NZ614184B2 - Baclofen and acamprosate based therapy of neurogical disorders - Google Patents
Baclofen and acamprosate based therapy of neurogical disorders Download PDFInfo
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- NZ614184B2 NZ614184B2 NZ614184A NZ61418412A NZ614184B2 NZ 614184 B2 NZ614184 B2 NZ 614184B2 NZ 614184 A NZ614184 A NZ 614184A NZ 61418412 A NZ61418412 A NZ 61418412A NZ 614184 B2 NZ614184 B2 NZ 614184B2
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Abstract
Provided are compositions comprising a combination of baclofen and acamprosate and their use in the treatment of neurological disorders. Further provided are compositions comprising the combination with one or more additional active ingredients 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. The neurological disorders for treatment with the compositions include Alzheimer’s disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), neuropathies, alcoholism or alcohol withdrawal, Huntington's disease (HD) and spinal cord injury. yphylline, quinacrine, carbenoxolone, aminocaproic acid, cabergoline, diethylcarbamazine, cinacalcet, cinnarizine, eplerenone, fenoldopam, leflunomide, levosimendan, sulodexide, terbinafine, zonisamide, etomidate, phenformin, trimetazidine, mexiletine, ifenprodil, moxifloxacin, bromocriptine or torasemide. The neurological disorders for treatment with the compositions include Alzheimer’s disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), neuropathies, alcoholism or alcohol withdrawal, Huntington's disease (HD) and spinal cord injury.
Description
BACLOFEN AND ACAMPROSATE BASED THERAPY OF NEUROLOGICAL
DISORDERS
FIELD OF THE INVENTION
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
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).
Incidence of Alzheimer 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).
The incidence rate of AD increases dramatically with age. United Nation
population projections estimate that the number of people older than 80 years will
approach 370 million by the year 2050. Currently, it is estimated that 50% of people
older than age 85 years are afflicted with AD. Therefore, more than 100 million people
worldwide will suffer from dementia in 50 years. The vast number of people requiring
constant care and other services will severely affect medical, monetary and human
resources (6).
Memory impairment is the early feature of the disease and involves episodic
memory (memory for day-today events). Semantic memory (memory for verbal and
visual meaning) is involved later in the disease. 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.
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 not only is this important for clinical management but provides further
implication of specific medication treatments for functionally different forms (8).
The pathological hallmark of AD includes amyloid plaques containing beta-
amyloid (Abeta), neurofibrillary tangles (NFT) containing Tau and neuronal and
synaptic dysfunction and loss (9-1 1). 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 breakdown have been identified as key events contributing to the APP toxicity in
the amyloid cascade (17). On contrary, Tau protein has received much less attention
from the pharmaceutical industry than amyloid, because of both fundamental and
practical concerns. Moreover, synaptic density change is the pathological lesion that
best correlates with cognitive impairment than the two others. Studies have revealed
that the amyloid pathology appears to progress in a neurotransmitter-specific manner
where the cholinergic terminals appear most vulnerable, followed by the glutamatergic
terminals and finally by the GABAergic terminals (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 receptors
overactivation (18). Abnormal accumulation of glutamate in synaptic cleft leads to the
overactivation of glutamate receptors that results in pathological processes and finally in
neuronal cell death. This process, named excitotoxicity, is commonly observed in
neuronal tissues during acute and chronic neurological disorders.
It is becoming evident that excitotoxicity is involved in the pathogenesis of
multiple disorders of various etiology such as: spinal cord injury, stroke, traumatic brain
injury, hearing loss, alcoholism and alcohol withdrawal, alcoholic neuropathy, or
neuropathic pain as well as neurodegenerative diseases such as multiple sclerosis,
Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, and
Huntington's disease (19-21). The development of efficient treatment for these diseases
remains major public health issues due to their incidence as well as lack of curative
treatments.
Two kinds of medication are used for improving or slowing down symptoms of
AD which lay on some acetylcholinesterase modulators and blockers of NMDA
glutamate receptors (26-27).
NMDAR antagonists that target various sites of this receptor have been tested to
counteract excitotoxicity. Uncompetitive NMDAR antagonists target the ion channel
pore thus reducing the calcium entry into postsynaptic neurons. Some of them reached
the approval status. As an example, Memantine is currently approved in moderate to
severe Alzheimer's disease. It is clinically tested in other indications that include a
component of excitotoxicity such as alcohol dependence (phase II), amyotrophic lateral
sclerosis (phase III), dementia associated with Parkinson (Phase II), epilepsy,
Huntington's disease (phase IV), multiple sclerosis (phase IV), Parkinson's disease
(phase IV) and traumatic brain injury (phase IV). This molecule is however of limited
benefit to most Alzheimer's disease patients, because it has only modest symptomatic
effects. Another approach in limiting excitotoxicity consists in inhibiting the
presynaptic release of glutamate. Riluzole, currently approved in amyotrophic lateral
sclerosis, showed encouraging results in ischemia and traumatic brain injury models
(22-25). 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 US20 10076075.
WO2009133128, WO2009133141, WO2009133142 and WO2011054759 disclose
drug combinations for use in the treatment of AD.
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 (C NS) a nd the peripheral nervous system (PNS).
SUMMARY OF INVENTION
In one or more aspects the present invention may advantageously 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 toxicity, based on a combination of
Baclofen and Acamprosate.
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 Alzheimers 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.
An aspect of this invention therefore relates to compositions comprising a
combination Baclofen and Acamprosate, for use in the treatment of a neurological
disorder, particularly AD and related disorders, Multiple Sclerosis (MS ), Amyotrophic
Lateral Sclerosis (A LS), Parkinson's disease (P D), neuropathies (f or instance
neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal,
Huntington's disease (H D) a nd spinal cord injury.
The composition 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.
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.
The invention also relates to any pharmaceutical composition per se comprising
a combination of at least two compounds as defined above.
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.
In a preferred embodiment, the compound is used as such or in the form a salt,
hydrate, ester, ether or sustained release form thereof. A particularly preferred salt for
use in the present invention is Acamprosate calcium.
In another preferred embodiment, a prodrug or derivative is used.
A further aspect 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.
Another aspect 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.
A further aspect of this invention relates to a method for treating Alzheimer 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.
A preferred aspect 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.
A more preferred aspect of this invention relates to a method for treating Alzheimer
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.
In one aspect, the present invention provides a composition comprising baclofen
and acamprosate, or salts, prodrugs or sustained release formulations thereof, wherein the
dose of acamprosate is less than 200mg.
In another aspect, the present invention provides use of a composition comprising
baclofen and acamprosate, or salts, prodrugs or sustained release formulation thereof, in
the manufacture of a medicament for the treatment of a neurodegenerative disease,
neuropathy, or spinal cord injury in a subject in need thereof.
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 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
Figure 1: Validation of the experimental model of human amyloids toxicity on
endothelial cells used for drug screening. One hour of VEGF pre-treatment at 10nM
significantly protected the capillary network from this amyloid injury (+ 70% of
capillary network compared to amyloid intoxication).
Figure2: Effect of Baclofen (BCL) and Acamprosate (A CP) combination therapy on
the total length of capillary network in beta-amyloid intoxicated HBMEC cultures. The
human amyloid peptide (A 2.5µM) produces a significant intoxication, above 40%,
1-42
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Acamprosate and Baclofen (A) whereas, at those concentrations,
Acamprosate (B) and Baclofen (C) alone have no significant effect on intoxication. <3>:
p<0.05, significantly different from A b ΐ intoxication; * : p<0.05, significantly
different from vehicle; "ns" no significant effect (ANOVA + Dunnett Post-Hoc test).
Figure 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 (Abi 2.5 m 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).
Figure 4; Validation of the experimental model of human pamyloid's toxicity on
neuronal cells used for drug screening. One hour of Estradiol (150 nM) or BDNF
(50ng/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 b 2 intoxication.
Figure 5; Effect of Acamprosate (ACP) and Baclofen (BCL) combination therapy on
LDH release in human Abi toxicity on rat primary cortical cells. The human amyloid
peptide (Abi _ 10 m M ) produces a significant intoxication compared to vehicle-treated
neurons. This intoxication is significantly prevented by the combination of Acamprosate
and Baclofen (A) whereas, at those concentrations, Acamprosate (B) and Baclofen (C)
alone have no significant effect on intoxication. :p<0.05, significantly different from
Abi intoxication; * : p<0.05, significantly different from vehicle; "ns" no significant
effect. (ANOVA + Dunnett Post-Hoc test).
Figure 6 : Effect of Cinacalcet (CNC) and Sulfisoxazole (SFX) combination therapy on
LDH release in human Abi toxicity on rat primary cortical cells. The human amyloid
peptide (Abi . 10 m 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.
Figure 7; Effect of Acamprosate (ACP) and Baclofen (BCL) combination therapy on
the total length of neurites network in beta-amyloid intoxicated cortical neurons. The
human amyloid peptide (A b 2.5 m M ) produces a significant intoxication, above 15 %,
compared to vehicle-treated cells. This intoxication is significantly prevented by the
combination of Acamprosate and Baclofen whereas, at those concentrations,
Acamprosate and Baclofen alone have no significant effect on intoxication. : p<0.05,
significantly different from Abi intoxication; * : p<0.05, significantly different from
vehicle (ANOVA + Dunnett Post-Hoc test).
Figure 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 % of protection) by the combination
of Acamprosate (0.2mg/kg/day) and Baclofen (3mg/kg/day). 0 : p<0.05, significantly
different from A b intoxication; * : p<0.05, significantly different from vehicle
(ANOVA + Dunnett Post-Hoc test).
Figure 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 (0.2mg/kg) and Baclofen (3mg/kg). 0 : p<0.05,
significantly different from A b intoxication; * : p<0.05, significantly different from
vehicle (ANOVA + Dunn' stest).
Figure 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 performances as measured by step-through latency,
above 44 %, compared to control. This deleterious effect is significantly prevented (78.8
% of protection effect) by the combination of Acamprosate (0.2mg/kg) and Baclofen
(3mg/kg) whereas, at those concentrations, Acamprosate and Baclofen alone have a
lower effect on intoxication. 0 : p<0.05, significantly different from A b 2 - intoxication;
* : p<0.05, significantly different from vehicle (ANOVA + Dunn's test).
Figure 11: Effect of Acamprosate and Baclofen combination therapy on neuron's
density in hippocampus. The amyloid peptide produces a significant decrease neuronal
density as measured by the number of neurons per millimeter in hippocampus, above 2 1
% , compared to control. This neuronal injury is significantly prevented (63.2 % of
injured neurons are protected) by the combination of Acamprosate (0.2mg/kg) and
Baclofen (3mg/kg). 0 : p<0.05, significantly different from A b 2 - intoxication; * :
p<0.05, significantly different from vehicle (ANOVA + Dunnett Post-Hoc test).
Figure 12: Effect of Acamprosate and Baclofen combination therapy on the blood brain
barrier integrity. The amyloid peptide affect the blood brain barrier (BBB) inducing a
significant increase of its permeability, above 5 1 % , compared to control. Those
damages on the blood brain barrier are significantly prevented (66.6 % of the integrity
restored) by the combination of Acamprosate (0.2mg/kg) and Baclofen (3mg/kg). :
p<0.05, significantly different from A b intoxication; * : p<0.05, significantly
different from vehicle (ANOVA + Dunnett Post-Hoc test).
Figure 13: Effect of Acamprosate and Baclofen combination therapy on the synaptic
density as reflected by the synaptophysin concentration. The amyloid peptide affect the
synapse function inducing a significant decrease the synaptophysin concentration in
brain, above 34 %, compared to control. Those damages on the synaptic density are
significantly prevented (76 %) by the combination of Acamprosate (0.2mg/kg/day) and
Baclofen (3mg/kg/day). 0 : p<0.05, significantly different from A b intoxication; * :
-35
p<0.05, significantly different from vehicle (ANOVA + Dunnett Post-Hoc test).
Figure 14: Protective effect of Acamprosate and Baclofen combination therapy on the
oxidative stress in hippocampus. The amyloid peptide induces a significant increase of
oxidative stress in hippocampus as measured by lipid peroxydation, above 59 % ,
compared to control. This oxidative stress is significantly prevented (65.9%) by the
combination of Acamprosate (0.2mg/kg/day) and Baclofen (3mg/kg/day). 0 : p<0.05,
significantly different from A b - 5 intoxication; * : p<0.05, significantly different from
vehicle (ANOVA + Dunnett Post-Hoc test).
Figure 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 nM) whereas, at those
concentrations, Baclofen and Acamprosate alone have no significant effect on
intoxication. < : p<0.001, significantly different from glutamate intoxication; (ANOVA
+ Dunnett Post-Hoc test).
Figure 16: Effect of Donepezil, 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
(51.5% versus 71.8%). This deleterious effect is significantly prevented (98 % of
protection) by the combination of Donepezil (0,25mg/kg/day), Acamprosate
(32 m g/kg/day) and Baclofen (480 g/kg/day), whereas at those concentrations drugs
alone have no significant effect. 0 : p<0.01, significantly different from A b
intoxication; * : p<0.01, significantly different from vehicle (ANOVA + Dunnett Post-
Hoc test).
Figure 17: Comparison of protective effect of Acamprosate and its derivative
Homotaurine pre-treatment in human Abi _ toxicity assays on rat primary cortical cells.
A i produces a significant intoxication compared to vehicle-treated neurons. The
intoxication is equally significantly prevented by Homotaurine and Acamprosate (99%,
8nM). 0 : p<0.0001: significantly different from A b 2 intoxication.
Figure 18: E ffect 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).
DETAILED DESCRIPTION OF THE INVENTION
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.
The invention is suited for treating any neurological disorders, whether central or
peripheral, particularly disorders wherein nerves or neurons injuries, Pamyloid, BBB
breakdown or glutamate excitotoxicity are involved. Specific examples of such
disorders include neurodegenerative diseases, neuropathies, spinal cord injury, and
substances abuse such as alcoholism.
Neurodegenerative disorders refer to diseases, such as Alzheimer's and related
disorders, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Parkinson's
Disease (PD), Huntington's Disease (HD), encompassing a progressive loss of function
and death of neurons.
Neuropathies refer to conditions where nerves of the peripheral nervous system are
damaged, this include damages of the peripheral nervous system provoked by genetic
factors, inflammatory disease, or by chemical substance including drugs (vincristine,
oxaliplatin, ethyl alcohol). The treatment of neuropathies also includes the treatment of
neuropathic pain.
The invention is particularly suited for treating AD and related disorders. In the
context of this invention, the term "related disorder" includes senile dementia of AD
type (SDAT), Lewis body dementia, vascular dementia, mild cognitive impairment
(MCI) and age-associated memory impairment (AAMI).
As used herein, "treatment" includes the therapy, prevention, prophylaxis,
retardation or reduction of symptoms provoked by or of the causes of the above diseases
or disorders. The term treatment includes in particular the control of disease progression
and associated symptoms. The term treatment particularly includes a protection against
the toxicity caused by Amyloid Beta, or a reduction or retardation of said toxicity,
and/or ii) a protection against glutamate excitotoxicity, or a reduction or retardation of
said toxicity, in the treated subjects. The term treatment also designates an improvement
of cognitive symptom or a protection of neuronal cells.
Within the context of this invention, the designation of 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.
The term "combination or combinatorial treating/therapy" designates 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.
The term "prodrug" as used herein refers to any functional derivatives (or
precursors) of a compound of the present invention, which, when administered to a
biological system, generates said compound as a result of e.g., spontaneous chemical
reaction(s), enzyme 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 (29-33). 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 (30; 34-40). For example, Arbaclofen Placarbil is
listed in ChemID plus Advance database (website: chem.sis.nlm.nih.gov/chemidplus/)
and Arbaclofen Placarbil is a well-known prodrug of Baclofen (41-42).
The term "derivative" of a compound includes any molecule that is functionally
and/or structurally related to said compound, such as an acid, amide, ester, ether,
acetylated variant, hydroxylated variant, or an alkylated (C1-C6) variant of such a
compound. The term derivative also includes structurally related compound having lost
one or more substituent as listed above. For example, Homotaurine is a deacetylated
derivative of Acamprosate. Preferred derivatives of a compound are molecules having a
substantial degree of similarity to said compound, as determined by known methods.
Similar compounds along with their index of similarity to a parent molecule can be
found in numerous databases such as PubChem
(http://pubchem.ncbi.nlm.nih.gov/search/) or DrugBank (http://www.drugbank.ca/). In a
more preferred embodiment, derivatives should have a Tanimoto similarity index
greater than 0.4, preferably greater than 0.5, more preferably greater than 0.6, even
more preferably greater than 0.7 with a parent drug. The Tanimoto similarity index is
widely used to measure the degree of structural similarity between two molecules.
Tanimoto similarity index can be computed by software such as the Small Molecule
Subgraph Detector (43-44) available online (http://www.ebi.ac.uk/thornton-
srv/software/SMSD/). Preferred derivatives should be both structurally and functionally
related to a parent compound, i.e., they should also retain at least part of the activity of
the parent drug, more preferably they should have a protective activity against A b or
glutamate toxicity.
The term derivatives also include metabolites of a drug, e.g., a molecule which
results from the (biochemical) modification(s) or processing of said drug after
administration to an organism, usually through specialized enzymatic systems, and
which displays or retains a biological activity of the drug. Metabolites have been
disclosed as being responsible for much of the therapeutic action of the parent drug. In a
specific embodiment, a "metabolite" as used herein designates a modified or processed
drug that retains at least part of the activity of the parent drug, preferably that has a
protective activity against A b toxicity or glutamate toxicity.
The term "salt" refers to a pharmaceutically acceptable and relatively non-toxic,
inorganic or organic acid addition salt of a compound of the present invention.
Pharmaceutical salt formation consists in pairing an acidic, basic or zwitterionic drug
molecule with a counterion to create a salt version of the drug. A wide variety of
chemical species can be used in neutralization reaction. Pharmaceutically acceptable
salts of the invention thus include those obtained by reacting the main compound,
functioning as a base, with an inorganic or organic acid to form a salt, for example, salts
of acetic acid, nitric acid, tartric acid, hydrochloric acid, sulfuric acid, phosphoric acid,
methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid or
citric acid. Pharmaceutically acceptable salts of the invention also include those in
which the main compound functions as an acid and is reacted with an appropriate base
to form, e.g., sodium, potassium, calcium, magnesium, ammonium, or choline salts.
Though most of salts of a given active principle are bioequivalents, some may have,
among others, increased solubility or bioavailability properties. Salt selection is now a
common standard operation in the process of drug development as teached by H . Stahl
and C.G Wermuth in their handbook (45).
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.
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.
In a particular embodiment, a sustained-release formulation of the compound is
used.
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.
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 capillary network or LDH release in beta-amyloid
intoxicated nervous cells, and represent new therapeutic approaches of AD. 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 that 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.
Furthermore, these combinations effectively protect neuronal cells from various
afflictions such as glutamate toxicity, oxidative stress and prevent BBB
permeabilization or neuronal cells induced apoptosis which are involved in several
neurological disorders.
The present invention therefore proposes a novel therapy of neurological disorders,
based on Baclofen and Acamprosate compositions. More particularly, the present
invention therefore proposes a novel therapy of 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.
In this regard, in a particular embodiment, the invention relates to a composition
comprising Baclofen and Acamprosate.
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.
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 related
disorders, MS, PD, ALS, HD, neuropathies (for instance neuropathic pain or alcoholic
neuropathy), alcoholism or alcohol withdrawal, or spinal cord injury.
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 1
Specific examples of prodrugs of Baclofen are given in Hanafi et al, 201 1 (41),
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. Baclofen placarbil as mentioned before is also a well-
k own 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:
WO20 10102071, US2009197958, WO2009096985, WO2009061934, WO2008086492,
US2009216037, WO2005066122, US201 1021571, WO2003077902, WO2010120370.
Useful prodrugs for acamprosate such as pantoic acid ester neopentyl sulfonyl
esters, neopentyl sulfonyl esters prodrugs or masked carboxylate neopentyl sulfonyl
ester prodrugs of acamprosate are notably listed in WO2009033069, WO2009033061,
WO2009033054 WO2009052191, WO2009033079, US 2009/0099253, US
2009/00694 1 , US 2009/0082464, US 2009/0082440, and US 2009/0076 147.
Baclofen and Acamprosate 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 2
CAS NUMBER
DRUG NAME
Aminocaproic Acid 602
Bromocriptine 256143
Cabergoline 814097
56973
Carbenoxolone
Cinacalcet 2262560
Cinnarizine 2987
Diethylcarbamazine 901
Dyphylline 4795
1077249
Eplerenone
Etomidate 331252
Fenoldopam 672270
Ifenprodil 232102 or 232104
Leflunomide 757066
Levosimendan 1415051
Methimazole 600
53704 or 318284
Mexiletine
Moxifloxacin 3548122
Phenformin 1143
Prilocaine 7216 or 142897 or 142898
Quinacrine 836
Sulfisoxazole 1275
Sulodexide 578211
9 11616
Terbinafine
Torasemide 562116 or 728104
501 17 or 131710
Trimetazidine
Zonisamide 682914
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.
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.
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 Alzheimer's
disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Parkinson's Disease,
Huntington's Disease, neuropathies (for instance neuropathic pain or alcoholic
neuropathy), alcoholism or alcohol withdrawal, and spinal cord injury. These
compositions efficiently prevent toxicity of amyloid b (A b ) peptide or glutamate
excitotoxicity on neuronal cells. Moreover, in vivo, these compositions lead to an
improvement of several cognitive symptoms as well as to a protection of neuronal cells.
Hence they represent novel and potent methods for treating such disorders.
The experimental section further shows that the above mentioned compositions are also
efficient i) in synergistically protecting in vitro neuronal cells from glutamate
excitotoxicity, and ii) in conferring clinical benefit in in vivo models for diseases related
to glutamate excitotoxicity.
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.
Preferred compositions of the invention, for use in the treatment of a neurological
disorder such as Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD,
neuropathies (for instance neuropathic pain or alcoholic neuropathy), alcoholism or
alcohol withdrawal, or spinal cord injury, comprise one of the following drug
combinations, for combined, separate or sequential administration:
- Baclofen and Acamprosate,
- Baclofen and Acamprosate and Diethylcarbamazine,
- Baclofen and Acamprosate and Cinacalcet,
- Baclofen and Acamprosate and Sulfisoxazole,
- Baclofen and Acamprosate and Torasemide,
- Baclofen and Acamprosate and Ifenprodil,
- Baclofen and Acamprosate and Mexiletine,
- Baclofen and Acamprosate and Eplerenone,
- Baclofen and Acamprosate and Levosimendan,
- Baclofen and Acamprosate and Terbinafine, or
- Baclofen and Acamprosate and Leflunomide.
As disclosed in the experimental section, combinatorial therapies of the
invention provide substantial therapeutic and biological effect to improve Alzheimer's
disease or related disorders in human subjects. They induce a strong neuroprotective
effect against A b toxicity and give positive results in behavioural performances and
biochemical assays in vivo. Results show that compositions of the invention i)
efficiently correct molecular pathways triggered, in vivo, by A b aggregates and ii) lead
to an improvement of neurophysiological impairments observed in diseased animals as
neuron survival or synapse integrity.
Moreover, the results presented show also that the above combinations therapies
have an important synergistic neuroprotecting effect against glutamate excitotoxicity
(figures 15) a pathway which is implicated in various neurological diseases as AD, MS,
PD, ALS, HD, neuropathies (for instance neuropathic pain or alcoholic neuropathy),
alcoholism or alcohol withdrawal, or spinal cord injury. These therapies give positive
results in in vivo or in vitro models for these diseases.
In addition, in vivo results show that compositions of the invention efficiently
restore Brain Blood Barrier integrity and prevent, retard, or lessen apoptosis triggering,
which are known to be impaired in several neurological diseases.
Furthermore, the particularly high synergisitic interaction observed for these two
drugs allows the use drug concentrations showing no effect when used in single drug
treatment. Moreover, as shown in the experimental section, Baclofen and Acamprosate
combination causes an enhanced therapeutic benefit on Alzheimer's disease compared
to other therapeutic combinations. These compositions efficiently prevent the toxic
effects of amyloid b protein or peptide on human cells and in an in vivo model and
represent novel and potent methods for treating such disorder.
An object of this invention thus also resides in a composition as defined above
for treating a neurological disorder such as Alzheimer's disease (AD), AD related
disorders, MS, PD, ALS, HD, neuropathies (for instance alcoholic neuropathy or
neuropathic pain), alcoholism or alcohol withdrawal, or spinal cord injury.
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.
A further object of this invention resides in the use of a composition as defined
above for the manufacture of a medicament for treating a neurological disorder such as
Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies (for
instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol withdrawal,
or spinal cord injury.
The invention further provides a method for treating a neurological disorder such
as Alzheimer's disease (AD), AD related disorders, MS, PD, ALS, HD, neuropathies
(for instance neuropathic pain or alcoholic neuropathy), alcoholism or alcohol
withdrawal, or spinal cord injury, comprising administering to a subject in need thereof
an effective amount of a composition as disclosed above.
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.
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 injuryin a subject in need thereof,
comprising administering simultaneously, separately or sequentially to the subject an
effective amount of Baclofen and Acamprosate.
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.
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.
In a particular embodiment, the method comprises mixing Baclofen and
Acamprosate in an appropriate excipient or carrier.
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.
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
or association or combination with additional drugs or treatments beneficial to the
treated neurological condition in the subjects.
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 show also that the above combinations
therapies have an important synergistic neuroprotecting effect when combined with
donepezil (figure 16). Thereby, illustrative therapies which can be used with
combinations of the invention are Donepezil (CAS: 1200144), Gabapentine (CAS:
4782969; 601423), Rivastigmine (1234412) or Memantine (CAS: 19982-
08-2).
In this regard, in a particular embodiment, the drug(s) or compositions according to
the present invention may be further combined with Ginkgo Biloba extracts. Suitable
extracts include, without limitation, Ginkgo biloba extracts, improved Ginkgo biloba
extracts (for example enriched in active ingredients or lessened in contaminant) or any
drug containing Ginkgo biloba extracts.
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.
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 recovery from any as yet unforeseen side-effects. The drugs may also be
formulated together such that one administration delivers all drugs.
The administration of each drug of the combination may be by any suitable
means that results in a concentration of the drug that, combined with the other
component, is able to ameliorate the patient condition or efficiently treat the disease or
disorder.
While it is possible for the drugs the combination to be administered as the pure
chemical it is preferable to present them as a pharmaceutical composition, also referred
to in this context as pharmaceutical formulation. Possible compositions include those
suitable for oral, rectal, topical (including transdermal, buccal and sublingual), or
parenteral (including subcutaneous, intramuscular, intravenous and intradermal)
administration.
More commonly these pharmaceutical formulations are prescribed to the patient
in "patient packs" containing a number dosing units or other means for administration of
metered unit doses for use during a distinct treatment period in a single package, usually
a blister pack. Patient packs have an advantage over traditional prescriptions, where a
pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the
patient always has access to the package insert contained in the patient pack, normally
missing in traditional prescriptions. The inclusion of a package insert has been shown to
improve patient compliance with the physician's instructions. Thus, the invention
further includes a pharmaceutical formulation, as herein before described, in
combination with packaging material suitable for said formulations. In such a patient
pack the intended use of a formulation for the combination treatment can be inferred by
instructions, facilities, provisions, adaptations and/or other means to help using the
formulation most suitably for the treatment. Such measures make a patient pack
specifically suitable for and adapted for use for treatment with the combination of the
present invention.
The drug may be contained, in any appropriate amount, in any suitable carrier
substance. 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., intravenously, intramuscularly), rectal, cutaneous,
nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the
composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates,
suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams,
plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables,
implants, sprays, or aerosols.
The pharmaceutical compositions may be formulated according to conventional
pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy
(20th ed.), ed. A . R . Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia
of Pharmaceutical Technology, eds. J. Swarbrick and J . C . Boylan, 1988-1999, Marcel
Dekker, New York).
Pharmaceutical compositions according to the invention may be formulated to
release the active drug substantially immediately upon administration or at any
predetermined time or time period after administration.
The controlled release formulations include (i) formulations that create a
substantially constant concentration of the drug within the body over an extended period
of time; (ii) formulations that after a predetermined lag time create a substantially
constant concentration of the drug within the body over an extended period of time; (iii)
formulations that sustain drug action during a predetermined time period by maintaining
a relatively, constant, effective drug level in the body with concomitant minimization of
undesirable side effects associated with fluctuations in the plasma level of the active
drug substance; (iv) formulations that localize drug action by, e.g., spatial placement of
a controlled release composition adjacent to or in the diseased tissue or organ; and (v)
formulations that target drug action by using carriers or chemical derivatives to deliver
the drug to a particular target cell type.
Administration of drugs in the form of a controlled release formulation is
especially preferred in cases in which the drug has (i) a narrow therapeutic index (i.e.,
the difference between the plasma concentration leading to harmful side effects or toxic
reactions and the plasma concentration leading to a therapeutic effect is small; in
general, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD50)
to median effective dose (ED50)); (ii) a narrow absorption window in the gastro
intestinal tract; or (iii) a very short biological half-life so that frequent dosing during a
day is required in order to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled
release in which the rate of release outweighs the rate of metabolism of the drug in
question. Controlled release may be obtained by appropriate selection of various
formulation parameters and ingredients, including, e.g., various types of controlled
release compositions and coatings. Thus, the drug is formulated with appropriate
excipients into a pharmaceutical composition that, upon administration, releases the
drug in a controlled manner (single or multiple unit tablet or capsule compositions, oil
solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches,
and liposomes).
Solid Dosage Forms for Oral Use
Formulations for oral use include tablets containing the 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); and lubricating agents, glidants, and antiadhesives (e.g., stearic acid, silicas, or
talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents,
plasticizers, humectants, buffering agents, and the like.
The tablets may be uncoated or they may be coated by known techniques,
optionally to delay disintegration and absorption in the gastrointestinal tract and thereby
providing a sustained action over a longer period. The coating may be adapted to release
the active drug substance in a predetermined pattern (e.g., in order to achieve a
controlled release formulation) or it may be adapted not to release the active drug
substance until after passage of the stomach (enteric coating). The coating may be a
sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,
carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or
polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer,
cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl
methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or
ethylcellulose). A time delay material such as, e.g., glyceryl monostearate or glyceryl
distearate may be employed.
The solid tablet compositions may include a coating adapted to protect the
composition from unwanted chemical changes, (e.g., chemical degradation prior to the
release of the active drug substance). The coating may be applied on the solid dosage
form in a similar manner as that described in Encyclopedia of Pharmaceutical
Technology.
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.
Formulations for oral use may also be presented as chewable tablets, or as hard
gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g.,
potato starch, microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or
an oil medium, for example, liquid paraffin, or olive oil. Powders and granulates may be
prepared using the ingredients mentioned above under tablets and capsules in a
conventional manner.
Controlled release compositions for oral use may, e.g., be constructed to release
the active drug by controlling the dissolution and/or the diffusion of the active drug
substance.
Dissolution or diffusion controlled release can be achieved by appropriate
coating of a tablet, capsule, pellet, or granulate formulation of drugs, or by
incorporating the drug into an appropriate matrix. A controlled release coating may
include one or more of the coating substances mentioned above and/or, e.g., shellac,
beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate,
glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,
polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate,
methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation, the matrix material
may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol,
carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate,
polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
A controlled release composition containing one or more of the drugs of the
claimed combinations may also be in the form of a buoyant tablet or capsule (i.e., a
tablet or capsule that, upon oral administration, floats on top of the gastric content for a
certain period of time). A buoyant tablet formulation of the drug(s) can be prepared by
granulating a mixture of the drug(s) with excipients and 20-75% w/w of hydrocolloids,
such as hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropylmethylcellulose. The obtained granules can then be compressed into
tablets. On contact with the gastric juice, the tablet forms a substantially water-
impermeable gel barrier around its surface. This gel barrier takes part in maintaining a
density of less than one, thereby allowing the tablet to remain buoyant in the gastric
juice.
Liquids for Oral Administration
Powders, dispersible powders, or granules suitable for preparation of an aqueous
suspension by addition of water are convenient dosage forms for oral administration.
Formulation as a suspension provides the active ingredient in a mixture with a
dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable
suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose,
sodium alginate, and the like.
Parenteral Compositions
The pharmaceutical composition may also be administered parenterally by
injection, infusion or implantation (intravenous, intramuscular, subcutaneous, or the
like) in dosage forms, formulations, or via suitable delivery devices or implants
containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
The formulation and preparation of such compositions are well known to those skilled
in the art of pharmaceutical formulation.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in
single-dose ampoules), or in vials containing several doses and in which a suitable
preservative may be added (see below). The composition may be in form of a solution, a
suspension, an emulsion, an infusion device, or a delivery device for implantation or it
may be presented as a dry powder to be reconstituted with water or another suitable
vehicle before use. Apart from the active drug(s), the composition may include suitable
parenterally acceptable carriers and/or excipients. The active drug(s) may be
incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for
controlled release. The composition may include suspending, solubilizing, stabilizing,
pH-adjusting agents, and/or dispersing agents.
The pharmaceutical compositions according to the invention may be in the form
suitable for sterile injection. To prepare such a composition, the suitable active drug(s)
are dissolved or suspended in a parenterally acceptable liquid vehicle. Among
acceptable vehicles and solvents that may be employed are water, water adjusted to a
suitable 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.
Controlled release parenteral compositions may be in form of aqueous
suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil
suspensions, or emulsions. Alternatively, the active drug(s) may be incorporated in
biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.
Materials for use in the preparation of microspheres and/or microcapsules are, e.g.,
biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate),
poly(2-hydroxyethyl-L-glutamnine). Biocompatible carriers that may be used when
formulating a controlled release parenteral formulation are carbohydrates (e.g.,
dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in
implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g.,
poly(caprolactone), poly(glycolic acid) or poly(ortho esters)).
Alternative routes
Although less preferred and less convenient, other administration routes, and
therefore other formulations, may be contemplated. In this regard, for rectal application,
suitable dosage forms for a composition include suppositories (emulsion or suspension
type), and rectal gelatin capsules (solutions or suspensions). In a typical suppository
formulation, the active drug(s) are combined with an appropriate pharmaceutically
acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated
gelatin, and various water-soluble or dispersible bases like polyethylene glycols.
Various additives, enhancers, or surfactants may be incorporated.
The pharmaceutical compositions may also be administered topically on the skin
for percutaneous absorption in dosage forms or formulations containing conventionally
non-toxic pharmaceutical acceptable carriers and excipients including microspheres and
liposomes. The formulations include creams, ointments, lotions, liniments, gels,
hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, and other kinds of
transdermal drug delivery systems. The pharmaceutically acceptable carriers or
excipients may include emulsifying agents, antioxidants, buffering agents,
preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents,
ointment bases, perfumes, and skin protective agents.
The preservatives, humectants, penetration enhancers may be parabens, such as
methyl or propyl p-hydroxybenzoate, and benzalkonium chloride, glycerin, propylene
glycol, urea, etc.
The pharmaceutical compositions described above for topical administration on
the skin may also be used in connection with topical administration onto or close to the
part of the body that is to be treated. The compositions may be adapted for direct
application or for application by means of special drug delivery devices such as
dressings or alternatively plasters, pads, sponges, strips, or other forms of suitable
flexible material.
Dosages and duration of the treatment
It will be appreciated that the drugs of the combination may be administered
concomitantly, either in the same or different pharmaceutical formulation or
sequentially. If there is sequential administration, the delay in administering the second
(or additional) active ingredient should not be such as to lose the benefit of the
efficacious effect of the combination of the active ingredients. A minimum requirement
for a combination according to this description is that the combination should be
intended for combined use with the benefit of the efficacious effect of the combination
of the active ingredients. The intended use of a combination can be inferred by
facilities, provisions, adaptations and/or other means to help using the combination
according to the invention.
Therapeutically effective amounts of the drugs in a combination of this invention
include, e.g., amounts that are effective for reducing Alzheimer's disease symtpoms,
halting or slowing the progression of the disease once it has become clinically manifest,
or prevention or reduction of the risk of developing the disease.
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.
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.
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.
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, 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.
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.
One remarkable advantage of the invention is that each compound may be used at
low doses in a combination therapy, while producing, in combination, a substantial
clinical benefit to the patient. The combination therapy may indeed be effective at doses
where the compounds have individually low 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.
At such sub-therapeutic dosages, the compounds would exhibit no side effect,
while the combination(s) according to the invention are fully effective in treating
Alzheimer's disease.
A preferred dosage corresponds to amounts from 1% up to 50% of those usually
prescribed for long-term maintenance treatment.
The most preferred dosage may correspond to amounts from 1% up to 10% of
those usually prescribed for long-term maintenance treatment.
Specific examples of dosages of drugs for use in the invention are provided
below:
Acamprosate between 1 and 1000 mg/day, preferably less than 400 mg per day,
more preferably less than 200 mg/day, even more preferably less than 50
mg/day, such dosages being particularly suitable for oral administration.
- Baclofen between 0.01 to 150 mg per day, preferably less than 100 mg per day,
more preferably less than 50 mg/day, even more preferably less than 25 mg/day,
such dosages being particularly suitable for oral administration.
Aminocaproic Acid orally from about 0 .1 g to 2.4 g per day,
- Bromocriptine orally from about 0.01 to 10 mg per day,
- Diethylcarbamazine orally from about 0.6 to 600 mg per day,
Cabergoline orally from about 1 to 10 g per day,
Cinacalcet orally from about 0.3 to 36 mg per day,
Cinnarizine orally from about 0.6 to 23 mg per day,
- Dyphylline orally from about 9 to 320 mg per day,
- Eplerenone orally from about 0.25 to 10 mg per day,
- Ifenprodil orally from about 0.4 to 6 mg per day,
- Leflunomide orally from about 0.1 to 10 mg per day,
- Levosimendan orally from about 0.04 to 0.8 mg per day,
- Mexiletine orally from about 6 to 120 mg per day,
- Moxifloxacin orally from about 4 to 40 mg per day,
- Phenformin orally from about 0.25 to 15 mg per day,
Quinacrine orally from about 1 to 30 mg per day,
Sulfisoxazole orally from about 20 to 800 mg per day,
Sulodexide orally from about 0.05 to 40 mg per day,
Terbinafine orally from about 2.5 to 25 mg per day,
- Torasemide orally from about 0.05 to 4 mg per day,
Trimetazidine orally from about 0.4 to 6 mg per day,
- Zonisamide orally from about 0.5 to 50 mg per day.
When the composition comprises, as active ingredient, only Baclofen and
Acamprosate, these two compounds may be used in different ratios, e.g., at a weight
ratio Acamprosate/Baclofen comprised between from 0.05 to 1000 (W:W), preferably
between 0.05 to 100 (W:W), more preferably between 0.05 to 50 (W:W).
It will be understood that the amount of the drug actually administered will be
determined by a physician, in the light of the relevant circumstances including the
condition or conditions to be treated, the exact composition to be administered, the age,
weight, and response of the individual patient, the severity of the patient's symptoms,
and the chosen route of administration. Therefore, the above dosage ranges are intended
to provide general guidance and support for the teachings herein, but are not intended to
limit the scope of the invention.
The following examples are given for purposes of illustration and not by way of
limitation.
EXAMPLES
The care and husbandry of animals as well as the experimentations are performed
according to the guidelines of the Committee for Research and Ethical Issue of the
I.A.S.P. (1983).
A) TREATMENT OF DISEASES RELATED TO A b TOXICITY
In this series of experiments, candidate combinations have been tested for their
ability to prevent or reduce the toxic effects of human Abi . . Abi is the full length
42 -42
peptide that constitutes aggregates found in biopsies from human patients afflicted with
AD. The 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 animals and ii) on molecular hallmarks (apoptosis induction, oxidative
stress induction, inflammation pathway induction) of AD.
. BACLOFEN-ACAMPROSATE COMBINATION THERAPIES PREVENT
TOXICITY OF HUMAN A IN VITRO
1.1 Effect on the toxicity of human A Q 2peptide on human HBME cells.
Human brain microvascular endothelial cell cultures were used to study the protection
afforded by candidate compound(s) on Abi toxicity.
Human brain microvascular endothelial cerebral cells (HBMEC, ScienCell Ref: 1000,
frozen at passage 10) were rapidly thawed in a waterbath at +37°C. The supernatant was
immediately put in 9 ml Dulbecco's modified Eagle's medium (DMEM; Pan Biotech
ref: P04-03600) containing 10% of foetal calf serum (FCS; GIBCO ref 10270-106). Cell
suspension was centrifuged at 180 x g for 10 min at +4°C and the pellets were
suspended in CSC serum-free medium (CSC serum free, Cell System, Ref: SF-4Z0-
500-R, Batch 51407-4) with 1.6% of Serum free RocketFuel (Cell System, Ref: SF-
4Z0R, Batch 54102), 2% of Penicillin 10.000 U/ml and Streptomycin lOmg/ml
(PS ; Pan Biotech ref: P06-07100 batch 133080808) and were seeded at the density of
000 cells per well in 96 well-plates (matrigel layer biocoat angiogenesis system, BD,
Ref 354150, Batch A8662) in a final volume of IOOmI . On matrigel support, endothelial
cerebral cells spontaneously started the process of capillary network morphogenesis
(33).
Three separate cultures were performed per condition, 6 wells per condition.
Test compounds and Human treatment
Briefly, Abi peptide (Bachem, ref: H1368 batch 1010533) was reconstituted in define
culture medium at 20m M (mother solution) and was slowly shacked at +37 °C for 3
days in dark. The control medium was prepared in the same conditions.
After 3 days, human amyloid peptide was used on HBMEC at 2.5 M diluted in control
medium (optimal incubation time). The Abi peptide was added 2 hours after HBMEC
seeding on matrigel for 18 hours incubation.
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
lhour before the A i application (in a final volume per culture well of IOOmI ) . One
hour after test compounds or VEGF incubation (two hours after cell seeding on
matrigel), IOOmI of Abi peptide was added to a final concentration of 2.5 m M diluted
in control medium in presence of test compounds or VEGF (in a 200 m ΐ total
volume/well), in order to avoid further drug dilutions.
Organization of cultures plates
VEGF- 165 known to be a pro-angiogenic isoform of VEGF-A, was used for all
experiment in this study as reference compound. VEGF- 165 is one of the most abundant
VEGF isoforms involved in angiogenesis. VEGF was used as reference test compound
at IOhM (Figure 1).
The following conditions were assessed:
• Negative Control medium alone + 0.1% DMSO
• Intoxication: (2.5 m M ) for 18h
• Positive control. VEGF-165 (IOhM ) ( 1 reference compound/culture) lhr
before the Abi (2.5 m M ) addition for a 18h incubation time.
• Test compounds: Test compound(s) lhr before the Abi (2.5 m M )
addition for a 18h incubation time.
Capillary network quantification
Per well, 2 pictures with 4x lens were taken using InCell AnalyzerTM 1000 (GE
Healthcare) in light transmission. All images were taken in the same conditions.
Analysis of the angiogenesis networks was done using Developer software (GE
Healthcare). The total length of capillary network was assessed.
Data processing
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). Statistic 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
Baclofen-Acamprosate combination gives a significant protective effect against toxicity
of human Abi peptide in HBMEC model (a reduction of 24 % of Abi peptide
.42 .42
injury is observed), as shown in Figure 2 . The results clearly show that the intoxication
by human amyloid peptide (Abi 2.5 m 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.
Conversely, combination of Baclofen and Terbinafine (which is presented here only for
the sake of comparison) affords a weaker protection (a reduction of 15 % of Abi
peptide injury is observed) against Abi (figure 3).
Thus, although these two combinations allow a protection against Abi _ , the
combination Baclofen - Acamprosate stands out clearly. Indeed, these drugs at
concentrations having no effect alone allow significant protection of human HBME
cells against Abi 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.
Moreover, 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).
1.2 Effect on the toxicity of human peptide on primary cortical neuron cells.
Culture of primary cortical neurons
Rat cortical neurons were cultured as described by Singer et al. (47). Briefly pregnant
female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar) and
the foetuses were removed from the uterus. The cortex was removed and placed in ice-
cold medium of Leibovitz (L15) containing 2% of Penicillin 10.000 U/ml and
Streptomycin lOmg/ml and 1% of bovine serum albumin (BSA). Cortexes were
dissociated by trypsin for 20 min at 37°C (0.05%). The reaction was stopped by the
addition of Dulbecco's modified Eagle's medium (DMEM) containing DNasel grade II
and 10% of foetal calf serum (FCS). Cells were then mechanically dissociated by 3
serial passages through a 10 ml pipette and centrifuged at 515 x g for 10 min at +4°C.
The supernatant was discarded and the pellet of cells was re-suspended in a defined
culture medium consisting of Neurobasal supplemented with B27 (2%), L-glutamine
(0.2mM), 2% of PS solution and lOng/ml of BDNF. Viable cells were counted in a
Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a
density of 30 000 cells/well in 96 well-plates (wells were pre-coated with poly-L-lysine
(K^g/ml)) and were cultured at +37°C in a humidified air (95%)/C02 (5%)
atmosphere.
Three independent cultures will be performed per condition, 6 wells per condition.
Test compounds and Human amyloid^l-42 treatment
Briefly, A ΐ 2 peptide was reconstituted in define culture medium at 40m M (mother
solution) and was slowly shaken at +37 °C for 3 days in dark. The control medium was
prepared in the same conditions.
After 3 days, the solution was used on primary cortical neurons as follows:
After 10 days of neuron culture, test compounds were solved in culture medium (+0.1
% DMSO) and then pre-incubated with neurons for lhour before the Abi application
(in a final volume per culture well of 100 m ΐ). One hour after test compound(s)
incubation, IOOmI of Abi peptide was added to a final concentration of 10 m M diluted
in presence of drug(s), in order to avoid further test compound(s) dilutions. Cortical
neurons were intoxicated for 24 hours. Three separate cultures were performed per
condition, 6 wells per condition.
BDNF (50ng/ml) and Estradiol- b (150nM) were used as positive control and reference
compounds respectively. Three separate cultures will be performed per condition, 12
wells per condition.
Organization of cultures plates Estradiol- b at 150nM was used as a positive control
(Figure 4).
Estradiol- b was solved in culture medium and pre-incubated for 1 h before the amyloid-
b i application.
The following conditions were assessed:
- CONTROL PLAQUE: 12 wells/condition
• Negative Control: medium alone + 0.1% DMSO
• Intoxication: (10 m M ) for 24h
· Reference compound: Estradiol (150nM) lhr.
- DRUG PLA TE: 6 wells/condition
• Negative Control: medium alone + 0.1% DMSO
• Intoxication: (10 m M ) for 24h
• Test compound(s): test compound(s) - lhr followed by (10
m M ) for 24h
Lactate dehydrogenase (LDH) activity assay
24 hours after intoxication, the supernatant was taken off and analyzed with
Cytotoxicity Detection Kit (LDH, Roche Applied Science, ref: 11644793001, batch:
11800300). This colorimetric assay for the quantification of cell toxicity is based on the
measurement of lactate dehydrogenase (LDH) activity released from the cytosol of
dying cells into the supernatant.
Data processing
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). Statistic 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
The combination of Baclofen and Acamprosate induces a significant protective effect
against the toxicity of human Abi _ peptide (improvement of 34% of cell survival) in
primary cortical neuron cells as shown in figure 5 . The results clearly show that the
intoxication by human amyloid peptide (Abi 10 m M ) is significantly prevented by the
combination, whereas at those concentrations, Baclofen or Acamprosate, alone, have no
significant effect on intoxication.
Conversely, although active in this model, the combination of Sulfisoxazole and
Cinacalcet affords a weaker protection against Abi (19%, figure 6).
Thus, while those two combinations allow a protection against Abi the combination
.4 ,
Baclofen - Acamprosate stands out clearly. Indeed, at concentrations having no effect
alone, the drugs cause a significant protection of primary cortical neuron cells against
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.
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-Terbinafme), or other active combination
therapies (Sulfisoxazole-Cinacalcet).
A comparison of Acamprosate and Homotaurine protection activity on cortical cells has
been done (Figure 17). Those results shown that the derivative of acamprosate, called
Homotaurine, allow an effective protection against Abi In the context of this
_4 ·
invention, Baclofen or Acamprosate can thus be substituted by their derivatives,
provided that those derivatives are efficient in assay described herein.
1.3. Protection against the toxicity of A Q1 in a neurite growth and syna se
functionality model.
Rat cortical neurons were cultured as described by Singer et al. (35). Briefly pregnant
female rats of 15 days gestation were killed by cervical dislocation (Rats Wistar) and
the foetuses were removed from the uterus. The cortex was removed and placed in ice-
cold medium of Leibovitz (L15) containing 2% of Penicillin 10.000 U/ml and
Streptomycin lOmg/ml and 1% of bovine serum albumin (BSA). Cortices were
dissociated by trypsin for 20 min at 37°C (0.05%). The reaction was stopped by the
addition of Dulbecco's modified Eagle's medium (DMEM) containing DNasel grade II
and 10% of foetal calf serum (FCS). Cells were then mechanically dissociated by 3
serial passages through a 10 ml pipette and centrifuged at 515 x g for 10 min at +4°C.
The supernatant was discarded and the pellet of cells was re-suspended in a defined
culture medium consisting of Neurobasal supplemented with B27 (2%), L-glutamine
(0.2mM), 2% of PS solution and lOng/ml of BDNF. Viable cells were counted in a
Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a
density of 30 000 cells/well in 96 well-plates (wells were pre-coated with poly-L-lysine
(^g/ml)) and were cultured at +37°C in a humidified air (95%)/C02 (5%)
atmosphere.
After 10 days of culture, cells are incubated with drugs. After 1 hour, cells are
intoxicated by 2.5 m M of beta-amyloid (1-42; Bachem) in defined medium without
BDNF but together with drugs. Cortical neurons are intoxicated for 24 hours. BDNF
(lOng/ml) is used as a positive (neuroprotective) control. Three independent cultures
were performed per condition, 6 wells per condition.
Neurites length and Synapse quantitation
After 24 hours of intoxication, the supernatant is taken off and the cortical neurons are
fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min. After
permeabilization with 0.1% of saponin, cells are blocked for 2 h with PBS containing
1% foetal calf serum. Then, cells are incubated with monoclonal antibody anti
microtubule-assiociated-protein 2 (MAP-2; Sigma) or with anti synaptophysin (SYN,
S5798, Sigma) together with anti PSD95 (P246, Sigma) antibodies in order to quantify
synapses. These antibodies stain specifically cell bodies and neurites of neurons of
neurons (MAP2) or pre and post synaptic elements (SYN and PSD95, respectively).
These antibodies are revealed with Alexa Fluor 488 goat anti-mouse IgG (Molecular
probe). Nuclei of neurons were labeled by a fluorescent marker (Hoechst solution,
SIGMA).
Per well, 10 pictures are taken using InCell Analyzer TM 1000 (GE Healthcare) with
20x magnification. All pictures are taken in the same conditions. Analysis of the neurite
network is done using Developer software (GE Healthcare) in order to assess the total
length of neurite network.
Results
The combination of Baclofen and Acamprosate induces a significant protective effect
against the toxicity of human Abi peptide (improvement of 80% of neurites network)
in primary cortical neuron cells as shown in figure 7 . The results clearly show that the
intoxication by human amyloid peptide (Abi 2.5 m M ) is significantly prevented by the
combination, whereas at those concentrations, Baclofen or Acamprosate, alone, have no
significant effect on intoxication.
Furthermore, the total length of neurite network 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 Abi peptide
but also a neurite growth comparable to a sane cortical neuron cell.
II. BACLOFEN-ACAMPROSATE COMBINATION THERAPIES PREVENT
TOXICITY OF HUMAN A 2 - IN VIVO
Animals
Male Swiss mice, are used throughout the study. Animals are housed in plastic cages,
with free access to laboratory chow and water, except during behavioural experiments,
and kept in a regulated environment, under a 12 h light/dark cycle (light on at 8:00
a.m.). 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
Drug(s) is/are daily administred by gavage (per os). The b 25-35 peptide and scrambled
b 25-35 peptide (control) have been dissolved in sterile bidistilled water, and stored at -
°C until use. The b -amyloid peptides are then administered intracerebroventricularly
(i.e.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
Drug(s) treatment
On day -1, i.e. 24 h before the peptide injection, drug combinations or the
A 25-35
vehicle solution are administered per os by gavage twice daily (at 8:00 am and 6:00
pm).
On day 0 (at 10:00 am), mice are injected i.c.v. with A b 25-35 peptide or scrambled A b
-35 peptide (control) in a final volume of 3 m ΐ (3 mM).
Between day 0 and day 7, drugs, drugs combination or the vehicle solution are
administered per os by gavage once or twice daily (at 8:00 am and 6:00 pm). One
animal group receives donepezil (reference compound - 1 mg/kg/day) per os by gavage
in a single injection (at 8:00 am). Drugs are solubilized in water and freshly prepared
just before each gavage administration.
On day 7, all animals are tested for the spontaneous alternation performance in the Y-
maze test, an index of spatial working memory.
On day 7 and 8, the contextual long-term memory of the animals is assessed using the
step-down type passive avoidance procedure.
On day 8, animals are sacrificed. Their brain is dissected and kept at -80°C for further
analysis.
Combinations enhance behavioral and cognitiveperformances of intoxicated animals
Spontaneous alternation performances-Y Maze Test
On day 7, all animals are tested for spontaneous alternation performance in the Y-maze,
an index of spatial working memory. The Y-maze is made of grey polyvinylchloride.
Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top,
and converging at an equal angle. Each mouse is placed at the end of one arm and
allowed to move freely through the maze during an 8 min session. The series of arm
entries, including possible returns into the same arm, are checked visually. An
alternation is defined as entries into all three arms on consecutive occasions. The
number of maximum alternations is therefore the total number of arm entries minus two
and the percentage of alternation is calculated as (actual alternations / maximum
alternations) x 100. Parameters include the percentage of alternation (memory index)
and total number of arm entries (exploration index). Animals that show an extreme
behavior (Alternation percentage < 25% or> 85% or number of arm entries < 10) are
discarded. Usually, it accounts for 0-5% of the animals. This test incidentally serves to
analyze at the behavioral level the impact and the amnesic effect induced in mice by the
A b 25-35 injection.
Passive avoidance test
The apparatus is a two-compartment (15 x 20x 15 cm high) box with one illuminated
with white polyvinylchloride walls and the other darkened with black polyvinylchloride
walls and a grid floor. A guillotine door separates each compartment. A 60 W lamp
positioned 40 cm above the apparatus lights up the white compartment during the
experiment. Scrambled footshocks (0.3 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 footshock
is delivered for 3 s. The step-through latency, that is, the latency spent to enter the
darkened compartment, and the number of vocalizations is recorded. The retention test
is carried out 24 h after training. Each mouse is placed again into the white
compartment. After 5 s the doors is raised, the step-through latency and the escape
latency, i.e. the time spent to return into the white compartment, are recorded up to 300
Positive results are observed in behavioural performances and biochemical assays
performed 7 days after b 25-35 peptide icv injection.
The combination of Baclofen and Acamprosate induces a significant protective effect
on behavioral and cognitive performances of intoxicated animals as shown in figures 8,
9 & 10.
In figure 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 control, the impairment is significantly
prevented on mice treated with Baclofen and Acamprosate.
Similarly, figures 9 & 10 show that intoxicated animals exhibit impaired behavioral and
cognitive performances according to their score 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 (Figure 9) and step
through latency (Figure 10) is significantly increased by combinations of the invention
with an enhanced effect of the combination compared to drugs alone.
Memory impairment is the early feature of Alzheimer 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.
Furthermore, the figure 16 shows that extremely low dose of Baclofen (480m g/kg/day),
Acamprosate (32 g kg day) and Donepezil (0,25mg/kg/day) can be combined to allow
a complete protection of behavioral and cognitive performances of mice as measured by
Y-maze test. Whereas donepezil, at this concentration, have 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. Combinations of the invention can thus be further combined
with other therapies in order to potentiate their action.
Combinations improve neurophysiological concern of neurological diseases
Combinations therapies are tested in the in vivo model of A b intoxication. Their effects
on several parameters which are affected in neurological diseases are assessed:
Caspases 3 and 9 expression level, considered as an indicator of apoptosis,
- Lipid peroxidation, considered as a marker for oxidative stress level,
GFAP expression assay, considered as a marker of the level of brain
inflammation,
Brain Blood Barrier integrity,
Overall synapse integrity (synaptophysin ELISA).
- Quantification of viable neurons in the CA1
Brain Blood Barrier integrity
Experimental design about animal intoxication by A b is the same that in part III.
The potential protective effect of the combination therapies on the blood brain barrier
(BBB) integrity is analyzed in mice injected intracerebroventricularly (i.c.v.) with
oligomeric amyloid-P25-35 peptide (A b 25-35) or scrambled A b 25-35 control peptide
(Sc.AP), 7 days after injection.
On day 7 after the A b injection, animals are tested to determine the BBB integrity
by using the EB (Evans Blue) method. EB dye is known to bind to serum albumin after
peripheral injection and has been used as a tracer for serum albumin.
EB dye (2% in saline, 4 ml/kg) is injected intraperitoneal (i.p.) 3 h prior to the
transcardiac perfusion. Mice are then anesthetized with i.p. 200 m ΐ of pre-mix ketamine
80 mg/kg, xylazine 10 mg/kg, the chest is opened. Mice are perfused transcardially with
250 ml of saline for approximately 15 min until the fluid from the right atrium becomes
colourless. After decapitation, the brain is removed and dissected out into three regions:
cerebral cortex (left + right), hippocampus (left + right), diencephalon. Then, each brain
region is weighed for quantitative measurement of EB-albumin extravasation.
Samples are homogenized in phosphate-buffered saline solution and mixed by vortexing
after addition of 60% trichloroacetic acid to precipitate the protein. Samples are cooled
at 4°C, and then centrifuged 30 min at 10,000 g, 4°C. The supernatant is measured at
610 nm for absorbance of EB using a spectrophotometer.
EB is quantified both as
/ ¾ of brain tissue by using a standard curve, obtained by known
concentration of EB-albumin.
g mg of protein.
Overall synapse integrity (synaptophysin ELISA)
Synaptophysin has been chosen as a marker of synapse integrity and is assayed using
a commercial ELISA kit (USCN , Ref. E90425Mu). Samples are prepared from
hippocampus tissues and homogenized in an extraction buffer specific to as described
by manufacturer and reference literature.
Tissues are rinsed in ice-cold PBS (0.02 mol/1, pH 7.0-7.2) to remove excess blood
thoroughly and weighed before nitrogen freezing and -80°C storage. Tissues are cut into
small pieces and homogenized in 1ml ice-cold phosphate buffer saline (PBS) solution
with a glass homogenizer. The resulting suspension is sonicated with an ultrasonic cell
disrupter or subjected to two freeze-thawing cycles to further break the cell membranes.
Then, homogenates are centrifugated for 5 min at 5,000 g and the supernatant is assayed
immediately.
All samples are assayed in triplicates.
Quantification of proteins is performed with the Pierce BCA (bicinchoninic acid)
protein assay kit (Pierce, Ref. #23221) to evaluate extraction performance and allow
normalization.
The total protein concentrations are then calculated from standard curve dilutions and
serve to normalize ELISA results.
Quantification of viable neurons in the CAl
On day 8, each mouse is anesthetized with 200 m ΐ 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 °C,
After post-fixation, brains are washed in a phosphate buffer saline (PBS) solution, then
cerebellum is removed and the forebrains are placed on a vibratom plateform (Leica
VT100OS, Leica, Wetzlar, Germany) for slicing.
Brains are cut in coronal sections (20 mih thickness) using a vibratom (Leica VT100OS,
Leica, Wetzlar, Germany). Serial sections are placed on 24-well plate with PBS. They
are then selected to include the hippocampal formation and 9 sections are placed in
gelatin-coated glass-strip (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 are mounted with Mountex medium
(BDH Laboratory Supplies, Poole, Dorset, UK).
After mounting, slides are kept at RT for 24 h drying. Examination of the CA 1 area are
performed using a light microscope (Dialux 22, Leitz), with slices digitalized through a
CCD camera (Sony XC-77CE, Sony, Paris, France) with the NIHImage® vl.63
software (NIH). CA1 measurement and pyramidal cells counts are processed using
ImageJ® (NIH). Data are expressed as mean of nine slices of CA 1 pyramidal cells per
millimeter for each group (left and right hippocampus CAI counting) (49).
Oxidative stress assay
Mice are sacrificed by decapitation and both hippocampi are rapidly removed, weighted
and kept in liquid nitrogen until assayed. After thawing, hippocampus are homogenized
in cold methanol (1/10 w/v), centrifuged at 1,000 g during 5 min and the supernatant
placed in eppendorf tube. The reaction volume of each homogenate are added to FeS04
1 mM, H2S04 0.25 M, xylenol orange 1 mM and incubated for 30 min at room
temperature. After reading the absorbance at 580 nm (A580 1), 10 m ΐ of cumene
hydroperoxyde 1 mM (CHP) is added to the sample and incubated for 30 min at room
temperature, to determine the maximal oxidation level. The absorbance is measured at
580 nm (A580 2). The level of lipid peroxidation is determined as CHP equivalents
(CHPE) according to: CHPE = A580 1/A580 2 x [CHP] and expressed as CHP
equivalents per weight of tissue and as percentage of control group data.
Caspase pathway induction assay and GFAP expression assay
Mice are sacrificed by decapitation and both hippocampi are rapidly removed, rinsed in
ice-cold PBS (0.02 mol/1, pH 7.0-7.2) to remove excess blood thoroughly weighted and
kept in liquid nitrogen until assayed. Tissues are cut into small pieces and homogenized
in 1ml ice-cold PBS with a glass homogenizer. The resulting suspension is sonicated
with ultrasonic cell disrupter or subjected to two freeze-thawing cycles to further break
the cell membranes. Then, homogenates are centrifugated at 5,000g during 5 min and
the supernatant is assayed immediately.
Experiments are conducted with commercial assay: Caspase-3 (USCN - E90626Mu),
Caspase-9 (USCN - E90627Mu), GFAP (USCN - E90068).
Quantification of proteins is performed with the Pierce BCA (bicinchoninic acid)
protein assay kit (Pierce, Ref. #23221) to evaluate extraction performance and allow
normalization.
The combination of Baclofen and Acamprosate induces a significant protective effect
on neurophysiological functions of intoxicated animals as shown in figures 11, 12, 13 &
With a protection of more than 60 % compared to non-treated intoxicated animals, the
combination is effective for the protection of neurons (Figure 11) and synaptic density
(Figure 13).
Similarly, figure 12 show that the combination of Baclofen and Acamprosate protect the
BBB integrity (76%) compared with non-treated intoxicated animals.
Finally, this combination therapy is efficient in reducing the overall oxidative stress
induced by A b in brain of treated animals when compared with the non-treated
intoxicated animals (figure 14).
As shown in the Part A of examples, several neurological functions impaired in
numerous neurological disorders, including neurodegenerative disorders such as
Alzheimer disease and related disorders have been protected and symptoms retarded or
reduced by the combination Baclofen - Acamprosate.
B) PREVENTION OF GLUTAMATE TOXICITY ON NEURONAL CELLS
In this further set of experiment, candidate compounds have been tested for their
ability to prevent or reduce the toxic effects of glutamate toxicity on neuronal cells.
Glutamate toxicity is involved in the pathogenesis of neurological diseases or disorder
such as Multiple Sclerosis, Alzheimer's Disease, Amyotrophic Lateral Sclerosis,
Parkinson's Disease, Huntington's Disease, neuropathies, alcoholism or alcohol
withdrawal, or spinal cord injury.The drugs are first tested individually, followed by
assays of their combinatorial action.
Methods
The efficacy of drug combinations of the invention is assessed on primary cortical
neuron cells. The protocol which is used in these assays is the same as described in
section A .1. above.
Glutamate toxicity assays
The neuroprotective effect of compounds is assessed by quantification of the neurite
network (Neurofilament immunostaining (NF)) which specifically reveals the
glutamatergic neurons.
After 12 days of neuron culture, drugs of the candidate combinations are solved in
culture medium (+0.1% DMSO). Candidate combinations are then pre-incubated with
neurons for 1 hour before the Glutamate injury. One hour after incubation with,
Glutamate is added for 20 min, to a final concentration of 40m M , in presence of
candidate combinations, in order to avoid further drug dilutions. At the end of the
incubation, medium is changed with medium with candidate combination but without
glutamate. The culture is fixed 24 hours after glutamate injury. MK801
(Dizocilpinehydrogen maleate, 770867 - 20m M ) is used as a positive control.
After permeabilization with saponin (Sigma), cells are blocked for 2h with PBS
containing 10% goat serum, then the cells are incubated with mouse monoclonal
primary antibody against Neurofilament antibody (NF, Sigma). This antibody is
revealed with Alexa Fluor 488 goat anti-mouse lgG.
Nuclei of cells are labeled by a fluorescent marker (Hoechst solution, SIGMA), and
neurite network quantified. Six wells per condition are used to assess neuronal survival
in 3 different cultures.
Results
The combination Baclofen - Acamprosate give a protective effect against glutamate
toxicity for cortical neuronal cells. As exemplified in figure 15, combinations of the
invention strongly protect neurons from glutamate toxicity under experimental
conditions described above. It is noteworthy that an effective protection is noticed using
drug concentrations at which drugs used alone have lower protective effect.
Combination of Baclofen and Acamprosate induce an improvement of more than 200 %
compared to acamprosate alone and more than 47 % compared to baclofen used alone.
C) IMPROVEMENT OF OTHER DISORDERS RELATED TO
GLUTAMATE EXCITOXICITY USING COMBINATIONS OF THE
INVENTION
The above mentioned in vitro protective effect against glutamate toxicity of drugs and
drug combinations of the invention combined with the protective effects exemplified
herein in several AD models, prompted the inventors to test these drugs and
combinations in some models of other diseases in the pathogenesis of which glutamate
toxicity is also involved, as MS, ALS and neuropathic pain.
I) PROTECTIVE EFFECT OF COMBINATIONS IN AN IN VIVO MODEL OF
MULTIPLE SCLEROSIS.
A model in which myelin-oligodendrocyte glycoprotein immunized (MOG-
immunized) mice develop chronic progressive EAE is used to demonstrate the
beneficial effect of compositions of the invention in multiple sclerosis treatment.
Animals and chemicals
C57L/6J female mice (8 weeks old) are purchased from Janvier (France); after two
weeks of habituation, female mice (10 weeks old) develop chronic paralysis after
immunization with MOG (Myelin Oligodendrocyte Glycoprotein) peptide. The
experimental encephalomyelitis is induced with the Hooke Kit MOG /CFA
Emulsion PTX (Pertussis toxin) for EAE Induction (EK-01 10, EK-0115; Hooke
laboratories). The control kit is CK-01 15 (Hooke laboratories).
Experimentalprocedure
The experimental encephalomyelitis is induced by following procedure:
The day 0, two subcutaneous injections of 0.1 ml each are performed; one on upper
back of the mouse and one in lower back. Each injection contains 100 g of MOG
peptide (MEVGWYRSPF SRVVHLYRNGK), 200m g of inactivated Mycobacterium
tuberculosis H37Ra and is emulsified in Complete Freund's adjuvant (CFA) (Hooke
laboratories). The emulsion provides antigen needed to expand and differentiate MOG-
specific autoimmune T cells.
Two intraperitoneal injections of 500 ng of Pertussis toxin in PBS (Hooke kit) are
performed 2 hours (Day 0) and 24 hours (Day 1) after the MOG injection. Pertussis
toxin enhances EAE development by providing additional adjuvant.
Mice develop EAE 8 days after immunization and stay chronically paralyzed for the
duration of the experiment. After the immunization, mice are daily observed for clinical
symptoms in a blind procedure. Animals are kept in a conventional pathogen-free
facility and all experiments are carried out in accordance with guidelines prescribed by,
and are approved by, the standing local committee of bioethics.
Experimental groups and drug treatment:
Groups of female mice as disclosed are homogenized by weight before the
immunization:
- Control group: vehicle injection in the same conditions of EAE mice (from Day
- 1 to Day 28, placebo is given daily)
- EAE group: MOG injection (day 0) + Pertussis toxin injections (Day 0 and 1) -
from Day - 1 to Day 28, placebo is given orally daily
- EAE + positive control: MOG injection (Day 0) + Pertussis toxin injections(Day
0 and 1) - from Day - 1 to Day 28, dexamethazone is given orally daily.
- EAE + treatment group: MOG injection (Day 0) + Pertussis toxin injections
(Day 0 and 1). The treatments start one Day before immunization and last until
Day 28.
The clinical scores are measured at Days 0812162126-28.
Statistica software (Statsoft Inc.) is utilized throughout for statistical analysis. ANOVA
analysis and Student's t test are employed to analyse clinical disease score. P < 0.05 is
considered significant.
Delays of disease occurrence, clinical score and delay of death, have been compared
between each group to the reference 'immu" group with Kaplan-Meier curves and a Cox
model ( package 'survival'). Resulting -values are unilateral and test the hypothesis
to be better than the reference 'immu' group.
The total clinical score is composed of the tail score, the hind limb score, the fore limb
score and the bladder score described as below:
Tail score:
Score=0
A normal mouse holds its tail erect when moving.
Score=l If the extremity of the tail is flaccid with a tendency to fall.
Score=2 If the tail is completely flaccid and drags on the table.
Hind limbs score:
Score=0 A normal mouse has an energetic walk and doesn't drag his paws
Score=l Either one of the following tests is positive:
A - Flip test: while holding the tail between thumb and index finger, flip the
animal on his back and observe the time it takes to right itself. A healthy
mouse will turn itself immediately. A delay suggests hind-limb weakness.
B - Place the mouse on the wire cage top and observe as it crosses from one
side to the other. If one or both limbs frequently slip between the bars we
consider that there is a partial paralysis.
Score=2 Both previous tests are positive.
Score=3 One or both hind limbs show signs of paralysis but some movements are
preserved; for example: the animal can grasp and hold on to the underside of
the wire cage top for a short moment before letting go.
Score=4
When both hind legs are paralyzed and the mouse drags them when moving.
Fore limbs score:
Score=0
A normal mouse uses its front paws actively for grasping and walking and
holds its head erect.
Score=l Walking is possible but difficult due to a weakness in one or both of the
paws, for example, the front paws are considered weak when the mouse has
difficulty grasping the underside of the wire top cage. Another sign of
weakness is head drooping.
Score=2 When one forelimb is paralyzed (impossibility to grasp and the mouse turns
around the paralyzed limb). At this time the head has also lost much of its
muscle tone.
Score=3 Mouse cannot move, and food and water are unattainable.
Bladder score:
The global score for each animal is determined by the addition of all the above
mentioned categories. The maximum score for live animals is 10.
Results-Combinations therapies are efficient in a MS model
A significant improvement of global clinical score is observed in "EAE+ treatment
group" mice for the Baclofen and Acamprosate combination.
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
(Figure 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 Baclofen-Acamprosate
combination on the neuronal protection including on demyelination and its implications.
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 pamyloid, BBB breakdown, glutamate excitotoxicity or demyelination.
II. PROTECTIVE EFFECT OF COMBINATIONS IN MODELS OF ALS.
The effect of Combination therapies according to the present invention on ALS have
been demonstrated in vitro, in a coculture model, and in vivo, in a mouse model of ALS.
Protocols and results are presented in this section.
II.1 Protective effect against glutamate toxicity in primary cultures of nerve-muscle
co-culture
Primary cocultures of nerve-and muscle cells
Human muscle is prepared according to a previously described method from portions of
biopsie of healthy patient (48). Muscle cells are established from dissociated cells
(10000 cells per wells), plated in gelatin-coated 0.1% on 48 wells plate and grown in a
proliferating medium consisting of mix of MEM medium and M l 99 medium.
Immediately after satellite cells fusion, whole transverse slices of 13-day-old rat Wistar
embryos spinal cords with dorsal root ganglia (DRG) attached are placed on the muscle
monolayer 1 explant per well (in center area). DRG are necessary to achieve a good
ratio of innervations. Innervated cultures are maintained in mix medium. After 24h in
the usual co-culture neuritis are observed growing out of the spinal cord explants. They
make contacts with myotubes and induce the first contractions after 8 days. Quickly
thereafter, innervated muscle fibres located in proximity to the spinal cord explants, are
virtually continuously contracting. Innervated fibres are morphologically and spatially
distinct from the non-innervated ones and could easily be distinguished from them.
One co-culture is done (6 wells per conditions).
Glutamate injury
On day 27, co-cultures are incubated with candidate compounds or Riluzole one hour
before glutamate intoxication (60 m M ) for 20 min. Then, co-cultures are washed and
candidate compounds or Riluzole are added for an additional 48h. After this incubation
time, unfixed cocultures are incubated with a-bungarotoxin coupled with Alexa 488 at
concentration 500 nmol/L for 15 min at room temperature. Then, cocultures fixed by
PFA for 20 min at room temperature. After permeabilization with 0.1% of saponin, co-
cultures are incubated with anti-neurofilament antibody (NF).
These antibodies are detected with Alexa Fluor 568 goat anti-mouse IgG (Molecular
probe). Nuclei of neurons are labeled by a fluorescent marker (Hoechst solution).
Endpoints are (1) Total neurite length, (2) Number of motor units, (3) Total motor unit
area, which are indicative of motorneurone survival and functionality.
For each condition, 2 x 10 pictures per well are taken using InCell Analyzer TM 1000
(GE Healthcare) with 20x magnification. All the images are taken in the same
conditions.
Results
Baclofen and Acamprosate combination effectively protect motomeurones and motor
units in the coculture model.
II.2 —Combinations therapies are efficient in ALS Mouse model
Experiments are performed on male mice. Transgenic male mice B6SJL-
Tg(SODl)2Gur/J mice and their control (respectively SN2726 and SN2297 from
Jackson Laboratories, Ben Harbor, USA and distributed by Charles River in France) are
chosen in this set of experiments to mimic ALS.
Diseased mice express the SOD1-G93A transgene, designed with a mutant human
SODl gene (a single amino acid substitution of glycine to alanine at codon 93) driven
by its endogenous human SODl promoter. Control mice express the control human
SODl gene.
Randomisation of the animals:
The group assignation and the randomisation of the animals are based on the body
weight; for each group, the randomisation is done one day before the first treatment.
Drug administration
Mice are dosed with candidate drug treatment diluted in vehicle from 60th day after
birth till death. Diluted solutions of drug candidates are prepared with water at room
temperature just before the beginning of the administration.
· In drinking water :
Riluzole is added in drinking water at a final concentration of 6mg/ml (adjusted to each
group mean body weight) in 5% cyclodextrin. As a mouse drinks about 5 ml/day, the
estimated administrated dose is 30mg/kg/day which is a dose that was shown to increase
the survival of mice.
- Cyclodextrine is used as vehicle at the final concentration of 5 %, diluted in water at
room temperature from stock solution (cyclodextrin 20%).
• Oral administration (per os) :
- Drug combinations are administrated per os, daily.
- Cyclodextrine is used as vehicle at the final concentration of 5 %, diluted in water at
room temperature from stock solution (cyclodextrin 20%).
Clinical observation
The clinical observation of each mouse is performed daily, from the first day of
treatment (60 days of age) until the death (or sacrifice). Clinical observation consists in
studying behavioural tests: onset of paralysis, "loss of splay", "loss of righting reflex",
and general gait observation:
- Onset of paralysis: The observation consists of paralysis observation of each limb.
Onset of paralysis corresponds to the day of the first signs of paralysis.
- The loss of splay test consists of tremors or shaking notification and the position of
hind limb (hanging or splaying out) when the mouse is suspended by the tail.
- The loss of righting reflex test evaluates the ability of the mouse to right itself within
sec of being turned on either side. The righting reflex is lost when the mouse is
unable to right itself. The loss of righting reflex determines the end stage of disease: the
mouse unable to right itself is euthanized.
Results-Combinations therapies are efficient in ALS in vivo model
An improvement of the disease is observed for the diseased animals treated with for the
Baclofen and Acamprosate combination.
Ill) PROTECTIVE EFFECT OF COMBINATIONS IN OXALIPLATINE
INDUCED NEUROPATHY AS AN IN VIVO MODEL FOR NEUROPATHIC PAIN
Combinatorial therapies of the present invention are tested in vivo, in suitable models of
peripheral neuropathy, i.e., acute model of oxaliplatin-induced neuropathy and chronic
model of oxaliplatin-induced neuropathy. The animals, protocols and results are
presented in this section.
Animal Husbandry
Sprague-Dawley rats (CERJ, France), weighing 150 - 175 g at the beginning of the
experimental of the Oxaliplatin treatment (D ) are used. Animals are housed in a limited
access animal facility in a temperature (19.5°C - 24.5°C) and relative humidity (45 % -
65 %) controlled room with a 12 h - light/dark cycle, with ad libitum access to standard
pelleted laboratory chow and water throughout the study. Animals are housed 4 or 5 per
cage and a one week-acclimation period is observed before any testing.
Experimental design
Four following groups of rats are used in all experiments:
Control groups :
Group 1 : Vehicle of Oxaliplatin (distilled water), i.p. / Vehicle of candidate
combination(s) (Distilled water), p.o. daily.
Group 2 : Oxaliplatin (distilled water), i.p. / Vehicle of candidate combination(s)
(Distilled water), p.o. daily.
Group 3 : Oxaliplatin 3 mg/kg i.p. / single drug in Distilled water, p.o. daily x 9 .
Tested composition groups :
Group 4 : Oxaliplatin 3 mg/kg i.p. / candidate combination(s) in Distilled water, p.o.
daily x 9 .
Group 5 : Oxaliplatin 3 mg/kg i.p. / Gabapentin (100 mg/kg) in Distilled water, p.o. on
testing days (i.e. Di & D );
Vehicle and test items are delivered daily from D-1 to D7 (the day before the last testing
day) whereas Gabapentin is administered on testing days (120 minutes before the test).
All treatments are administered in a coded and random order when it is possible. Doses
are expressed in terms of free active substance.
Neuropathy induction
Acute neuropathy is induced by a single intraperitoneal injection of oxaliplatin
(3 mg/kg).
Chronic peripheral neuropathy is induced by repeated intraperitoneal injections of
oxaliplatin (3 mg/kg, i.p.) on days 0, 2, 4 and 7 (CD= 12 mg/kg, i.p.). Chronic
neuropathy in humans is cumulative as well and is most commonly seen in patients who
have received total doses of oxaliplatin > or =540 mg/m which corresponds to -15
mg/kg as cumulative dose in rats (Cersosimo RJ. 2005).
The oxaliplatin-induced painful neuropathy in rat reproduces the pain symptoms in
oxaliplatin-treated patients:
The thermal hyperalgesia is the earliest symptom. It can be measured with the
acetone test or with the tail-immersion test;
The mechanical hyperalgesia appears later. It can be quantified with the Von
Frey test or the paw pressure test.
Animal dosing and testing
All drug combinations are administered from the day before the first intraperitoneal
injection of oxaliplatin 3 mg/kg (D-1) and pursued daily orally until D7. During the
testing days (i.e. Dl and D7), the drug combinations are administered after the test.
Animals from the reference-treated group (gabapentin) are dosed only during the testing
days.
Acetone test
Cold allodynia is assessed using the acetone test by measuring the responses to thermal
non-nociceptive stimulation on Dl (around 24h after the first injection of oxaliplatin 3
mg/kg (acute effect of oxaliplatin), and D8 (chronic effect of oxaliplatin).
In the acetone test, latency of hindpaw withdrawal is measured after application of a
drop of acetone to the plantar surface of both hindpaws (reaction time) and the intensity
of the response is scored (cold score). Reaction time to the cooling effect of acetone is
measured within 20 sec (cut-off) after acetone application. Responses to acetone are
also graded to the following 4-point scale: 0 (no response); 1 (quick withdrawal, flick of
the paw); 2 (prolonged withdrawal or marked flicking of the paw); 3 (repeated flicking
of the paw with licking or biting).
For each experimental group, results are expressed as:
- The reaction time defined as the time expressed in sec required to elicit paw reaction
(mean of 6 measures for each rat together ± SEM).
- The cumulative cold score defined as the sum of the 6 scores for each rat together ±
SEM. The minimum score being 0 (no response to any of the 6 trials) and the maximum
possible score being 18 (repeated flicking and licking or biting of paws on each of the
six trials).
Statistical analyses
Student test, unilateral, type 3 is performed. The significance level is set as p < 0.05; all
the groups are compared to the diseased+vehicle group (oxaliplatin treated group).
Means and standard error mean are shown on the figures.
Results
Oxaliplatin induced a significant decrease in reaction time of paw withdrawal after
acetone application (diseased group + vehicle) during the time course. This decrease is
progressive and significant from day 1 (acute model of oxaliplatin-induced neuropathy)
to day 8 (chronic model) as compared to the vehicle group.
Anti-allodynic effect in acute model and chronic model of oxaliplatin-induced
neuropathy
Baclofen and Acamprosate combination are tested in both models of oxaliplatin-
induced neuropathy. It induces a significant decrease in the cumulative cold score and a
significant increase of reaction time as compared to the oxaliplatin-vehicle treated
group. In conclusion, this drug combination protects from chronic and acute
neuropathy.
The reference in this specification to any prior publication (or information
derived from it), or to any matter which is known, is not, and should not be taken as an
acknowledgment or admission or any form of suggestion that that prior publication (or
information derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification relates.
Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will be understood to imply the inclusion of a stated integer or step or
group of integers or steps but not the exclusion of any other integer or step or group of
integers or steps.
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H:\r ec\In terwoven\N RPortbl\ D CC\R EC\8107939_1. docx-4/08/2015
Claims (3)
1. A composition comprising baclofen and acamprosate, or salts, prodrugs or sustained release formulations thereof, wherein the dose of acamprosate is less than 200mg.
2. The composition according to claim 1, further comprising 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, donepezil, rivastigmine and memantine or salts or sustained release formulations thereof.
3. The composition according to claim 1 or 2, comprising at least one of the following combinations of compounds: baclofen and acamprosate, baclofen and acamprosate and diethylcarbamazine, baclofen and acamprosate and cinacalcet, baclofen and acamprosate and sulfisoxazole, baclofen and acamprosate and ifenprodil, baclofen and acamprosate and mexiletine, baclofen and acamprosate and eplerenone, baclofen and acamprosate and levosimendan, baclofen and acamprosate and terbinafine, baclofen and acamprosate and leflunomide, baclofen, acamprosate and donepezil, baclofen, acamprosate and rivastigmine, or baclofen, acamprosate and memantine, or salts or sustained release formulations thereof. H:\r ec\In terwoven\N RPortbl\D CC\R EC\8107939_1. docx-4/
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EP11305217 | 2011-03-01 | ||
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US61/468,658 | 2011-03-29 | ||
US201161493606P | 2011-06-06 | 2011-06-06 | |
EP11305687 | 2011-06-06 | ||
EP11305687.3 | 2011-06-06 | ||
US61/493,606 | 2011-06-06 | ||
PCT/EP2012/053570 WO2012117076A2 (en) | 2011-03-01 | 2012-03-01 | Baclofen and acamprosate based therapy of neurogical disorders |
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