and
acamprosate-based compositions are actually efficient in improving
cognitive performance, as well as in correcting
electrophysiological disturbances observed in patients diagnosed
with mild AD.
A. Baclofen-Acamprosate Combination Therapies Prevent Toxicity of
Human A.beta..sub.1-42 In Vitro
[0249] 1. Effect on the Toxicity of Human A.beta..sub.1-42 Peptide
on Human Brain Microvascular Endothelial Cells (HBMEC).
HBMEC Culture Conditions
[0250] HBMEC cultures were used to study the protection afforded by
candidate compound(s) on A.beta..sub.1-42 toxicity.
[0251] HBMEC (ScienCell Ref: 1000, frozen at passage 10) were
rapidly thawed in a water bath at +37.degree. C. The supernatant
was immediately put in 9 ml Dulbecco's modified Eagle's medium
(DMEM; Pan Biotech ref: P04-03600) containing 10% fetal calf serum
(FCS; GIBCO ref 10270-106). Cell suspension was centrifuged at
180.times.g for 10 min at +4.degree. C. and the pellets were
suspended in CSC serum-free medium (CSC serum-free medium. Cell
System, Ref: SF-4Z0-500-R, Batch 51407-4) with 1.6% serum-free
RocketFuel (Cell System, Ref: SF-4Z0-500-R, Batch 54102), 2% of
Penicillin 10,000 U/ml and Streptomycin 10 mg/ml (PS; Pan Biotech
ref: P06-07100 batch 133080808) and were seeded at the density of
20,000 cells per well in 96-well plates (matrigel layer biocoat
angiogenesis system, BD, Ref 354150, Batch A8662) in a final volume
of 100 .mu.l. On matrigel support, endothelial cerebral cells
spontaneously started the process of capillary network
morphogenesis [57].
[0252] Three separate cultures were performed per condition, 6
wells per condition.
Test Compounds and Human A.beta..sub.1-42 Treatment
[0253] Briefly, A.beta..sub.1-42 peptide (Bachem, ref: H1368 batch
1010533) was reconstituted in defined culture medium at 20 .mu.M
(mother solution) and was slowly shaken at +37.degree. C. for 3
days in the dark. The control medium was prepared in the same
conditions.
[0254] After 3 days, human amyloid peptide was used on HBMEC at 2.5
.mu.M diluted in control medium (optimal incubation time). The
A.beta..sub.1-42 peptide was added 2 hours after HBMEC seeding on
matrigel for 18 hours incubation.
[0255] One hour after HBMEC seeding on matrigel, test compounds and
VEGF-165 were solved in culture medium (+0.1% DMSO) and then
pre-incubated with HBMEC for 1 hour before the A.beta..sub.1-42
application (in a final volume per culture well of 100 .mu.l). One
hour after test compounds or VEGF incubation (two hours after cell
seeding on matrigel), 1001l of A.beta..sub.1-42 peptide was added
to a final concentration of 2.5 .mu.M diluted in control medium in
the presence of test compounds or VEGF (in a 200 .mu.l total
volume/well), in order to avoid further drug dilutions
Organization of Culture Plates
[0256] VEGF-165, known to be a pro-angiogenic isoform of VEGF-A,
was used for all experiments 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 10 nM
(FIG. 1).
[0257] The following conditions were assessed: [0258] Negative
Control: medium alone+0.1% DMSO. [0259] Intoxication:
amyloid-.beta..sub.1-42 (2.5 .mu.M) for 18 h. [0260] Positive
control: VEGF-165 (10 nM) (1 reference compound/culture) 1 h before
the A.beta..sub.1-42 (2.5 .mu.M) addition for an 18 h incubation
time. [0261] Test compounds: Test compound(s) 1 h before the
A.beta..sub.1-42 (2.5 .mu.M) addition for an 18 h incubation
time.
Capillary Network Quantification
[0262] Per well, 2 pictures with 4.times. lens were taken using
InCell Analyzer.TM. 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
[0263] Data were expressed in percentage of control conditions (no
intoxication, no amyloid=100%) in order to express the amyloid
injury. All values were expressed as mean+/-SEM (s.e. mean) of the
3 cultures (n=6 wells per condition). Statistical analyses were
done on the different conditions (ONE-WAY ANOVA followed by the
Dunnett's test when it was allowed, Statview software version
5.0).
Results
[0264] The baclofen-acamprosate combination gives a significant
protective effect against toxicity of human A.beta..sub.1-42
peptide in an HBMEC model (a reduction of 24% of A.beta..sub.1-42
peptide injury is observed), as shown in FIG. 2. The results
clearly show that the intoxication by human amyloid peptide
(A.beta..sub.1-42 2.5 .mu.M) is significantly prevented by the drug
combination whereas, at those concentrations, the drugs alone have
no significant effect on intoxication in the experimental
conditions described above.
[0265] Conversely, the combination of baclofen and terbinafine
(which is presented here only for the sake of comparison) affords a
weaker protection (a reduction of 15% of A.beta..sub.1-42 peptide
injury is observed) against A.beta..sub.1-42 (FIG. 3).
[0266] Thus, although these two combinations allow a protection
against A.beta..sub.1-42, the combination of baclofen-acamprosate
clearly stands out. Indeed, these drugs at concentrations having no
effect alone allow significant protection of human HBMEC against
A.beta..sub.1-42 when used in combination. Furthermore, the
baclofen-acamprosate combination is more effective than the
baclofen-terbinafine combination. Such an effect of baclofen and
acamprosate represents a remarkable improvement, by 60%, in
comparison to, e.g., the effect of the combination of
baclofen-terbinafine.
[0267] Moreover, the concentration of baclofen used in the
baclofen-acamprosate combination is much lower than the
concentration of baclofen used in the baclofen-terbinafine
combination (25-fold reduction).
[0268] 2. Effect on the Toxicity of Human a A.beta..sub.1-42
Peptide on Primary Cortical Neuron Cells.
[0269] a) Baclofen-Acamprosate Combination has a Neuroprotective
Effect
Culture of Primary Cortical Neurons
[0270] Rat cortical neurons were cultured as described by Singer et
al. [58]. 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 10 mg/ml and 1% of bovine serum
albumin (BSA). Cortices were dissociated by trypsin for 20 min at
37.degree. C. (0.05%). The reaction was stopped by the addition of
Dulbecco's modified Eagle's medium (DMEM) containing DNaseI 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.times.g for 10 min at +4.degree. 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 (0.2 mM), 2% of PS solution and 10 ng/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 (10 .mu.g/ml)) and were cultured at +37.degree. C. in
a humidified air (95%)/CO.sub.2 (5%) atmosphere.
[0271] Three independent cultures will be performed per condition,
6 wells per condition.
Test Compounds and Human Amyloid-.beta..sub.1-42 Treatment
[0272] Briefly, A.beta..sub.1-42 peptide was reconstituted in
defined culture medium at 40 .mu.M (mother solution) and was slowly
shaken at +37.degree. C. for 3 days in the dark. The control medium
was prepared in the same conditions.
[0273] After 3 days, the solution was used on primary cortical
neurons as follows.
[0274] After 10 days of neuron culture, test compounds were solved
in culture medium (+0.1% DMSO) and then pre-incubated with neurons
for one hour before the A.beta..sub.1-42 application (in a final
volume per culture well of 100 .mu.l). One hour after test
compound(s) incubation, 100 .mu.l of A.beta..sub.1-42 peptide was
added to a final concentration of 10 .mu.M diluted in the presence
of the drug(s), in order to avoid further test compound(s)
dilutions. Cortical neurons were intoxicated for 24 h. Three
separate cultures were performed per condition, 6 wells per
condition.
[0275] BDNF (50 ng/ml) and Estradiol-.beta. (150 nM) were used as
positive control and reference compounds respectively. Three
separate cultures were performed per condition, 12 wells per
condition.
Organization of Cultures Plates
[0276] Estradiol-.beta. at 150 nM was used as a positive control
(FIG. 4).
[0277] Estradiol-.beta. was solved in culture medium and
pre-incubated for 1 h before the amyloid-.beta..sub.1-42
application.
[0278] The following conditions were assessed: [0279] CONTROL
PLAQUE: 12 wells/condition [0280] Negative Control: medium
alone+0.1% DMSO, [0281] Intoxication: amyloid-.beta..sub.1-42 (10
.mu.M) for 24 h, and [0282] Reference compound: Estradiol (150 nM)
t h. [0283] DRUG PLATE: 6 wells/condition [0284] Negative Control:
medium alone+0.1% DMSO, [0285] Intoxication:
amyloid-.beta..sub.1-42 (10 .mu.M) for 24 h, and [0286] Test
compound(s): test compound(s)--1 h followed by
amyloid-.beta..sub.1-42 (10 .mu.M) for 24 h.
Lactate Dehydrogenase (LDH) Activity Assay
[0287] 24 hours after intoxication, the supernatant was taken off
and analyzed with the 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 activity released from
the cytosol of dying cells into the supernatant.
Data Processing
[0288] Data were expressed in percentage of control conditions (no
intoxication, no amyloid=100%) in order to express the amyloid
injury. All values were expressed as mean+/-SEM (s.e. mean) of the
3 cultures (n=6 wells per condition). Statistical analyses were
done on the different conditions (one-way ANOVA followed by the
Dunnett's test when it was allowed, Statview software version
5.0).
Results
[0289] The combination of baclofen and acamprosate induces a
significant protective effect against the toxicity of human
A.beta..sub.1-42 peptide (improvement of 34% of cell survival) in
primary cortical neuron cells, as shown in FIG. 5. The results
clearly show that intoxication by human amyloid peptide
(A.beta..sub.1-42 10 .mu.M) is significantly prevented by the
combination, whereas at those concentrations, baclofen or
acamprosate alone have no significant effect on intoxication.
[0290] Conversely, although active in this model, the combination
of sulfisoxazole and cinacalcet affords a weaker protection against
A.beta..sub.1-42 (19%, FIG. 6).
[0291] Thus, while those two combinations allow a protection
against A.beta..sub.1-42, the combination baclofen-acamprosate
stands out clearly. Indeed, at concentrations having no effect
alone, the drugs cause a significant protection of primary cortical
neuron cells against A.beta..sub.1-42 when used in combination.
Furthermore, the baclofen-acamprosate combination is much more
effective than the sulfisoxazole-cinacalcet combination Such an
effect of baclofen and acamprosate represents a remarkable
improvement by 60% in comparison to, e.g., the effect of the
combination of sulfisoxazole and cinacalcet.
[0292] Taken together these results show an unexpected and
remarkable positive effect of baclofen-acamprosate combinations in
several in vitro models of Alzheimer's disease. The effect observed
is highly superior to that provoked by other baclofen-based
combination therapies (e.g., baclofen-terbinafine), or other active
combination therapies (sulfisoxazole-cinacalcet).
[0293] A comparison of acamprosate and homotaurine protection
activity on cortical cells has been done (FIG. 17). Those results
show that the derivative of acamprosate, called homotaurine, allows
an effective protection against A.beta..sub.1-42. In the context of
this invention, baclofen or acamprosate can thus be substituted by
their derivatives, provided that those derivatives are efficient in
the assay described herein.
[0294] b) Cellular Pathways Triggered in Neurons by
A.beta..sub.1-42 Oligomers are Reversed Upon Baclofen-Acamprosate
Treatment
[0295] Mitochondrial dysfunction is thought to play a critical role
in AD by producing two major cellular consequences: oxidative
stress and cell death by apoptosis. The effect of
baclofen-acamprosate treatment on oxidative stress and apoptosis
generated by A.beta. oligomers has been assessed by measuring the
oxidation of methionine residues (methionine sulfoxide, MetO) and
by measuring the release of mitochondrial cytochrome C (Cyto C, as
a marker of early apoptosis) into the cytoplasm of intoxicated
neuronal cells. The effect on A.beta.-induced apoptosis was further
confirmed by measuring the level of caspase 3, a marker of late
apoptosis.
[0296] Other hallmarks of AD are i) the accumulation of
hyperphosphorylated Tau proteins (pTau) resulting in the formation
of neurofibrillary tangles and ii) excitotoxicity due to an excess
of glutamate. The effect of baclofen-acamprosate treatment on Tau
phosphorylation within A.beta.-intoxicated neuronal cells and on
glutamate accumulation in culture medium was assessed (FIG.
21).
Culture of Primary Cortical Neurons, A.beta..sub.1-42 Oligomer
Formation and Cell Intoxication
[0297] Cell culture, formation of A.beta..sub.1-42 oligomer and
cell intoxication were mainly performed as above. Variable
intoxication times and A.beta. concentrations were used depending
on the experiment and are summarized in Table 5 below.
TABLE-US-00005 TABLE 5 A.beta..sub.1-42 Days Seeding Intoxication
of density time Assay culture (cells/well) Concentration (h) MetO
11 15,000 1.25 .mu.M 4 Cyto c 11 30,000 1.25 .mu.M 4 Caspase 3 11
30,000 10 .mu.M 24 Tau 11 30,000 2.5 .mu.M 16 phosphorylation
Glutamate release 13 30,000 2.5 .mu.M 4
Methionine Sulfoxide (MetO), Cytochrome c (Cyto c), Caspase 3, and
Phosphorylated Tau (pTau) Assays:
[0298] After A.beta..sub.1-42 intoxication, cells were fixed,
permeabilized, and non-specific sites were blocked with a solution
of phosphate buffered saline (PBS; PanBiotech) containing 0.1% of
saponin (Sigma) and 1% FCS. Then, cells were incubated with one of
the assay specific primary antibodies (Rabbit polyclonal anti MetO,
1/100, Euromedex, France; Rabbit polyclonal anti Cyto c, 1/100,
Abcam; Rabbit polyclonal anti Caspase 3, 1/500, Sigma; Mouse
monoclonal anti PHF-Tau, clone AT100, 1/100, Thermo Scientific) and
with MAP2 primary antibody (Mouse monoclonal anti MAP2, 1/400,
Sigma Aldrich or Chicken polyclonal anti MAP2, 1/400, Abcam), which
are revealed with the fitting secondary antibodies (Alexa Fluor 488
goat anti mouse IgG; Alexa Fluor 568 goat anti rabbit IgG; Alexa
Fluor 568 goat anti chicken IgG, all the three from Invitrogen and
used at the 1/400 dilution).
[0299] Nuclei were counter-stained with Hoechst (Sigma). For MetO
and Cyto c on the one hand, and pTau on the other hand, 20 and 10
pictures with 20.times. and 40.times. magnifications respectively
were taken per well using the InCell Analyzer.TM. 1000 (GE
Healthcare, France)
[0300] Analysis was done using Developer software (GE Healthcare)
assessing the overlap between MAP2 on one side, and MetO, Cyto c,
Caspase 3 or pTau staining on the other side. Results were
expressed as the number of overlapping stained cells per field and
reported as a percentage of vehicle treated control.
Glutamate Release Assays:
[0301] After 4 h of A.beta..sub.1-42 intoxication, cell media
supernatants were analysed with the Amplex Red Glutamic Acid assay
kit (Invitrogen) according to the manufacturer's instructions.
Results
[0302] Results presented in FIG. 21 show that the
baclofen-acamprosate combination corrects the major hallmarks of
AD. Indeed, treatment with the baclofen-acamprosate combination
leads to a protection against oxidative stress, as demonstrated by
a significantly lower content in methionine sulfoxide residues
(MetO) in A.beta.-intoxicated cells treated with
baclofen-acamprosate when compared to non-treated intoxicated cells
(FIG. 21 A). A marked reduction of the release of mitochondrial
cytochrome C is also noticed in baclofen-acamprosate treated
A.beta.-intoxicated cells thereby showing that the combination is
efficient in protecting the neuronal cells from A.beta. induced
apoptosis. (FIG. 21 B). Such effect was confirmed by the
observation of a significant lowering of Caspase 3 upon
baclofen-acamprosate treatment (not shown). Treatment with
baclofen-acamprosate also significantly prevents Tau
hyperphosphorylation in A.beta..sub.1-42-treated neurons (FIG. 21
C), as well as glutamate accumulation in culture medium (FIG. 21
D), a feature mirroring excitotoxicity due to excess of glutamate
in AD.
[0303] Baclofen-acamprosate combination is thus efficient in
counteracting oxidative stress, apoptosis, hyperphosphorylated Tau
accumulation, and glutamate excitotoxicity induced in neuronal
cells by AB oligomers. These surprising properties can, at least in
part, account for the observed neuroprotective properties of the
composition and for the actual correction of the disease. Such
properties are of a particular interest for the treatment of AD and
related disorders, but also in the treatment of other neurological
disorders which share some of these features with AD. For instance,
the control of the events leading to the accumulation of pTau by
the administration of the baclofen-acamprosate combination is
particularly relevant when considering the treatment of Tau
pathologies such as frontotemporal dementia.
[0304] c) Protection Against the Toxicity of A.beta..sub.1-42 in a
Neurite Growth and Synapse Functionality Model.
Culture of Primary Cortical Neurons, A.beta..sub.1-42 Oligomer
Formation and Cell Intoxication
[0305] Rat cortical neurons were cultured as described by Singer el
al. [58]. 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 10 mg/ml and 1% of bovine serum
albumin (BSA). Cortices were dissociated by trypsin for 20 min at
37.degree. C. (0.05%). The reaction was stopped by the addition of
Dulbecco's modified Eagle's medium (DMEM) containing DNaseI 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.times.g for 10 min at +4.degree. 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 (0.2 mM), 2% of PS solution and 10 ng/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 (10 .mu.g/ml)) and were cultured at +37.degree. C. in
a humidified air (95%)/CO.sub.2 (5%) atmosphere.
[0306] After 10 days of culture, cells are incubated with drugs.
After 1 hour, cells are intoxicated by 2.5 .mu.M of beta-amyloid
(1-42; Bachem) in defined medium without BDNF but together with
drugs. Cortical neurons are intoxicated for 24 hours. BDNF (10
ng/ml) is used as a positive (neuroprotective) control. Three
independent cultures were performed per condition, 6 wells per
condition.
Neurite Length and Synapse Quantitation
[0307] After 24 h 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-associated-protein 2 (MAP-2; Sigma) or with
anti-synaptophysin (SYN, S5798, Sigma) together with anti-PSD95
(P246, Sigma) antibodies in order to quantify synapses. These
antibodies specifically stain cell bodies and neurites of neurons
(MAP2) or pre- and postsynaptic elements (SYN and PSD95,
respectively).
[0308] These antibodies are revealed with Alexa Fluor 488 goat
anti-mouse IgG (Molecular Probes). Nuclei of neurons were labeled
by a fluorescent marker (Hoechst solution, SIGMA).
[0309] Per well, 10 pictures are taken using InCell Analyzer.TM.
1000 (GE Healthcare) with 20.times. 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 the neurite network.
Results
[0310] The combination of baclofen and acamprosate induces a
significant protective effect against the toxicity of human
A.beta..sub.1-42 peptide (improvement of 80% of neurite network) in
primary cortical neuron cells as shown in FIG. 7. The results
clearly show that the intoxication by human amyloid peptide
(A.beta..sub.1-42 2.5 .mu.M) is significantly prevented by the
combination, whereas at those concentrations, baclofen or
acamprosate alone have no significant effect on intoxication.
[0311] Furthermore, the total length of the neurite network in
primary cortical neuron cells treated with this combination is no
more significantly different from control cells. Hence, this
combination allows an effective protection of cortical neuron cells
against the toxicity of human A.beta..sub.1-42 peptide but also a
neurite growth comparable to a sane cortical neuron cell.
[0312] d) Protection of Synapses of Hippocampal Neurons Against the
Toxicity of A.beta..sub.1-42 Oligomer Formation and Cell
Intoxication.
[0313] Culture of primary hippocampal neurons, A.beta..sub.1-42
oligomer formation and cell intoxication Hippocampus brain area is
a key player in the building processes of memory; also, hippocampus
atrophy is one of the most accurate pieces of evidence used in the
diagnosis of AD. A.beta. is known to have a synaptotoxic activity
which underlies the early cognitive decline in AD. The ability of
combinations of the invention to protect hippocampal neuronal
plasticity through protection of synapses from A.beta..sub.1-42
toxicity has then been evaluated. Rat hippocampal neurons were
cultured as described by Harrison (1990) [59]. Pregnant females
(Wistar, Janvier) at 17 days of gestation were killed by cervical
dislocation. Fetuses were collected and immediately placed in
ice-cold Leibovitz (L15; Panbiotech) containing 2% of Penicillin
10,000 U/ml and Streptomycin 10 mg/ml (PS; Panbiotech) and 1% of
bovine serum albumin (BSA; Panbiotech). Hippocampi were dissociated
by trypsin (0.05%, Panbiotech) for 20 min at 37.degree. C. The
reaction had been stopped by the addition of Dulbecco's modified
Eagle's medium (DMEM; Panbiotech) containing DNAase I grade II (0.5
mg/ml; Panbiotech) and 10% of foetal calf serum (FCS; Invitrogen).
Cells were then mechanically dissociated by 3 passages through a 10
ml pipette. Cells were centrifuged at 515 g for 10 min at
+4.degree. C. The supernatant was discarded and pellet of cells was
re-suspended in a defined culture medium consisting of Neurobasal
(Invitrogen) supplemented with B27 (2%; Invitrogen), L-glutamine (2
mM; Panbiotech), 2% of PS solution and 10 ng/ml of brain-derived
neurotrophic factor (BDNF, Panbiotech). Viable cells were counted
in a Neubauer cytometer, using the trypan blue exclusion test, then
seeded at a density of 20,000 per well in a 96-well plates
pre-coated with poly-L-Lysine (Greiner) and cultured at 37.degree.
C. in an air (95%)-CO2 (5%) incubator. The medium was changed every
2 days.
[0314] After 18 days of culture, cells were incubated with R/S
baclofen and acamprosate combination (80 nM and 0.32 nM
respectively). After 2 days of culture, hippocampal neurons were
intoxicated with A.beta..sub.1-42 human peptide (Bachem) at 0.3
.mu.M during 48 hours in the presence of the drug combination.
[0315] BDNF (50 ng/ml) has been used as positive control and
reference compound.
[0316] Three separate cultures were performed per condition, 6
wells per condition.
PSD95 and Synaptophysin Immunostaining, Synaptic Loss
Quantification.
[0317] After 48 hours of intoxication, cells were permeabilized and
non-specific sites were blocked with a solution of PBS containing
0.1% of saponin and 1% of FCS for 15 min and then were incubated
with mouse monoclonal primary antibody against Post Synaptic
Density 95 kDa (PSD95, Abcam) and with rabbit polyclonal primary
antibody against Synaptophysin (Sigma) overnight at 4.degree. C.
These antibodies were revealed with Alexa Fluor 488 goat anti-mouse
(Molecular probe) and Alexa Fluor 568 goat anti-rabbit (Molecular
probe) for 1 hour. Nuclei of cells were labelled by a fluorescent
marker (Hoechst solution, SIGMA). The total area of PSD95,
Synaptophysin and the colocalization were evaluated. Results were
expressed in .mu.m.sup.2 per field. For each well of culture, 40
pictures per well were taken using InCell Analyzer.TM. 2000 (GE
Healthcare) with 60.times. magnification. Colocalization of the two
labellings corresponds to intact synapses. The area was
automatically evaluated with Developer system analysis (GE
Healthcare). PSD95 and synaptophysin total surface overlap was
quantified (in .mu.m.sup.2) and computed for each condition.
[0318] Data were expressed in percentage of control conditions (no
intoxication, no A.beta..sub.1-42=100%) in order to express the
amyloid injury. All values were expressed as mean+/-SEM (s.e.
mean). Statistical analyses were done on the different conditions
(ANOVA followed by Dunnett's test or t-test).
Results
[0319] Results show that A.beta..sub.1-42 (0.3 .mu.M) intoxication
induces a significant lowering of PSD95 and synaptophysin
colocalization surface (almost 34%, FIG. 20, black bar) when
compared to non-intoxicated cells cultures. Incubation with the
baclofen-acamprosate (80 nM and 0.32 nM respectively) combination
is found to significantly reverse A.beta..sub.1-42 oligomer
toxicity toward synapse junctions: a 62% increase in colocalization
area is observed when compared to non-treated cells (FIG. 20, light
grey bar). Hence the baclofen-acamprosate combination is efficient
in maintaining synaptic junctions between hippocampal cells even in
the presence of A.beta. oligomers.
[0320] Baclofen-acamprosate therapy is thus efficient in
maintaining synapse function in the presence of A.beta. oligomers;
together with the neuritis growth protection as mentioned above,
this therapy can be thus considered as of particular interest for
protecting synaptic plasticity and cellular networking which is
impaired in AD.
[0321] e) Molecular Targets Important for Baclofen-Acamprosate
Mechanism of Action in Neuroprotection Against A.beta.
Toxicity.
Culture of Primary Cortical Neurons, Ligand Cell Intoxication
[0322] Culture of rat primary cortical cells, A.beta..sub.1-42
peptide preparation and cell intoxication were performed as stated
in I.A.2.a). Non-toxic concentrations of CGP54626 (10 .mu.M, GABABR
antagonist, Tocris Biosciences), muscimol (1 .mu.M, GABA.sub.AR
agonist, Sigma Aldrich), strychnine (2.5 .mu.M, Glycine receptor
antagonist, Tocris Biosciences), (S)-3,5-dihydroxyphenylglycine
(DHPG, mGluR1/5 agonist 10 .mu.M, Tocris Biosciences) and
(2R,4R)-amino-2,4-pyrrolidinedicarboxylic acid (APDC, mGluR2/3
agonist, 0.3 .mu.M, Sigma Aldrich) were dissolved in 0.1% DMSO
(except for DHPG dissolved in water) and added 2 h (except for
DHPG, 1 h) before ACP (8 nM) or BCL (400 nM) to 11 days rat primary
neuronal cell cultures.
[0323] BDNF at 50 ng/ml was used as a positive control.
Cell Survival Evaluation After 24 hours incubation with
A.beta..sub.1-42 peptide, cortical neurons were fixed by a cold
solution of alcohol/acetic acid during 5 minutes. Then, cells were
permeabilized and non-specific sites were blocked with a solution
of PBS containing 0.1% of saponin (Sigma) and 1% of fetal calf
serum for 15 min. Cells were then incubated with mouse monoclonal
primary microtubule-associated protein 2 antibody (MAP-2, Sigma)
for 2 hours in the same solution at the dilution of 1/400. An
incubation of 1 hour with Alexa Fluor 488 goat anti-mouse
(Molecular Probes) at 1/400 as a secondary antibody was thereafter
performed. Nuclei of cells were labelled by a fluorescent marker
(Hoechst solution, Sigma).
[0324] Total neuronal survival was evaluated by numbering MAP-2
positive neuronal cell bodies. Ten pictures per well of culture
were taken using InCell Analyzer.TM. 2000 (GE Healthcare) with
20.times. magnification. The number of neurons was automatically
evaluated with Developer system analysis (GE Healthcare).
[0325] Data are expressed in percentage of control conditions (no
intoxication, no amyloid=100%) in order to express the amyloid
injury. All values are expressed as mean+/-SEM (s.e. mean). An
ANOVA followed by Dunnett's test was done on each condition.
Results
[0326] Data gathered in these experiments show that CGP54626, an
orthosteric antagonist of GABA.sub.B receptors, blocks, at
non-toxic doses, the neuroprotective effect of baclofen (not
shown), which confirms the importance of GABA.sub.B receptor
activation for the neuroprotective action of baclofen within
baclofen-acamprosate combination. In addition, muscimol, an agonist
of GABA.sub.A receptors, was found to block the neuroprotective
effect of acamprosate (FIG. 22 A), which demonstrates the role of
the antagonistic activity of acamprosate on GABA.sub.A receptors
for the protection of neuronal cultures against A.beta..sub.1-42
cytotoxicity. An agonistic effect on ionotropic glycine receptors
is also found necessary for neuroprotection since strychnine, an
antagonist of inhibitory glycine-gated channels, reverses the
neuroprotective effect of the acamprosate (FIG. 22 B).
Preincubation with DHPG and APDC, agonists of group I and II
metabotropic glutamate receptors respectively, results in the
abolition of neuroprotection normally afforded by acamprosate,
which demonstrates the importance of the antagonistic effect of
acamprosate on these molecular targets for its neuroprotective
effect (FIGS. 22 C and D).
[0327] From the above, it can be deduced that the particularly
effective neuroprotective effect reported for the
baclofen-acamprosate combination is the result of a concerted
action on, at least, the above 5 molecular targets. Acamprosate is
moreover of particular interest because of its simultaneous actions
on at least GABA.sub.A receptors and ionotropic glycine receptors
as well as group I and II metabotropic glutamate receptors which,
as demonstrated above, play an essential role in neuroprotection
against A.beta. peptides.
[0328] 3. Concentration Ranges of Baclofen-Acamprosate Combination
afford a Protection in the Three Main Features of A.beta. Peptide
Toxicity.
[0329] As mentioned in the set of the above experiments, the
inventors have found that the baclofen-acamprosate combination is
efficient in counteracting events triggered by the oligomers of
A.beta. at the synaptic, at the neuronal and at the endothelial
levels.
[0330] The inventors have been able to determine ranges of
concentrations of baclofen and acamprosate that allow an effective
protection, at the same time, of neurons, synapse function and
endothelial function (FIG. 23). Such a range for a simultaneous
activity has been determined for these three features to be from 80
nM to 1 .mu.M for baclofen and from 320 .mu.M to 4 nM for
acamprosate.
[0331] A particularly enhanced efficacy of the treatment can then
be expected within these ranges of concentrations due to the
conjunction of effects that is obtained. It can be thus considered
that reaching these ranges of plasmatic concentrations of baclofen
and acamprosate in the brain is of particular interest.
[0332] 4. Examples of Synergistic Combinations
[0333] The inventors have further found that the
baclofen-acamprosate combination is active and shows a marked
synergistic effect according to methods commonly recognized among
pharmacology community [60-62] in at least one of the above in
vitro models for AD (Table 6) in the tested concentrations. This
ensures a particular clinical benefit. At certain concentrations,
the combination provides a synergistic protection in all three
models, which is even more particularly advantageous.
TABLE-US-00006 TABLE 6 Combination activity ACP/BCL Neuritis
(concentration, M) Angiogenesis Neuroprotection network 1.44
10.sup.-10/2.00 10.sup.-06 Synergy Synergy 1.44 10.sup.-10/3.60
10.sup.-08 Synergy Synergy Synergy 1.44 10.sup.-10/4.00 10.sup.-07
.dagger-dbl. Synergy Synergy 1.44 10.sup.-10/8.00 10.sup.-08
Synergy Synergy Synergy 3.20 10.sup.-10/2.00 10.sup.-06 Synergy
Synergy 3.20 10.sup.-10/3.60 10.sup.-08 Synergy Synergy Synergy
3.20 10.sup.-10/4.00 10.sup.-07 Synergy Synergy Synergy 3.20
10.sup.-10/8.00 10.sup.-08 Synergy Synergy Synergy 1.60
10.sup.-09/1.60 10.sup.-08 .dagger-dbl. Synergy NA 1.60
10.sup.-09/3.60 10.sup.-08 .dagger-dbl. Synergy Synergy 1.60
10.sup.-09/4.00 10.sup.-07 Synergy Synergy Synergy 1.60
10.sup.-09/8.00 10.sup.-08 Synergy Synergy Synergy 6.40
10.sup.-11/1.60 10.sup.-08 Synergy Synergy NA 6.40 10.sup.-11/2.00
10.sup.-06 Synergy NA 6.40 10.sup.-11/3.60 10.sup.-08 Synergy
Synergy NA 6.40 10.sup.-11/4.00 10.sup.-07 .dagger-dbl. Synergy NA
6.40 10.sup.-11/8.00 10.sup.-08 Synergy Synergy NA 6.40
10.sup.-11/3.20 10.sup.-09 NA NA Synergy 8.00 10.sup.-09/3.60
10.sup.-08 .dagger-dbl. Synergy Synergy is determined according
Loewe or Bliss methods [60-62] .dagger-dbl.: no effect; : no
synergy; NA: not available
B. Baclofen-Acamprosate Combination Therapies Prevent Toxicity of
Human A.beta. in In Vivo Models
[0334] 1. Intracerebroventricular Administration of
A.beta..sub.25-35 in Swiss Mice.
Animals
[0335] Male Swiss mice, 6 weeks old, 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.). 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.
Combinatory treatment and administration of A.beta..sub.25-35
[0336] Drug(s) is/are daily administered by gavage (per os). The
A.beta..sub.25-35 peptide and scrambled A.beta..sub.25-35 peptide
(control) have been dissolved in sterile bidistilled water, and
stored at -20.degree. C. until use. The .beta.-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 a significant
influence on survival, behavioral responses or cognitive
functions.
[0337] On Day -1, i.e., 24 h before the A.beta..sub.25-35 peptide
injection, baclofen, acamprosate, a combination thereof or the
vehicle solution are administered twice per os by gavage at 8:00 am
and 6:00 pm.
[0338] On Day 0 (at 10:00 am), mice are injected i.c.v. with
A.beta..sub.25-35 peptide or scrambled A.beta..sub.25-35 peptide
(control) in a final volume of 3 .mu.l (3 mM).
[0339] Between Day 0 and Day 7, baclofen, acamprosate, a
combination thereof or the vehicle solution are administered per os
by gavage twice daily (at 8:00 am and 6:00 pm). A dose designated
as bid (bis in die) means that said dose is administered twice
daily. Drugs are solubilized in water and freshly prepared just
before each gavage administration. One animal group receives
donepezil (reference compound, 1 mg/kg/day) in a single injection
intraperitoneally (at 8:00 am).
[0340] On Day 7, all animals are tested for the spontaneous
alternation performance in the Y-maze test, an index of spatial
working memory.
[0341] On Days 7 and 8, the contextual long-term memory of the
animals is assessed using the step-down type passive avoidance
procedure.
[0342] On Day 8, animals are sacrificed. Their brains are dissected
and kept at -80.degree. C. for further analysis.
[0343] When the baclofen-acamprosate combination is tested in a
combination with reference compounds for AD (for instance,
currently approved treatments for AD are donepezil, galantamine,
rivastigmine or memantine), said reference compounds are
administered intraperitoneally, between Day 0 and Day 7, at 8:00
am.
[0344] a) Combinations Enhance Behavioral and Cognitive
Performances of Intoxicated Animals
Spontaneous Alternation Performances Y-Maze Test
[0345] 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
converges 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).times.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, this 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.beta..sub.25-35 injection.
Passive Avoidance Test
[0346] The apparatus is a two-compartment (15.times.20.times.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 foot shocks (0.3 mA
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 foot
shock 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 door 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.
Results
[0347] Positive results are observed in behavioural performances
and biochemical assays performed 7 days after A.beta..sub.25-35
peptide i.c.v. injection.
[0348] The combination of baclofen and acamprosate induces a
significant protective effect on behavioral and cognitive
performances of intoxicated animals as shown in FIGS. 8, 9 and
10.
[0349] In FIG. 8, with only 53.8% of alternation, intoxicated mice
exhibit a strongly impaired spatial working memory compared to
control. With an improvement of more than 48% of their percentage
of alternation compared to intoxicated controls, the impairment is
significantly prevented in mice treated with baclofen and
acamprosate.
[0350] Similarly, FIGS. 9 and 10 show that intoxicated animals
exhibit impaired behavioral and cognitive performances according to
their scores in escape latency and step-through latency
respectively. In both tests, the combination of baclofen and
acamprosate allows a significant correction of the impairment. The
escape latency of mice treated with this combination is no more
significantly different from control mice (FIG. 9) and step-through
latency (FIG. 10) is significantly increased by combinations of the
invention, with an enhanced effect of the combination compared to
the drugs alone.
[0351] Memory impairment is the early feature of Alzheimer's
disease and these results clearly show that the toxic effect of
amyloid peptide on behavioral and cognitive performances (including
memory) is significantly prevented by the combinations of the
invention.
[0352] Furthermore, FIG. 16 shows that extremely low doses of
baclofen (480 .mu.g/kg bid), acamprosate (32 .mu.g/kg bid) and
donepezil (0.25 mg/kg/day) can be combined to allow complete
protection of behavioral and cognitive performances of mice as
measured by the Y-maze test. While donepezil, at this
concentration, has no significant effect (32% protection) on
spatial working memory, its use in conjunction with the baclofen
and acamprosate combination allows a complete protection (98%) of
intoxicated mice's cognitive performances. The combination index of
donepezil in combination with baclofen-acamprosate is 0.687 which
determines a marked synergy between the compounds (as determined by
the method of Loewe [60, 61]). The human equivalent dose of
donepezil used in this combination is more than 4 times lower than
the lowest and almost 20 times lower than the highest dose
currently used in humans for AD treatment.
[0353] An improvement is also observed in the performances in the
Y-maze test when combining baclofen (480 .mu.g/kg bid), acamprosate
(32 .mu.g/kg bid) and memantine (0.5 mg/kg/day), as shown in FIG.
19. At the concentrations used neither the baclofen-acamprosate
combination nor memantine has a significant effect. Noteworthy, the
dose of memantine used in these experiments is more than 7 times
lower than the human equivalent dose corresponding to the
maintenance treatment. Moreover the combination of
baclofen-acamprosate with memantine is synergistic, displaying a
combinatory index of 0.784 (as determined by the method of Loewe
[60, 61]).
[0354] Combinations of the invention can thus be further combined
with other therapies for AD in order to potentiate their action,
and to lower their potential side effects by using lower doses for
these drugs.
[0355] b) Combinations Improve Neurophysiological Concern of
Neurological Diseases
[0356] Combination therapies are tested in an in vivo model of
A.beta. intoxication. Their effects on several parameters which are
affected in neurological diseases are assessed: [0357] Caspases 3
and 9 expression level, considered as an indicator of apoptosis,
[0358] Lipid peroxidation, considered as a marker for oxidative
stress level, [0359] GFAP expression assay, considered as a marker
of the level of brain inflammation, [0360] Blood-Brain Barrier
integrity, [0361] Overall synapse integrity (synaptophysin ELISA),
and [0362] Quantification of viable neurons in the Cornus Ammonis
area1 (CA1) of hippocampus.
Blood-Brain Barrier Integrity
[0363] The experimental design about animal intoxication by A.beta.
is the same as previously stated.
[0364] The potential protective effect of the combination therapies
on blood-brain barrier (BBB) integrity is analyzed in mice injected
intracerebroventricularly (i.c.v.) with oligomeric
amyloid-.beta.25-35 peptide (A.beta..sub.25-35) or scrambled
A.beta..sub.25-35 control peptide (Sc.A.beta.), 7 days after
injection.
[0365] On day 7 after the A.beta..sub.25-35 injection, animals are
tested to determine 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 intraperitoneally (i.p.) 3 h
prior to the transcardiac perfusion. Mice are then anesthetized
with i.p. 200 .mu.l of pre-mix ketamine 80 mg/kg, xylazine 10
mg/kg, and the chests are 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 brains
are 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.
[0366] 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.degree. C., and then centrifuged 30 min at 10,000 g, 4.degree.
C. The supernatant is measured at 610 nm for absorbance of EB using
a spectrophotometer.
[0367] EB is quantified both as: [0368] .mu.g/mg of brain tissue by
using a standard curve, obtained by known concentration of
EB-albumin, and [0369] .mu.g/mg of protein.
Overall Synapse Integrity (Synaptophysin ELISA)
[0370] 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.
[0371] 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.degree. C. storage. Tissues are cut into small
pieces and homogenized in 1 ml 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.
[0372] All samples are assayed in triplicate.
[0373] Quantification of proteins is performed with the Pierce BCA
(bicinchoninic acid) protein assay kit (Pierce, Ref. 23227) to
evaluate extraction performance and allow normalization.
[0374] The total protein concentrations are then calculated from
standard curve dilutions and serve to normalize ELISA results.
Quantification of Viable Neurons in the CA1
[0375] On Day 8, each mouse is anesthetized with 200 .mu.l i.p. of
a pre-mix of ketamine 80 mg/kg and xylazine 10 mg/kg and
transcardially perfused with 100 ml of saline solution followed by
100 ml of 4% paraformaldehyde. The brains are removed and kept for
24 h post-fixation in 4% paraformaldehyde solution at 4.degree. C.
After post-fixation, brains are washed in a phosphate buffer saline
(PBS) solution, then cerebellums are removed and the brains are cut
in coronal sections (20 .mu.m thickness) using a vibratome (Leica
VT100OS, Leica, Wetzlar, Germany). Serial sections are placed on
24-well plates with PBS. They are then selected to include the
hippocampal formation and 9 sections are placed in gelatin-coated
glass strips (one slide per animal for cresyl violet). All slides
are dried at room temperature for 48 h to avoid unsticking. The
slides are stored at room temperature until cresyl violet staining.
Sections are stained with 0.2% cresyl violet reagent
(Sigma-Aldrich), then dehydrated with graded ethanol, treated with
toluene, and mounted with Mountex medium (BDH Laboratory Supplies,
Poole, Dorset, UK).
[0376] After mounting, slides are kept at RT for 24 h drying.
Examination of the CA1 area is performed using a light microscope
(Dialux 22, Leitz), with slices digitalized through a CCD camera
(Sony XC-77CE, Sony, Paris, France) with the NIH Image.RTM. v1.63
software (NIH). CA1 measurement and pyramidal cells counts are
processed using ImageJ.RTM. (NIH). Data are expressed as mean of
nine slices of CA1 pyramidal cells per millimeter for each group
(left and right hippocampus CA1 counting) [63].
Oxidative Stress Assay
[0377] Mice are sacrificed by decapitation and both hippocampi are
rapidly removed, weighed and kept in liquid nitrogen until assayed.
After thawing, hippocampi are homogenized in cold methanol (1/10
w/v), centrifuged at 1,000 g during 5 min and the supernatant
placed in Eppendorf tubes. The reaction volume of each homogenate
is added to FeSO4 1 mM, H2SO4 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 A1 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.times.[CHP] and expressed as CHP equivalents per
weight of tissue and as percentage of control group data.
Caspase Pathway Induction Assay and GFAP Expression Assay
[0378] Mice are sacrificed by decapitation and both hippocampi are
rapidly removed, rinsed in ice-cold PBS (0.02 mol/l, pH 7.0-7.2) to
remove excess blood thoroughly, weighed and kept in liquid nitrogen
until assayed. Tissues are cut into small pieces and homogenized in
1 ml ice-cold PBS 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 at 5,000 g during 5
min and the supernatant is assayed immediately.
[0379] Experiments are conducted with commercial assays: Caspase-3
(USCN--E90626Mu), Caspase-9 (USCN--E90627Mu). GFAP
(USCN--E90068).
[0380] Quantification of proteins is performed with the Pierce BCA
(bicinchoninic acid) protein assay kit (Pierce, Ref. 23227) to
evaluate extraction performance and allow normalization.
Results
[0381] The combination of baclofen and acamprosate induces a
significant protective effect on neurophysiological functions of
intoxicated animals as shown in FIGS. 11, 12, 13 and 14.
[0382] With a protection of more than 60% compared to non-treated
intoxicated animals, the combination is effective for the
protection of neurons (FIG. 11) and synaptic density (FIG. 13).
[0383] Similarly, FIG. 12 shows that the combination of baclofen
and acamprosate protects the BBB integrity (76%) compared with
non-treated intoxicated animals.
[0384] Finally, this combination therapy is efficient in reducing
the overall oxidative stress induced by A.beta. in the brains of
treated animals when compared with non-treated intoxicated animals
(FIG. 14).
[0385] 2. hAPPSL Transgenic Mice
[0386] Efficiency of the baclofen-acamprosate combination has been
tested in another murine model for AD. A mouse line overexpressing
the 751 amino acid form of human APP (hAPP) with London (V717I) and
Swedish (KM670/671NL) mutations (hAPP.sub.SL) under the control of
the murine Thy-1 promoter has been chosen. In this model,
significant deficits in spatial memory and learning in the water
maze task and in habituation in the hole-board task are noticed as
soon as 6 months of age. This cognitive decline is due to
dysfunction of synaptic transmission and mimics some aspects of the
early phases of human AD.
Animals
[0387] Experiments were conducted on male subjects. Animals were
bred at QPS (Austria), and C57BL6 age and gender-matched transgenic
littermates served as controls. Animals were housed in plastic
cages with free access to food and water, except during behavioural
experiments, and kept in a regulated environment (23.+-.1.degree.
C., 50-60% humidity) under a 12 h light/dark cycle.
Drug Treatment
[0388] Drug combination (acamprosate 0.2 mg/kg and baclofen 3
mg/kg) or the vehicle solution are administered per os by gavage
twice daily (at 8:00 am and 6:00 pm) in a volume of 5 ml/kg.
Treatment with the baclofen-acamprosate combination was started at
8 months of age (i.e., when the disease is well established) and
lasted 4 weeks until the end of functional tests. All behavioral
tests were performed 2 h after treatment.
Morris Water Maze (MWM) Acquisition Task
[0389] Mice were tested for acquisition and working memory.
Briefly, swimming was recorded using a Videotrack software
(Viewpoint, Champagne-au-Mont-d'Or, France), with trajectories
being analysed as latencies and distances. The software divides the
pool into four quadrants. Training (Acquisition) consisted of 4
swims per day for 4 days. The latency, expressed as mean.+-.s.e.m.,
was calculated for each training day. A probe test was performed 24
h after the last swim (retention phase). The platform was removed
and each animal was allowed a free 60 s swim. The time spent in
each quadrant was then determined. After the training test, animals
were tested for spatial working memory. Working memory was
specifically assayed by changing the platform location every day
(four swims per day during 2 days) and by using a training
inter-trial time of 2 min. The swimming times to find the platform
of the first (swim1), second (swim2), third (swim3) and fourth
trial (swim4) of Day 2 were calculated and averaged.
Statistical Analyses
[0390] A mixed ANOVA with Dunnett's test, including fixed effect
terms for treatment, time and the treatment by time interaction,
and a random effect term for animals was applied. Treatment effect
was assessed for each time point and for combined time points
(global effect). Tests were conducted at a 5% significance
level.
Results
[0391] At the age of 8 months, when hAPP.sub.SL transgenic mice's
cognition was already impaired and diseased installed, a one-month
treatment with the baclofen-acamprosate combination is found to
substantially improve cognitive deficits as they had been assessed
in the MWM acquisition and working memory tests (FIGS. 24 A) and B)
respectively).
[0392] Thus, notwithstanding the extensive in vitro results, the
results obtained in two different in vivo models confirm the
efficacy of the combination in counteracting the toxicity of
A.beta. and its behavioural effects as well as its effects on brain
physiology.
[0393] Several neurological functions impaired in numerous
neurological disorders, including neurodegenerative disorders such
as Alzheimer's disease and related disorders, have been protected
and symptoms retarded or reduced by the combination of
baclofen-acamprosate.
C. Baclofen-Acamprosate Combination Therapies have Positive Effects
in Human Subjects
[0394] The particular efficiency of the compositions of the
invention in different in vitro and animal models for AD, together
with the possibility of acting not only on a single but on a group
of phenomena described as being at the origin of the disease, have
prompted the inventors to test it in aged people, in a model of
chemically induced amnesia that is commonly used in clinical trials
for AD, and also in people suffering from mild AD. More
particularly, baclofen-acamprosate combination efficacy in
improving memory or memory related mental functions and in
improving electrophysiological features underlying working memory
has been assessed through clinical trials.
[0395] The studies were conducted in compliance with the following
protocols and in accordance with Good Clinical Practice (GCP) as
required by the European Medicines Agency ICH-E6 (RI) guideline
recommendations and the French law No. 2004-806, Aug. 9, 2004
relative to public health law.
[0396] 1. Improvement in Memory and Related Functions in Humans
Subjected to Chemically-Induced Cognitive Impairment.
[0397] AD patients show the signs of diminished cholinergic
synaptic activity, with diminished levels of acetylcholine. The
pharmacological model of this phenomenon consists of treating the
subjects with scopolamine, a blocker of muscarinic receptors. This
drug thereby induces a transitory and reversible cognitive
impairment upon administration [64]; it is therefore currently used
as a pharmacologic model for induced dementia.
Experimental Design
[0398] Twenty-one healthy male volunteers aged from 20 to 40 years
were enrolled in the study, which is a randomized, 2-way
cross-over, double blind, placebo-controlled study. The study had
two distinct periods, P1 and P2, each consisting of 40 hours
hospitalization and being spaced from each other by 7 days as a
wash-out period. During both these periods, the
baclofen-acamprosate combination is administered orally, the drugs
being administered concomitantly. Results were compared to those of
placebo treatment. A dose designated as bid (bis in die) means that
said dose is administered twice daily.
[0399] During P1, subjects were administered according to the
following treatment. [0400] at Day 1: baclofen (6 mg) and
acamprosate (0.4 mg) mix, or placebo, orally, in a sub-acute
administration, bid. [0401] at Day 2 (test day), H3 just before
scopolamine injection: single dose of baclofen (6 mg) and
acamprosate (0.4 mg) mix or placebo, orally. [0402] at Day 2, H3: a
sub-cutaneous injection of 0.5 mg of scopolamine.
[0403] According to the same above dosage schedule, subjects who
had received baclofen-acamprosate treatment in P1 received placebo
during P2, while those administered with placebo in P1 were treated
with the baclofen-acamprosate mix in P2.
Cognitive Test
[0404] Effects on impaired cognition were explored by measuring the
following features in the Cognitive Groton Maze Learning Task
(GMLT) test: [0405] the efficiency of performance: mps, [0406] the
total number of errors: ter, and [0407] the duration of task:
dur.
[0408] The GMLT test was performed for each treatment period on Day
1 (2 training sessions), on Day 2 (test day) HO, H2.5, H4, H5.5, H7
and H9 and on Day 3 H24.
Data Analyses
[0409] The data from the GMLT test were pooled together in a
"composite" GMLT score. Comparison between treatment groups for the
two periods was performed on change from baseline value by using a
mixed Analysis of Covariance (ANCOVA) model with SAS.RTM. Mixed
procedure (see Worldwide Website sas.com), including fixed effect
terms for period treatment, time and the treatment by time
interaction, a random effect term for subject and the baseline
value as covariate. Treatment effect was assessed for combined time
points (global effect) and for each time point.
Results: Baclofen-Acamprosate Combination Improves the Cognitive
Deficit Induced by Scopolamine.
[0410] For each of the periods P1 and P2, similar data were
obtained for each of mps, ter and dur components of the GMLT test.
The resulting "composite" GMLT score is presented in FIG. 25.
[0411] Scopolamine is known to act as early as 30 min after its
administration, its effect lasting for approximately 6 hours. Its
Tmax is known to be of about 3 hours [64]. These data correspond to
the transient collapse of performances in the GMLT test observed in
placebo-treated subjects (FIG. 25, circles, dotted line).
[0412] An improvement of cognitive performances was observed for
baclofen-acamprosate treated subjects (FIG. 25 squares, grey line)
compared to placebo dosed subjects. This improvement is
particularly significant in the time period around H5.5 that
corresponds to the Tmax of baclofen-acamprosate (FIG. 25, darkest
area in the pharmacokinetic scale of mix compounds).
[0413] Hence, the baclofen-acamprosate combination is efficient in
counteracting scopolamine induced cognitive impairment. Hence,
besides being considered as of interest in the cases of provoked
amnesia, combinations of the invention can also be considered as of
particular interest in reversing memory impairment related to
conditions implying diminished cholinergic synaptic activity.
Baclofen-acamprosate combinations are thus of particular interest
for the treatment of AD and related diseases.
[0414] 2. Baclofen-Acamprosate is Effective in Mild AD
Patients.
[0415] Another part of the studies performed on human subjects
consists of a three-month clinical study that is currently
performed on patients diagnosed with mild AD with the aim of
exploring the effect of baclofen-acamprosate combinations on
cognitive and behavioural impairments related to the disease. This
study is a 12-week, prospective pilot single-blind and placebo
sequential controlled, multi-center trial which assesses the
effects of different doses of the baclofen-acamprosate combination.
It is a "challenge/de-challenge/re-challenge" (CDR) type study,
which means that the baclofen-acamprosate combination is
administered on a given time period, then withdrawn, and then
re-administered. During the withdrawal (de-challenge) phase, the
medication is allowed to wash out of the organism. Then, an
improvement of the disease's symptoms during the challenge and
re-challenge phases, together with no improvement or a worsening in
the de-challenge phase, would be considered as a signature of the
efficiency of the combination. This study is conducted in an open
way and was single-blind for the patients and the neurophysiologist
assessor. Preliminary results obtained for 29 patients are
presented herein.
Experimental Design
[0416] Cognitive and behavioural impairments of the subjects are
measured by psychometric assessments during visits to a
neurophysiologist set at the beginning and the end of each of the 3
phases of the CDR study:
[0417] Visit 1 (V1) holds on the beginning of the challenge phase,
which lasts 4 weeks, and at the end of which performances of
subjects are evaluated during visit 2 (V2); then the de-challenge
phase of 4 weeks runs and ends with visit 3 (V3), after which the
re-challenge phase lasts 4 weeks more, to be ended by the last
evaluation of the subjects by the neurophysiologist during visit 4
(V4). At each of visits V1-V4, ADAS cog tests (Alzheimer's Disease
Assessment Scale-cognitive subscale) are performed to evaluate the
changes in cognition performances of the subjects.
[0418] In this study, three doses, administered twice daily (once
in the morning and once in the evening), of the mix of
baclofen-acamprosate are tested: [0419] Dose 1: 0.4 mg acamprosate
and 6 mg baclofen, given concomitantly; [0420] Dose 2: 1 mg
acamprosate and 15 mg baclofen, given concomitantly; and [0421]
Dose 3: 20 mg acamprosate and 12 mg baclofen, given
concomitantly.
Subject Recruitment
[0422] 29 patients diagnosed as suffering from mild AD, i.e.,
displaying a Mini Mental State Examination (MMSE) score between 20
and 26, were enrolled in the study (clinical characteristics of the
cohort, Table 7) for the testing of doses 1 and 2.
TABLE-US-00007 TABLE 7 n = 29 patients Age +/- sem 71.8 +/- 1.33
years Gender 13 Females 16 Males MMSE +/- sem 23.4 +/- 0.389 ADAS
Cog. at V1 +/- sem 11.9 +/- 0.661 sem: standard error of the mean;
MMSE: Mini Mental State Examination; ADAS cog: Alzheimer's Disease
Assessment Scale-cognitive subscale
[0423] Depending on the subjects, the screening/recruitment
sessions were held from 1 to 2 weeks before V1.
[0424] At V1, the 29 patients were randomized into two groups:
[0425] 15 patients in group 1 receiving dose 1, and [0426] 14
patients in group 2 receiving dose 2.
[0427] ADAS cog score assessed at V1 is taken as the baseline to
evaluate changes in performances of the subjects assessed during
subsequent visits V2, V3 and V4.
[0428] Each patient is their own control for the 3 successive
sequences of the CDR study.
[0429] The effect of the mix is assessed on the changes of ADAS cog
score at the end of each period in comparison with the previous
visit and the treatment periods are compared to the placebo
period.
[0430] The mean changes from baseline to the end of the first
4-week active treatment period and the mean changes observed
between each visit are assessed.
[0431] The change of each endpoint is measured between each visit:
[0432] from baseline (V1) to the end of the first 4-week active
treatment period (V2), [0433] from V2 to the end of the 4-week
placebo period (V3), [0434] from V3 to the end of the second 4-week
active treatment period (V4), and [0435] from V1 to V4.
[0436] Differences between V1 and V2 or V1 and V4 are statistically
tested using a one-tailed paired test of Student at a 10%
significance level.
Dosing of Plasmatic Concentrations of Baclofen and Acamprosate
[0437] To evaluate the overall exposure of the patients to the
drugs, baclofen and acamprosate plasmatic concentration has been
measured at the end of the study (V4), in the morning just before
drug administration (on an empty stomach, assumed to be the lowest
drug concentration) and two hours after the dosing, which roughly
corresponds to the Tmax of the drugs (assumed to be the highest
concentration). Actual plasmatic concentration of the drugs to
which the patient is exposed thus oscillates during the treatment
between these lowest and highest drug concentrations.
[0438] The plasmatic concentration of a given drug can be
determined using any method well-known by biopharmacists. Briefly,
an LC-MS/MS analysis has been performed on samples with the
material detailed in Table 8, but any equivalent material can be
used.
TABLE-US-00008 TABLE 8 acamprosate quantification baclofen
quantification Chromatograph Shimadzu LC-20AD liquid Shimadzu
LC-20AB liquid chromatography system chromatography system
SIL-20AC-HT autosampler SIL-20AC autosampler Matrix C18 stationary
phase Phenyl-hexyl stationary phase Mass API5500 (AB-Sciex) A TSQ
Quantum Ultra spectrometer mass (Thermo Electron Corporation)
Electrospray Negative Positive Ionisation mode Reference
Acamprosate calcium Baclofen standard Internal Acamprosate-D12
calcium Baclofen-D4 standard trihydrate
[0439] Watson.RTM. LIMS 7.2 (Thermo Electron, Philadelphia, Pa.)
software was used for regression, calculation, and statistical
calculations.
[0440] Analyst 1.5.2 (AB-Sciex) and LCQuan 2.5 (Thermo Electron
Corporation) software was used for LC-MS/MS instrument control,
data acquisition and integration. Any other software can be used as
a function of the mass spectrometer to be used.
[0441] Samples are human plasma samples collected during the
clinical trial. Blank plasma samples were used to prepare
calibration.
[0442] For acamprosate quantification, sampling volume was 100
.mu.l and proteins have been precipitated, and for baclofen,
sampling volume was 200 .mu.l and samples were submitted to solid
phase extraction.
Mini Mental State Examination (MMSE) Score
[0443] The MMSE is widely used to assess the cognitive functions
and the mnesic abilities [65] in the frame of the diagnosis of AD
and of the assessment of the severity of the disease. The test
comprises a series of 30 questions addressing 5 different areas
(orientation, registration, attention calculation, recall, and
language) with the final score being graded out of 30 points. A
final score below or equal to 26 points corresponds to a dementia
diagnosis. Normal scores range from 30 to 26, whereas a score
between 26 and 20 indicates mild dementia, a score between 19 and
10 indicates moderate dementia, and a score below 10 is considered
as indicating severe dementia. A variation of 2 points of the score
is usually considered as clinically relevant.
ADAS Cog Score
[0444] ADAS cognitive subscale deals with memory, language,
construction and praxis orientation and is commonly considered as a
standard for the evaluation of mild to moderate AD patients in the
clinical trials. It consists of 11 tasks which measure the
disturbances of memory, language, praxis, attention and other
cognitive abilities which are often referred to as the core
symptoms of AD. It generally shows good test-retest and inter-rater
reliability and performs satisfactorily against more detailed
measures of cognitive function.
[0445] Score range varies from 0 to 70. The more mistakes made, the
higher the score. A normal score is defined as a score lower than
10. A 70-point score is a sign of severe dementia [66].
[0446] a) Combination Therapies Improve Cognitive Performances of
Mild AD Patients.
[0447] Results presented herein were obtained from an intermediate
analysis of the ADAS cog data for the 24 first subjects to complete
the study who were treated by doses 1 or 2.
[0448] As shown in FIG. 26, the evolution of performances in the
ADAS cog tests all along the study shows a clear correlation with
the CDR design of the study, either when considering the whole set
of patients (i.e., whatever the dosage, FIG. 26), or considering
separately patients treated with dose 1 or dose 2 (not shown). It
clearly appears that treatment with baclofen-acamprosate during the
challenge and re-challenge phases results in an improvement of the
subject's cognitive performances at the end of both phases when
compared to those measured at the beginning of each phase. This
positive effect of the treatment is emphasized by the clear
worsening of the score during the de-challenge phase, wherein a
placebo is administered.
[0449] A strongly significant improvement of performances of
patients in the ADAS cog test is observed between V1 and V2
(p<0.01). The three-month study led finally to a global
significant improvement of patient condition despite the dividing
de-challenge phase (p<0.05).
[0450] b) Baclofen-Acamprosate Combination Acts on the
Electrophysiological Features of Cognition in Mild AD Patients.
[0451] Alterations in brain functions can be detected by
electrophysiological techniques such as electroencephalography
(EEG). Event related potentials (ERPs) are observed within the EEG
recordings in response to an experimental condition, and represent
the corresponding activated cognitive phenomena such as perception,
attention, decision-making process, answering, memory process,
language, etc. ERP characteristics and their components can vary as
a function of various factors such as stimulus relevance, task
performed, lesions of the nervous system, the use of drugs and so
on.
[0452] ERPs have been found to be altered in patients suffering
from AD, vascular dementia or dementia associated with Parkinsonian
symptoms. Numerous research studies have helped to establish ERPs
as a useful cognitive biomarker for the diagnosis of dementia,
tracking disease progression, and evaluating the pro-cognitive
effect of therapeutics. More particularly ERP measures allow
pointing out alteration of cognitive function at an early stage,
and can contribute to the diagnosis of AD with good sensitivity and
specificity.
[0453] The most frequently recorded potential in clinical practice
is P300 which comprises P3 and N2 subcomponents: [0454] P3 (or late
positive complex) is a large centroparietal positivity in the ERP
that occurs with a latency of approximately 300 ms after a
discordant stimulus. P3 can be divided in the two subcomponents P3a
and P3b. P3a is generally considered to be related to the degree of
focal attention whereas P3b is supposed to index the working memory
update. P3 amplitude notably refers to selective attention stimulus
occurrence probability motivation and vigilance. [0455] N2 is a
negative wave which precedes P3 and which might be linked to the
detection of the target stimulus and be the reflection of the
selective attention processes coming into action.
[0456] Turning back to AD, a P3b latency increase and amplitude
decrease are the most consensual objective parameters observed in
AD patients. It is useful for the monitoring of AD progression, and
even for assessing AD treatment response. Generally speaking,
latency refers to the time necessary for the making of a decision
and amplitude refers to the difficulty of the task, probability of
occurrence of the stimulus, or emotional state.
[0457] Recording of ERPs
[0458] ERP assessment is done at visits V1-V4 of the above clinical
trial.
[0459] Recording of cognitive ERPs is done according to the
auditory oddball paradigm. Patients lay down on an "examination
bed," with eyes opened, in a soundproof, darkened room. Tones (60
dB SPL, 100-ms duration) were presented binaurally through a
headset up to a total of 150 stimuli. Patients were instructed to
identify the odd 2000 Hz high-pitched stimuli (target sounds),
which had a 20% occurrence probability among the standard 1000 Hz
low-pitched stimuli (common sounds). The cognitive task required
paying attention to the odd stimuli and counting them. The stimulus
order of appearance was random and there was at least a 1140-ms gap
between each stimulus. Three tests were recorded with a 2-minute
pause followed by repeated instructions. The test was stopped once
the 90 (3.times.30) target stimuli have been played out and the
patient was asked to give out his/her count of the oddest sounds.
Separate averaging of single records corresponding to frequent and
rare stimuli were processed online. Grand average of the evoked
potentials was calculated from the three trials for Fz (frontal
vertex), Cz (Central vertex) and Pz (Parietal vertex)
electrodes.
[0460] Evoked potential amplitudes were measured relative to the
prestimulus baseline from the records collected at Fz, Cz and Pz:
[0461] P3 was the most positive wave after N2 between 279 and 440
ms. [0462] N100 wave was the most negative peak in the range 75 to
150 ms. [0463] N200 wave was the most negative peak between 196 and
300 ms.
[0464] In Alzheimer's disease latencies are hugely delayed and
amplitudes are reduced.
[0465] Latencies and amplitudes of the P3a and P3b subcomponents of
P3 and N200 were taken from the signals recorded from Fz, Cz and Pz
electrodes and used for statistical analyses. Delta of amplitudes
between N2 and P3a and delta of amplitudes between N2 and P3b were
also calculated during statistical analyses. ASCII-format files
were also recorded for PCA statistical analyses.
[0466] All these 24 latencies/amplitudes/deltas-of-amplitudes
parameters and the PCA analysis were assessed for each period
(challenge, de-challenge, re-challenge) and compared across all
these 3 periods.
[0467] The analysis was computed with R. All quantitative
parameters of the ERPs were presented in terms of mean, standard
deviation, median, extreme values, Q1, Q3, number of patients and
missing data.
Case Study: Baclofen-Acamprosate Combinatorial Treatment
Significantly Corrects ERP Features in an AD Subject
[0468] Results presented in FIG. 27 represent ERP measures gathered
from all three electrodes all along the CDR study for patient
601.
[0469] Patient 601 is a 74-year-old female Caucasian subject with
no known antecedent of familial dementia. She was diagnosed as
suffering from probable AD since June 2012, and presents a moderate
hypotrophy of the hippocampus together with a global cerebral
hypotrophy in neuroimaging, which ascertains the diagnosis. At V0
she obtained a MMSE score of 24 She was administered with the low
dose (dose 1) of the combination.
[0470] ERPs obtained at V1 for this patient are characterized by a
substantial delay of P3, around 900 ins for this subject (FIG.
27).
[0471] As soon as V2, a significant correction of both the delay (a
significant decrease) and the amplitude (a significant enhancement)
of the P3b wave is noticed. This positive effect of the mix seems
to be maintained even during the de-challenge phase: P3b at V3 is
found shifted to the left when compared to the one of V2 and also
presents a greater amplitude. Improvement of latency is still
observed at V4 at the end of the re-challenge phase. These results
suggest that the mix is efficient in improving working memory
update in AD patients. Noteworthy, an improvement is also observed
regarding the P3A and N2 components, which are more related to the
attention processes.
[0472] Hence, the baclofen-acamprosate combination is efficient in
correcting the alterations of brain electrophysiological functions
which are observed in mild AD subjects.
[0473] c) Conclusion--Clinical Trial
[0474] Results of clinical trials performed in humans show the
effectiveness of combinations of the invention in treating AD and
related disorders as shown by the observed protection against
memory process degradation both in a chemically induced amnesia
model and in patients diagnosed with mild AD. The correction of
memory processes in patients is asserted by electrophysiology data
that show a correction in brain functioning upon treatment in mild
AD patients.
[0475] A great variability is commonly observed in plasmatic
concentrations of a given drug in response to the same dosing
regimen in different subjects. This can be due notably to
variations in the efficiency of metabolism of drugs within each
patient. Despite this, it is noteworthy that during the clinical
trial patients have been exposed to plasmatic concentrations of
drugs of the same order of magnitude as the concentrations of
baclofen and acamprosate that have been shown to simultaneously
protect neurons from death and protect synaptic and endothelial
functions (Table 9).
TABLE-US-00009 TABLE 9 Baclofen Acamprosate** (ng/ml) (ng/ml) In
vitro ranges for a simultaneous 17.1-213.7 0.058-0.725 protection
of neurons, synaptic and endothelial functions (FIG. 23) Plasmatic
ranges observed in patients 57.6-261.4 0.58-1.20 after one month of
treatment* *The lower value corresponds to the mean concentration
observed in patients through clinical trial after one month of
treatment on an empty stomach, just before administration of the
composition; the higher value corresponds to the mean concentration
of the drugs observed at Tmax. **Acamprosate calcium
D. Treatment of Age-Associated Memory Impairment.
[0476] Memory is known as one of the earliest cognitive functions
to decline through the aging process in humans and rodents [67,
68].
[0477] Inventors have evaluated the efficacy of the compositions of
the invention in treating AD-related diseases such as
age-associated memory impairment. Working memory performances were
evaluated through the T-maze alternation test in treated and
non-treated animals (roughly the same test as spontaneous
alternation in part I.B)1)a)).
Animals--Treatment
[0478] 24-month-old C57L/6J male mice were used. Mice were held on
a reversed light cycle. Each experiment was performed between 8 am
and 3 pm and, therefore, under red light conditions.
[0479] Mice were administered by gavage, twice daily with either
the combination of the invention (acamprosate 0.2 mg/kg+baclofen 3
mg/kg) or the vehicle solution all along the experiment. Working
memory performances of treated and non-treated animals were assayed
17 weeks and 29 weeks after the beginning of the treatment.
[0480] On the day of the test, animals were treated 2 hours before
the test.
Experimental Procedure
[0481] The T-maze apparatus is made of gray Plexiglas with a main
stem (55 cm long.times.10 cm wide.times.20 cm high) and two arms
(30 cm long.times.10 cm wide.times.20 cm high) positioned at a
90-degree angle relative to the main stem. A start box (15 cm
long.times.10 cm wide) is separated from the main stem by a sliding
door. Sliding doors are also provided to close specific arms during
the forced-choice alternation task.
[0482] The experimental protocol consists of one single session,
which starts with 1 "forced-choice" trial, followed by 14
"free-choice" trials. In the first "forced-choice" trial, the
animal is confined 5 s in the start arm and then released while
either the left or right goal arm is blocked by closing the sliding
door. Then the animal explores the open arm and returns to the
start arm. At this point, the animal has completed the
forced-choice trial. Immediately after the return of the animal to
the start position, the left or right goal door is opened and the
animal is allowed to freely choose between the left and right goal
arm ("free choice" trials). Each time that the animal has chosen a
goal arm, the opposite arm is closed in order to oblige the animal
to return to the start arm. Once the animal returns to the start
arm, all goal doors are opened to allow another round of free
choice trial begins. The animal is considered as entered in a
choice arm when it places its four paws in the arm. A session is
terminated and the animal is removed from the maze as soon as 14
free-choice trials have been performed or 15 min have elapsed,
whatever event occurs first.
[0483] The apparatus was cleaned between each animal using alcohol
(70.degree.). Urine and feces were removed from the maze.
[0484] During the trials, animal handling and the visibility of the
operator were minimized as much as possible.
Calculation and Statistical Analysis
[0485] The percent spontaneous alternation was calculated as the
number of spontaneous alternations divided by the number of
free-choice trials. An alternation is defined as a succession of 2
different arms over consecutive choices (e.g., the sequence
right-left-right represents 2 alternations).
[0486] Analysis of variance (ANOVA) was performed on the results.
Dunnett's test was applied to determine significance of
differences.
Results
[0487] As shown in FIG. 28, composition of the invention
significantly improved (by more than 50%) memory of aged mice as
soon as 17 weeks after the beginning of the treatment. A
significant improvement is also observed after 29 weeks of
treatment (not shown).
[0488] Hence compositions of the invention are also efficient in
counteracting AD-related disorders such as age-associated memory
impairment.
[0489] II. Treatment of Diseases Related to Glutamate Toxicity
[0490] A. Prevention of Glutamate Toxicity on Neuronal Cells In
Vitro
[0491] In this further set of experiments, 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
disorders such as multiple sclerosis, Alzheimer's Disease,
frontotemporal dementia, 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 for their combinatorial
action.
Methods
[0492] 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 I.A.2
above.
Glutamate Toxicity Assays
[0493] The neuroprotective effect of compounds is assessed by
quantification of the neurite network (neurofilament immunostaining
(NF), which specifically reveals the glutamatergic neurons).
[0494] 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, glutamate is added
for 20 min, to a final concentration of 40 .mu.M, in the presence
of the candidate combinations, in order to avoid further drug
dilutions. At the end of the incubation, the medium is changed with
medium with the candidate combination but without glutamate. The
culture is fixed 24 hours after glutamate injury MK801
(Dizocilpinehydrogen maleate, 77086-22-7-20 .mu.M) is used as a
positive control.
[0495] After permeabilization with saponin (Sigma), cells are
blocked for 2 h 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 IgG.
[0496] 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
[0497] The combination of baclofen-acamprosate gives a protective
effect against glutamate toxicity for cortical neuronal cells. As
exemplified in FIG. 15, combinations of the invention strongly
protect neurons from glutamate toxicity under the experimental
conditions described above. It is noteworthy that an effective
protection is noticed using drug concentrations at which the drugs
used alone have a lower protective effect. Combination of baclofen
and acamprosate induces an improvement of more than 200% compared
to acamprosate alone and more than 47% compared to baclofen
alone.
[0498] B. Improvement of Other Disorders Related to Glutamate
Excitoxicity Using Combinations of the Invention
[0499] 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, such as MS, ALS and
neuropathic pain.
[0500] 1. Protective Effect of Combinations in an In Vivo Model of
Multiple Sclerosis.
[0501] 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
[0502] 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.sub.35-55/CFA
Emulsion PTX (Pertussis toxin) for EAE Induction (EK-0110, EK-0115;
Hooke Laboratories). The control kit is CK-0115 (Hooke
Laboratories).
Experimental Procedure
[0503] Experimental encephalomyelitis is induced by following
procedure:
[0504] On day 0, two subcutaneous injections of 0.1 ml each are
performed: one in the upper back of the mouse and one in the lower
back. Each injection contains 100 .mu.g of MOG.sub.35-55 peptide
(MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO:1), 200 .mu.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.
[0505] 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.
[0506] 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:
[0507] Groups of female mice as disclosed are homogenized by weight
before the immunization: [0508] Control group: vehicle injection in
the same conditions of EAE mice (from Day -1 to Day 28, placebo is
given daily), [0509] EAE group: MOG injection (Day 0)+pertussis
toxin injections (Day 0 and 1)-from Day -1 to Day 28, placebo is
given orally daily, [0510] 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, and [0511] 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.
[0512] Treatments are applied into two divided doses (i.e.,
bid).
[0513] The clinical scores are measured at Days
0-5-8-9-12-14-16-19-21-23-26-28.
[0514] Statistical 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.
[0515] 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 p-values are unilateral and test the
hypothesis to be better than the reference "immu" group.
[0516] 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:
TABLE-US-00010 [0517] Score = 0 A normal mouse holds its tail erect
when moving. Score = 1 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:
TABLE-US-00011 [0518] Score = 0 A normal mouse has an energetic
walk and doesn't drag its paws Score = 1 Either one of the
following tests is positive: A - Flip test: while holding the tail
between thumb and index finger, flip the animal on its 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:
TABLE-US-00012 [0519] Score = 0 A normal mouse uses its front paws
actively for grasping and walking and holds its head erect. Score =
1 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:
TABLE-US-00013 [0520] Score = 0 A normal mouse has full control of
its bladder. Score = 1 A mouse is considered incontinent when its
lower body is soaked with urine.
[0521] 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: Combination Therapies are Efficient in a MS Model
[0522] A significant improvement of global clinical score is
observed in "EAE+treatment group" mice for the baclofen and
acamprosate combination.
[0523] The combination of baclofen (30 mg/kg/day) and acamprosate
(2 mg/kg/day) induced a significant protective effect against the
development of chronic progressive EAE and hence confirmed the
beneficial effect of the composition in multiple sclerosis
treatment (FIG. 18). With more than 30% reduction of the symptoms,
the results clearly show that the combination induces a significant
reduction of disease development from Day 13. This result confirms
the remarkable positive effect of the baclofen-acamprosate
combination on neuronal protection including demyelination and its
implications.
[0524] Taken together, these results show that this combination
enables effective protection of neurons against many stresses
involved in the development of neurological disease such as .beta.
amyloid, BBB breakdown, glutamate excitotoxicity or
demyelination.
[0525] 2) Protective Effects of Combinations in Models of ALS.
[0526] The effect of combination therapies according to the present
invention on ALS have been demonstrated in vitro, in a co-culture
model, and in vivo, in a mouse model of ALS. Protocols and results
are presented in this section.
[0527] a) Protective Effect Against Glutamate Toxicity in Primary
Cultures of Nerve-Muscle Co-Culture
Primary Cocultures of Nerve and Muscle Cells
[0528] Human muscle is prepared according to a previously described
method from portions of a biopsy of a healthy patient [69]. Muscle
cells are established from dissociated cells (10,000 cells per
well), plated in gelatin-coated 0.1% on 48-well plates and grown in
a proliferating medium consisting of a mix of MEM medium and M199
medium.
[0529] Immediately after satellite cell 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
mixed medium. After 24 h 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.
[0530] One co-culture is done (6 wells per condition).
Glutamate Injury
[0531] On day 27, co-cultures are incubated with candidate
compounds or Riluzole one hour before glutamate intoxication (60
.mu.M) for 20 min. Then, co-cultures are washed and candidate
compounds or Riluzole are added for an additional 48 h. After this
incubation time, unfixed cocultures are incubated with
.alpha.-bungarotoxin coupled with Alexa 488 at a concentration of
500 nmol/L for 15 min at room temperature. Then, co-cultures are
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).
[0532] These antibodies are detected with Alexa Fluor 568 goat
anti-mouse IgG (Molecular Probes). Nuclei of neurons are labeled by
a fluorescent marker (Hoechst solution).
[0533] Endpoints are (1) Total neurite length, (2) Number of motor
units, and (3) Total motor unit area, which are indicative of motor
neuron survival and functionality.
[0534] For each condition, 2.times.10 pictures per well are taken
using InCell Analyzer.TM.1000 (GE Healthcare) with 20.times.
magnification. All the images are taken in the same conditions.
Results
[0535] The baclofen and acamprosate combination effectively
protects motor neurons and motor units in the coculture model.
[0536] b) Combination Therapies are Efficient in ALS Mouse
Model
[0537] Experiments are performed on male mice. Transgenic male
B6SJL-Tg(SOD1)2Gur/J mice and their control (respectively SN2726
and SN2297 from Jackson Laboratories, Ben Harbor, USA, distributed
by Charles River in France) are chosen in this set of experiments
to mimic ALS.
[0538] Diseased mice express the SOD1-G93A transgene, designed with
a mutant human SOD1 gene (a single amino acid substitution of
glycine to alanine at codon 93) driven by its endogenous human SOD1
promoter. Control mice express the control human SOD1 gene.
Randomisation of the Animals
[0539] The group assignation and the randomisation of the animals
are based on body weight; for each group, the randomisation is done
one day before the first treatment
Drug Administration
[0540] Mice are dosed with candidate drug treatment diluted in a
vehicle from the 60.sup.th day after birth till death. Diluted
solutions of drug candidates are prepared with water at room
temperature just before the beginning of the administration.
[0541] In Drinking Water:
[0542] Riluzole is added in drinking water at a final concentration
of 6 mg/ml (adjusted to each group's mean body weight) in 5%
cyclodextrin. As a mouse drinks about 5 ml/day, the estimated
administrated dose is 30 mg/kg/day, which is a dose that was shown
to increase the survival of mice.
[0543] Cyclodextrin is used as vehicle at the final concentration
of 5%, diluted in water at room temperature from stock solution
(cyclodextrin 20%).
[0544] Oral Administration (Per Os): [0545] Drug combinations are
administrated per os, daily. [0546] Cyclodextrin is used as a
vehicle at the final concentration of 5%, diluted in water at room
temperature from stock solution (cyclodextrin 20%).
Clinical Observation
[0547] The clinical observation of each mouse is performed daily,
from the first day of treatment (60 days of age) until death (or
sacrifice). Clinical observation consists of studying behavioural
tests: onset of paralysis, "loss of splay", "loss of righting
reflex", and general gait observation: [0548] 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. [0549] 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. [0550] The
loss of righting reflex test evaluates the ability of the mouse to
right itself within 30 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: Combination Therapies are Efficient in ALS In Vivo
Model
[0551] An improvement of the disease is observed for the diseased
animals treated with for the baclofen and acamprosate
combination.
[0552] 3. Protective Effect of Combinations in Oxaliplatin-Induced
Neuropathy as an In Vivo Model for Neuropathic Pain.
[0553] Combinatorial therapies of the present invention are tested
in vivo, in suitable models of peripheral neuropathy, i.e., an
acute model of oxaliplatin-induced neuropathy and a chronic model
of oxaliplatin-induced neuropathy. The animals, protocols and
results are presented in this section.
Animal Husbandry
[0554] Sprague-Dawley rats (CERJ, France), weighing 150-175 g at
the beginning of the experiment of the oxaliplatin treatment (Do)
are used. Animals are housed in a limited access animal facility in
a temperature (19.5.degree. C.-24.5.degree. 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
[0555] Five following groups of rats are used in all
experiments:
Control Groups:
[0556] Group 1: vehicle of oxaliplatin (distilled water),
i.p./Vehicle of candidate combination(s) (distilled water), p.o.
daily.
[0557] Group 2: oxaliplatin (distilled water), i.p./Vehicle of
candidate combination(s) (distilled water), p.o. daily.
[0558] Group 3: oxaliplatin 3 mg/kg i.p./single drug in distilled
water, p.o. daily.times.9.
Tested Composition Groups:
[0559] Group 4: oxaliplatin 3 mg/kg i.p./candidate combination(s)
in distilled water, p.o. daily.times.9.
[0560] Group 5: oxaliplatin 3 mg/kg i.p./gabapentin (100 mg/kg) in
distilled water, p.o. on testing days (i.e. D.sub.1 &
D.sub.8).
[0561] 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).
[0562] All treatments are administered in a coded and random order
when possible. Doses are expressed in terms of free active
substance.
Neuropathy Induction
[0563] Acute neuropathy is induced by a single intraperitoneal
injection of oxaliplatin (3 mg/kg).
[0564] 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.sup.2 which
corresponds to .about.15 mg/kg as a cumulative dose in rats
[70].
[0565] The oxaliplatin-induced painful neuropathy in rat reproduces
the pain symptoms in oxaliplatin-treated patients: [0566] The
thermal hyperalgesia is the earliest symptom. It