Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS COMPRISING BACTERIAL STRAINS
TECHNICAL FIELD
This invention is in the field of compositions comprising bacterial strains
isolated from the
mammalian digestive tract and the use of such compositions in the treatment of
disease.
BACKGROUND TO THE INVENTION
The human intestine is thought to be sterile in utero, but it is exposed to a
large variety of maternal
and environmental microbes immediately after birth. Thereafter, a dynamic
period of microbial
colonization and succession occurs, which is influenced by factors such as
delivery mode,
environment, diet and host genotype, all of which impact upon the composition
of the gut
microbiota, particularly during early life. Subsequently, the microbiota
stabilizes and becomes
adult-like [1]. The human gut microbiota contains more than 1,500 different
phylotypes
dominated in abundance levels by two major bacterial divisions (phyla), the
Bacteroidetes and
the Filmicutes [2-3]. The successful symbiotic relationships arising from
bacterial colonization
of the human gut have yielded a wide variety of metabolic, structural,
protective and other
beneficial functions. The enhanced metabolic activities of the colonized gut
ensure that otherwise
indigestible dietary components are degraded with release of by-products
providing an important
nutrient source for the host and additional health benefits. Similarly, the
immunological
importance of the gut microbiota is well-recognized and is exemplified in
genii-free animals
which have an impaired immune system that is functionally reconstituted
following the
introduction of commensal bacteria [4-6].
A growing body of preclinical literature has demonstrated bidirectional
signalling between the
brain and the gut microbiome, involving multiple neurocrine and endocrine
signalling systems.
Indeed, increased levels of Clostridium species in the microbiome have been
linked to brain
disorders [7], and an imbalance of the Bacteroidetes and Filmicutes phyla has
also been
implicated in brain development disorders [8]. Suggestions that altered levels
of gut commensals,
including those of Bifidobacterium, Lactobacillus, Sutterella, Prevotella and
Ruminococcus
genera and of the Alcaligenaceae family are involved in immune-mediated
central nervous
system (CNS) disorders, are questioned by studies suggesting a lack of
alteration in the
microbiota between patients and healthy subjects [9]. This indicates that, at
present, the practical
effect of the link between the microbiome and human brain diseases is poorly
characterised.
Accordingly, more direct analytical studies are required to identify the
therapeutic impact of
altering the microbiome on CNS disorders.
In recognition of the potential positive effect that certain bacterial strains
may have on the animal
gut, various strains have been proposed for use in the treatment of various
diseases (see, for
example, [9-121). Also, certain strains, including mostly Lactobacillus and
Bifidobacterium
strains, have been proposed for use in treating various inflammatory and
autoimmune diseases
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that are not directly linked to the intestines (see [13] and [14] for
reviews). In addition, a range
of probiotics have been investigated in animal models to deteimine a role of
the gut microbiome
in modulating emotional behaviour, and Bifidobacterium and Lactobacillus are
the main genera
showing beneficial effects, reducing anxiety and repetitive behaviours, and
increasing social
interaction [15-17]. However, the relationship between different diseases and
different bacterial
strains, and the precise effects of particular bacterial strains on the gut
and at a systemic level and
on any particular types of diseases, are poorly characterised, particularly
for central nervous
system diseases.
There is a growing body of evidence to suggest that the microbiota-gut-brain
axis is affected in
neurodevelopmental and neuropsychiatric disorders, such as autism spectrum
disorders (ASD).
Animal models have provided considerable insight into how the microbiota may
be involved in
ASD. Furtheimore, preclinical studies have demonstrated that targeting the gut
microbiota
through administration of beneficial live biotherapeutics display efficacy in
improving autistic-
related behaviour in animal models.
Reference [18] discusses possible methods of treating neurodevelopmental
disorders by
administering a composition comprising a bacterial species selected from
Bacteroides and/or
Enterococcus, but provides data only for Bacteroides. Reference [19] discusses
a similar use of
Bacteroides and Enterococcus, with data limited to Bacteroides fragilis,
Bacteroides vulgatus
and Enterococcus faecalis. Reference [78] discusses use of Enterococcus
faecium for treating
.. and preventing central nervous system disorders and conditions. Reference
[79] discloses the
utilisation of bacteria to produce GABA. Table 10 of that document sets out a
list of 2219
bacterial species which are capable of being engineered to produce GABA. The
only reference
to Enterococcus durans in Reference [79] is in Table 10; there is no
suggestion at all that strains
of the species E. durans are inherently capable of treating any CNS disorder.
There is a requirement in the art for new methods of treating central nervous
system disorders.
SUMMARY OF THE INVENTION
The inventors have developed new therapies for treating or preventing central
nervous system
disorders and conditions, such as central nervous system disorders and
conditions mediated by
the microbiota-gut-brain axis. In particular, the inventors have identified
that bacterial strains of
the species Enterococcus durans can be effective for treating or preventing
diseases and
conditions mediated by the microbiota-gut-brain axis. As described in the
examples, oral
administration of compositions comprising Enterococcus durans may reduce
symptoms
associated with dysfunction of the microbiota-gut-brain axis.
Therefore, the invention provides a composition comprising a bacterial strain
of the species
Enterococcus durans, for use in a method of treating or preventing a central
nervous system
disorder or condition. The central nervous system disorder or condition may be
mediated by the
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microbiota-gut-brain axis. The invention further provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing a
neurodevelopmental disorder or a neuropsychiatric condition. For example, the
inventors have
identified that treatment with bacterial strains from this species can provide
clinical benefits in
mouse models of central nervous system disorders, in particular those mediated
by the
microbiota-gut-brain axis. In particular, treatment with bacterial strains
from this species may
modulate signalling in the central, autonomic and enteric nervous systems; may
modulate the
activity of the hypothalamus-pituitary-adrenal (HPA) axis pathway; may
modulate
neuroendocrine and/or neuroimmune pathways; and/or may modulate the levels of
commensal
.. metabolites, inflammatory markers and/or gastrointestinal permeability of a
subject.
In particular embodiments, the invention provides a composition comprising a
bacterial strain of
the species Enterococcus durans, for use in a method of treating or preventing
a disorder or
condition selected from the group consisting of: autism spectrum disorders
(ASDs); child
developmental disorder; obsessive compulsive disorder (OCD); major depressive
disorder
(MDD); depression; seasonal affective disorder; anxiety disorders; chronic
fatigue syndrome
(myalgic encephalomyelitis); stress disorder; post-traumatic stress disorder;
schizophrenia
spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder;
dementia;
Alzheimer's; Parkinson's disease; epilepsy; chronic pain, such as central
sensitisation or
fibromyalgia; motor neuron disease; Huntington's disease; Guillain-Barre
syndrome and/or
meningitis. The effects shown for the bacterial strains from the species
Enterococcus durans on
the microbiota-gut-brain axis and on disorders or conditions mediated by the
microbiota-gut-
brain axis suggest therapeutic benefits for other diseases and conditions
mediated by the
microbiota-gut-brain axis, such as those listed above.
In other embodiments, the invention provides a composition comprising a
bacterial strain of the
species Enterococcus durans, for use in a method of treating comorbidities
that occur with
disorders and conditions mediated by the microbiota-gut-brain axis, such as
the diseases and
conditions listed above. In particularly preferred embodiments, the invention
provides a
composition comprising a bacterial strain of the species Enterococcus durans,
for use in a method
of treating gastrointestinal comorbidities associated with diseases and
conditions mediated by the
.. microbiota-gut-brain axis, such as the diseases and conditions listed
above. The mouse model
experiments used in this application for the assessment of the symptoms of
autism spectrum
disorders are known in the art to be applicable for the assessment of the
symptoms of other central
nervous system disorders including those listed above [20- 211.
In particularly preferred embodiments, the invention provides a composition
comprising a
bacterial strain of the species Enterococcus durans, for use in a method of
treating or preventing
autism spectrum disorders, such as autism. For example, the inventors have
identified that
treatment with Enterococcus durans strains can reduce symptom severity in a
mouse model of
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autism spectrum disorders and can reduce stereotyped, repetitive, compulsive
and anxious
behaviour. In preferred embodiments, the invention provides a composition
comprising a
bacterial strain of the species Enterococcus durans for use in reducing
stereotyped, repetitive,
compulsive or anxious behaviour, in particular in the treatment of autism
spectrum disorders.
The inventors have also identified that treatment with Enterococcus durans
strains can enhance
learning and memory function. In preferred embodiments, the invention provides
a composition
comprising a bacterial strain of the species Enterococcus durans for improving
learning or
memory function. The inventors have identified that treatment with
Enterococcus durans can
ameliorate feelings of despair and helplessness. In preferred embodiments, the
invention
provides a composition comprising a bacterial strain of the species
Enterococcus durans for
reducing feelings of despair or helplessness. The inventors have identified
that treatment with
Enterococcus durans can ameliorate neophobia and sociability deficit. In
preferred
embodiments, the invention provides a composition comprising a bacterial
strain of the species
Enterococcus durans for treating, preventing or ameliorating neophobia and /
or sociability
deficit. In preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in the treatment of
behavioural symptoms of
autism spectrum disorders. In preferred embodiments, the invention provides a
composition
comprising a bacterial strain of the species Enterococcus durans for use in
the treatment of the
gastrointestinal symptoms of autism spectrum disorders. In preferred
embodiments, the invention
provides a composition comprising a bacterial strain of the species
Enterococcus durans, for use
in the treatment of the behavioural and gastrointestinal symptoms of autism
spectrum disorders.
Treatment with Enterococcus durans strains may modulate signalling in the
central, autonomic
and enteric nervous systems; may modulate the activity of the HPA axis
pathway; may modulate
neuroendocrine and/or neuroimmune pathways; and/or may modulate the levels of
commensal
metabolites, inflammatory markers and/or gastrointestinal peimeability of a
subject, all of which
are implicated in the neuropathology of autism spectrum disorders. In certain
embodiments,
treatment with Enterococcus durans strains may modulate the levels of oxytocin
and/or
vasopressin hormones.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing obsessive
compulsive disorder (OCD). In preferred embodiments, the invention provides a
composition for
use in reducing stereotyped, repetitive, compulsive or anxious behaviour, or
improving learning
or memory function in the treatment of OCD. In preferred embodiments, the
invention provides
a composition for reducing feelings of despair or helplessness in the
treatment of OCD. In
preferred embodiments, the invention provides a composition for treating or
preventing
neophobia and / or sociability deficit in the treatment of OCD. Treatment with
Enterococcus
durans strains may modulate signalling in the central, autonomic and enteric
nervous systems;
may modulate the activity of the HPA axis pathway; may modulate neuroendocrine
and/or
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neuroimmune pathways; and/or may modulate the levels of commensal metabolites
and/or
gastrointestinal penneability of a subject, all of which are implicated in the
neuropathology of
OCD.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
5 strain of the species Enterococcus durans, for use in a method of
treating or preventing major
depressive disorder (MDD). For example, treatment with Enterococcus durans
strains may
provide clinical benefits in a mouse model of depression. In preferred
embodiments, the invention
provides a composition comprising a bacterial strain of the species
Enterococcus durans, for use
in the treatment of depression. In preferred embodiments, the invention
provides a composition
for use in reducing stereotyped, repetitive, compulsive or anxious behaviour,
or improving
learning or memory function in the treatment of depression. In preferred
embodiments, the
invention provides a composition for reducing feelings of despair or
helplessness in the treatment
of depression. In preferred embodiments, the invention provides a composition
for treating or
preventing neophobia and / or sociability deficit in the treatment of
depression. Treatment with
Enterococcus durans strains may modulate signalling in the central, autonomic
and enteric
nervous systems; may modulate the activity of the HPA axis pathway; may
modulate
neuroendocrine and/or neuroimmune pathways; and may modulate the levels of
commensal
metabolites, inflammatory markers and/or gastrointestinal penneability of a
subject, all of which
are implicated in the neuropathology of MDD. In certain embodiments, treatment
with
Enterococcus durans strains may increase the level of oxytocin homione and/or
reduce the level
of vasopressin honnone. In certain embodiments, treatment with Entrococcus
durans strains may
decrease gastrointestinal permeability.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing anxiety
disorders. For example, treatment with Enterococcus durans strains reduces
disease incidence
and disease severity in a mouse model of anxiety, as shown in the examples. In
preferred
embodiments, the invention provides a composition comprising a bacterial
strain of the species
Enterococcus durans, for use in the treatment of anxiety disorder.
Compositions using
Enterococcus durans may be particularly effective for treating anxiety
disorder. In preferred
embodiments, the invention provides a composition for use in reducing
stereotyped, repetitive,
compulsive or anxious behaviour, or improving learning or memory function in
the treatment of
anxiety. In preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans for reducing feelings of despair or
helplessness in the
treatment of anxiety. In preferred embodiments, the invention provides a
composition for treating
or preventing neophobia and / or sociability deficit in the treatment of
anxiety.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing stress
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disorders, such as post-traumatic stress disorder. For example, compositions
comprising a
bacterial strain of the species Enterococcus durans may reduce stress in mouse
models of stress
disorders. Treatment with Enterococcus durans strains may modulate signalling
in the central,
autonomic and enteric nervous systems; may modulate the activity of the HPA
axis pathway;
may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the
levels of
commensal metabolites, inflammatory markers and/or gastrointestinal
peimeability of a subject,
all of which are implicated in the neuropathology of stress disorders. In
certain embodiments,
treatment with Enterococcus durans strains may increase the level of oxytocin
hoimone and/or
reduce the level of vasopressin hoimone.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing
schizophrenia spectrum and psychotic disorders, such as schizophrenia.
Treatment with
Enterococcus durans strains may modulate signalling in the central, autonomic
and enteric
nervous systems; may modulate the activity of the HPA axis pathway; may
modulate
neuroendocrine and/or neuroimmune pathways; and may modulate the levels of
commensal
metabolites and/or gastrointestinal permeability of a subject, all of which
are implicated in the
neuropathology of schizophrenia spectrum and psychotic disorders. In certain
embodiments,
treatment with Entrococcus durans strains may decrease gastrointestinal
peimeability.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing bipolar
disorder. For example, compositions comprising a bacterial strain of the
species Enterococcus
durans may reduce occasions of mania and/or depression in mouse models of
bipolar disorder.
Treatment with Enterococcus durans strains may modulate signalling in the
central, autonomic
and enteric nervous systems; may modulate the activity of the HPA axis
pathway; may modulate
neuroendocrine and/or neuroimmune pathways; and may modulate the levels of
commensal
metabolites, inflammatory markers and/or gastrointestinal peimeability of a
subject, all of which
are implicated in the neuropathology of bipolar disorder. In certain
embodiments, treatment with
Enterococcus durans strains may modulate the levels of oxytocin and/or
vasopressin hoimones.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing
neurocognitive disorders, such as Alzheimer's disease. For example,
compositions comprising a
bacterial strain of the species Enterococcus durans may improve cognitive and
behavioural
functioning in mouse models of neurocognitive disorders. Treatment with
Enterococcus durans
strains may modulate signalling in the central, autonomic and enteric nervous
systems; may
modulate the activity of the HPA axis pathway; may modulate neuroendocrine
and/or
neuroimmune pathways; and may modulate the levels of commensal metabolites
and/or
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gastrointestinal permeability of a subject, all of which are implicated in the
neuropathology of
neurocognitive disorders.
In further preferred embodiments, the invention provides a composition
comprising a bacterial
strain of the species Enterococcus durans, for use in a method of treating or
preventing
Parkinson's disease. Treatment with Enterococcus durans strains may modulate
signalling in the
central, autonomic and enteric nervous systems; may modulate the activity of
the HPA axis
pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may
modulate the
levels of commensal metabolites, inflammatory markers and/or gastrointestinal
permeability of
a subject, all of which are implicated in the neuropathology of Parkinson's
disease. In certain
embodiments, treatment with Enterococcus durans strains may modulate the
levels of oxytocin
and/or vasopressin hormones.
In certain embodiments, the compositions of the invention are for use in a
method of modulating
the microbiota-gut-brain axis in the treatment or prevention of a disease or
condition mediated
by the microbiota-gut-brain axis. In particular, the compositions of the
invention may be used in
modulating the microbiota-gut-brain axis in the treatment or prevention of
autism spectrum
disorders; obsessive compulsive disorder; major depressive disorder; anxiety
disorders; stress
disorders; schizophrenia spectrum disorders; bipolar disorders; neurocognitive
disorders and
Parkinson's disease.
In preferred embodiments, the bacterial strain in the compositions of the
invention has a 16s
rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,
99.8% or
99.9% identical to SEQ ID NO: 1 or 2. Preferably, the sequence identity is to
SEQ ID NO:2.
Preferably, the bacterial strain for use in the invention has the 16s rRNA
sequence represented
by SEQ ID NO:2. In these embodiments, the reference to a strain of the
bacterial species
Enterococcus durans is to be interpreted as any bacterial strain which has the
sequence identity
defined in this paragraph.
In preferred embodiments of the invention, the bacterial strain in the
composition is of
Enterococcus durans. The bacterial strain may have a 16s rRNA sequence that is
at least 95%,
96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the 16s
rRNA
sequence of a bacterial strain of Enterococcus durans. Preferably, the
bacterial strain has a 16s
rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,
99.8% or
99.9% identical to SEQ ID NO:1 or 2. Preferably, the sequence identity is to
SEQ ID NO:2.
Preferably, the bacterial strain for use in the invention has the 16s rRNA
sequence represented
by SEQ ID NO:2.
In certain embodiments, the compositions of the invention are for oral
administration. Oral
administration of the strains of the invention can be effective for treating
central nervous system
disorders and conditions, in particular those mediated by the microbiota-gut-
brain axis. Also, oral
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administration is convenient for patients and practitioners and allows
delivery to and / or partial
or total colonisation of the intestine.
In certain embodiments, the composition of the invention comprises one or more
pharmaceutically acceptable excipients or carriers.
The invention also provides a biologically pure culture of the Enterococcus
durans strain
deposited under accession number NCIMB 43456. A pure culture is a population
of cells growing
in the absence of other strains.
In certain embodiments, the composition of the invention has been lyophilised.
The composition
of the invention can also comprise a lyophilised bacteria strain of the
species Enterococcus
durans. Lyophilisation is an effective and convenient technique for preparing
stable compositions
that allow delivery of bacteria.
In certain embodiments, the invention provides a food product comprising a
composition as
described above.
In certain embodiments, the invention provides a vaccine composition
comprising a bacterial
strain of the species Enterococcus durans as described above, or an antigen
from said bacterium.
Additionally, the invention provides a method of treating or preventing a
disease or condition
mediated by the microbiota-gut-brain axis, comprising administering a
composition comprising
a bacterial strain of the species Enterococcus durans, as described herein.
As those skilled in the art will recognise, the species Enterococcus durans
is, relatively speaking,
genetically similar to the bacterial species Enterococcus faecium. Despite
this, however, these
species are taxonomically and phenotypically distinct. There have been
numerous articles
published detailing their phenotypic differences, including references [30, 31
and 771. The
organisms also have been reported as having different antibiotic resistance
profiles and other
functional properties. Further, if the results of the behavioural animal
studies presented in the
examples of the present application are compared to the corresponding studies
in Reference [78],
it can be observed that there are notable differences in the outcomes observed
following the
administration of strains of Enterococcus durans in the present application
and Enterococcus
faecium in Reference [78], demonstrating an unexpected yet advantageously
complementary
effect on CNS disorders.
In developing the above invention, the inventors have identified and
characterised a bacterial
strain that is particularly useful for therapy. The Enterococcus durans strain
of the invention is
shown to be effective for treating the diseases described herein, such as
autism spectrum disorder.
Therefore, in another aspect, the invention provides a cell of the
Enterococcus durans strain
deposited under accession number NCIMB 43456, or a biotype or derivative
thereof. The
invention also provides compositions comprising such cells, or biologically
pure cultures of such
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cells. The invention also provides a cell of the Enterococcus durans strain
deposited under
accession number NCIMB 43456, or a biotype or derivative thereof, for use in
therapy, in
particular for the diseases described herein.
In especially preferred embodiments, the invention provides a composition
comprising the strain
deposited under accession number NCIMB 43456, for use in a method of treating
or preventing
a central nervous system disorder or condition. In especially preferred
embodiments, the
invention provides a composition comprising the strain deposited under
accession number
NCIMB 43456, for use in a method of treating or preventing a
neurodevelopmental disorder or a
neuropsychiatric condition. In especially preferred embodiments, the invention
provides a
composition comprising the strain deposited under accession number NCIMB
43456, for use in
a method of treating or preventing autism spectrum disorder, or preferably
autism. In especially
preferred embodiments, the invention provides a composition comprising the
strain deposited
under accession number NCIMB 43456, for use in a method of reducing
stereotyped, repetitive,
compulsive and/or anxious behaviour, and / or improving learning and / or
memory function, for
example in the treatment of autism. In preferred embodiments, the invention
provides a
composition comprising the strain deposited under accession number NCIMB 43456
for reducing
feelings of despair or helplessness for example in the treatment of depression
and / anxiety. In
preferred embodiments, the invention provides a composition comprising the
straid deposited
under accession number NCIMB 43456 for treating or preventing neophobia and /
or sociability
deficit.
Further numbered embodiments of the invention are provided below:
1. A composition comprising a bacterial strain of the species Enterococcus
durans, for use in a
method of treating or preventing a central nervous system disorder or
condition.
2. The composition of embodiment 1, wherein the central nervous system
disorder or condition
is mediated by the microbiota-gut-brain axis.
3. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating or preventing a neurodevelopmental disorder or a neuropsychiatric
condition.
4. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating or preventing a disorder or condition selected from the group
consisting of autism
spectrum disorders (ASDs); child developmental disorder; obsessive compulsive
disorder
(OCD); major depressive disorder (MDD); depression; seasonal affective
disorder; anxiety
disorders; chronic fatigue syndrome (myalgic encephalomyelitis); stress
disorder; post-
traumatic stress disorder; schizophrenia spectrum disorders; schizophrenia;
bipolar disorder;
psychosis; mood disorder; dementia; Alzheimer's; Parkinson's disease;
epilepsy; chronic
pain, such as central sensitisation or fibromyalgia; motor neuron disease;
Huntington's
disease; Guillain-Barre syndrome and meningitis.
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5. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating or preventing autism spectrum disorder.
6. The composition of embodiment 5, wherein the composition is for use in a
method of treating
or preventing autism.
5 7. The composition of any preceding embodiment, wherein the composition
prevents, reduces
or alleviates stereotyped, repetitive, compulsive and/or anxious behaviour and
/ or improves
learning and / or memory function.
8. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating or preventing obsessive compulsive disorder.
10 9. The composition of embodiment 8, wherein the composition prevents,
reduces or alleviates
repetitive, compulsive and/or anxious behaviour.
10. The composition of embodiments 1 or 2, wherein the composition is for use
in in a method
of treating or preventing MDD.
11. The composition of embodiment 10, wherein the composition treats or
prevents acute major
depressive episodes and/or prevents new episodes (recurrence prevention).
12. The composition of embodiments 10 or 11, wherein the composition prevents,
reduces or
alleviates the occurrence of mild, moderate or severe MDD episodes.
13. The composition of embodiments 1 or 2, wherein the composition is for use
in in a method
of treating or preventing anxiety disorders.
14. The composition of embodiment 13, wherein the anxiety disorder is
generalised anxiety
disorder (GAD); specific phobia; social anxiety disorder; separation anxiety
disorder;
agoraphobia; panic disorder and/or selective mutism.
15. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating or preventing neurocognitive disorders.
16. The composition of embodiment 15, wherein the neurocognitive disorder is
vascular
dementias; mixed fonn Alzheimer's disease and vascular dementia; Lewy body
disease;
frontotemporal dementia; Parkinson's dementia; Creutzfeldt-Jakob disease;
Huntington's
disease; and Wernicke-Korsakoff syndrome.
17. The composition of embodiments 1 or 2, wherein the composition is for use
in a method of
treating epilepsy.
18. The composition of any preceding embodiment, wherein the composition is
for use in a
method of modulating the microbiota-gut-brain axis.
19. The composition of any preceding embodiments, wherein the bacterial strain
has a 16s rRNA
sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%
or 99.9%
identical to the 16s rRNA sequence of a bacterial strain of Enterococcus
durans.
20. The composition of any preceding embodiments, wherein the bacterial strain
has a 16s rRNA
sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%
or 99.9%
identical to SEQ ID NO:1 or 2.
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11
21. The composition of embodiment 20, wherein the bacterial strain has a 16s
rRNA sequence
that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%
identical
to SEQ ID NO:2, or wherein the bacterial strain has the 16s rRNA sequence
represented by
SEQ ID NO:2.
22. A composition comprising a bacterial strain, wherein the bacterial strain
has a 16s rRNA
sequence that is least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or
99.9%
identical to SEQ ID NO:2, for use in a method of treating or preventing a
central nervous
system disorder or condition.
23. The composition of any preceding embodiment, wherein the composition is
for oral
administration.
24. The composition of any preceding embodiment, wherein the composition
comprises one or
more pharmaceutically acceptable excipients or carriers.
25. The composition of any preceding embodiment, wherein the bacterial strain
is lyophilised.
26. The composition of any preceding embodiment, wherein the bacterial strain
is viable and
capable of partially or totally colonising the intestine.
27. The composition of any preceding embodiment, wherein the composition
comprises a single
strain of Enterococcus durans.
28. The composition of any preceding embodiment, which comprises the
Enterococcus durans
bacterial strain as part of a microbial consortium.
29. A food product comprising the composition of any preceding embodiment, for
the use of any
preceding embodiment.
30. A vaccine composition comprising the composition of any preceding
embodiment, for the
use in a method of preventing a central nervous system disorder or condition
of any preceding
embodiment.
31. A method of treating or preventing a central nervous system disorder or
condition,
comprising administering a composition comprising a bacterial strain of the
species
Enterococcus durans to a patient in need thereof.
32. A cell of the Enterococcus durans strain deposited under accession number
NCIMB 43456,
or a biotype or derivative thereof, for use in therapy, or for the use of any
of embodiments 1-
28.
33. A composition comprising the cell of embodiment 32, for the use of
embodiment 32.
34. The composition of embodiment 33, comprising a phaimaceutically acceptable
carrier or
excipient.
35. A biologically pure culture of the Enterococcus durans strain deposited
under accession
number NCIMB 43456, or a derivative thereof.
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BRIEF DESCRIPTION OF DRAWINGS
* denotes p < 0.05 relative to the vehicle control group; ** denotes p <0.01
relative to the vehicle
control group; *** denotes p < 0.001 relative to the vehicle control group;
**** denotes p <
0.0001 relative to the vehicle control group.
Figure 1: Deposit receipt for NCIMB 43456.
Figure 2: Effect of treatment with strain NCIMB 43456 or vehicle on (A)
occludin mRNA
expression in the ileum and (B) occludin mRNA expression in the colon.
Figure 3: Effect of treatment with strain NCIMB 43456 or vehicle on IDO1 mRNA
expression
in the ileum and (B) IDO1 mRNA expression in the colon.
Figure 4: Effect of treatment with strain NCIMB 43456 or vehicle on TJP1 mRNA
expression
in the ileum and (B) TJP1 mRNA expression in the colon.
Figure 5: Effect of treatment with strain NCIMB 43456 or vehicle on Tphl mRNA
expression
in the ileum and (B) Tphl mRNA expression in the colon.
Figure 6: Effect of treatment with strain NCIMB 43456 or vehicle on short
chain fatty acid
(SCFA) production.
Figure 7: Effect of treatment with strain NCIMB 43456 or vehicle on gene
expression in the
hippocampus.
Figure 8: Effect of treatment with strain NCIMB 43456 or vehicle on gene
expression in the
amygdala.
Figure 9: Effect of treatment with strain NCIMB 43456 or vehicle on gene
expression in the pre-
frontal cortex.
Figure 10: Effect of treatment with strain NCIMB 43456 or vehicle on
stereotype-related
behaviour in the marble burying test for wild type mice and mouse models of
autism.
Figure 11: Effect of treatment with strain NCIMB 43456 or vehicle on
stereotype-related
behaviour in the grooming test for wild type mice and mouse models of autism.
Figure 12: Effect of treatment with strain NCIMB 43456 or vehicle on anxiety-
like behaviour in
the elevated plus maze test for wild type mice and mouse models of autism.
Figure 13: Effect of treatment with strain NCIMB 43456 or vehicle on anxiety-
like behaviour in
the open field arena test for wild type mice and mouse models of autism.
Figure 14: Effect of treatment with strain NCIMB 43456 or vehicle on social
behaviour in the
three-chamber test for wild type mice and mouse models of autism.
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Figure 15: Effect of treatment with strain NCIMB 43456 or vehicle on
depression-like behaviour
in the novel object recognition test for wild type mice and mouse models of
autism.
Figure 16: Effect of treatment with strain NCIMB 43456 or vehicle on
depression-like behaviour
in the forced swim test for wild type mice and mouse models of autism.
Figure 17: Effect of treatment with strain NCIMB 43456 or vehicle on
intestinal motility for
wild type mice and mouse models of autism.
Figure 18 Effect of treatment with strain NCIMB 43456 or vehicle on gut
permeability for wild
type mice and mouse models of autism.
Figure 19: Effect of treatment with strain NCIMB 43456 or vehicle on animal
body weight for
-- wild type mice and mouse models of autism.
Figure 20: Effect of treatment with strain NCIMB 43456 or vehicle on organ
weight and colon
length for wild type mice and mouse models of autism.
Figure 21: Effect of treatment with strain NCIMB 43456 or vehicle on brainstem
monoamine
levels for wild type mice and mouse models of autism.
-- Figure 22: Effect of treatment with strain NCIMB 43456 or vehicle on
amygdalar gene
expression for wild type mice and mouse models of autism.
DISCLOSURE OF THE INVENTION
Bacterial strains
The invention provides a composition comprising a bacterial strain of the
species Enterococcus
-- durans for use in therapy, for example, for use in treating or preventing a
central nervous system
disorder or condition, in particular a central nervous system disorder or
condition mediated by
the microbiota-gut-brain axis. The mouse model experiments used in this
application for the
assessment of the symptoms of autism spectrum disorders are known in the art
to be applicable
for the assessment of the symptoms of other central nervous system disorders
including those
-- listed above.
In certain embodiments, the compositions of the invention comprise
Enterococcus durans and
do not contain any other bacterial species. In certain embodiments, the
compositions of the
invention comprise a single strain of Enterococcus durans and do not contain
any other bacterial
strains or species.
-- Enterococcus durans is a Gram-positive, coccus bacterium in the genus
Enterococcus.
Enterococcus durans is a facultative anaerobe. The type strain of Enterococcus
durans is 98D =
ATCC 19432 = DSM 20633 = CCM 5612 = NCDO 596 = NCTC 8307 = NCIMB 700596 [22].
The GenBank accession number for the 16S rRNA gene sequence of Enterococcus
durans strain
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14
98D is NR 036922 (disclosed herein as SEQ ID NO:1). This exemplary
Enterococcus durans
strain is described in [23].
Other Enterococcus durans strains for use in the invention include: NCIMB 662
[24], NCIMB
8587 [24], NCIMB 8782 [24], NCIMB 11077 [25], NCIMB 701624 [26], NCIMB 701630
[26],
NCIMB 701632 [26], NCIMB 701724 (NCD01724), NCIMB 701951 [27]. In certain
embodiments, the compositions of the invention comprise one of these strains,
or a derivative or
biotype thereof The Enterococcus durans bacterium tested in the examples was
deposited with
the international depositary authority NCIMB, Ltd. (Ferguson Building,
Craibstone Estate,
Bucksburn, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life
Sciences
Innovation Building, Aberdeen, AB25 2Z5, Scotland) on 9 August 2019 as
"Enterococcus
durans" and was assigned accession number NCIMB 43456. A 16S rRNA sequence for
strain
NCIMB 43456 is provided in SEQ ID NO:2.
Prior to being deposited with NCIMB, Ltd, strain NCIMB 43456 was taxonomically
identified
as belonging to the Enterococcus durans species using techniques known to
those skilled in the
art and reported in the literature as being capable of taxonomically
distinguishing the
Enterococcus durans species from other species in the Enterococcus genus,
including E. faecium
and E. faecalis, including 16S rRNA and MALDI-TOF analysis.
Further, while the use of strains of Enterococcus faecium as a probiotic has
been known,
particularly in animal feeds, to the inventors' knowledge, the use of
Enterococcus durans as a
probiotic or in the treatment of disease has not been comprehensively
explored.
Bacterial strains closely related to the strain tested in the examples,
including biotypes of the
bacterium deposited under accession number NCIMB 43456 are also expected to be
effective
for treating or preventing autism spectrum disorders and central nervous
system disorders and
conditions, in particular central nervous system disorders and conditions
mediated by the
microbiota-gut-brain axis.
In certain embodiments, the bacterial strain for use in the invention has a
16s rRNA sequence
that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%
identical to
the 16s rRNA sequence of a bacterial strain of Enterococcus durans.
Preferably, the bacterial
strain for use in the invention has a 16s rRNA sequence that is at least 95%,
96%, 97%, 98%,
99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO:1 or 2.
Preferably, the
sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use
in the invention has
the 16s rRNA sequence represented by SEQ ID NO:2.
A biotype is a closely related strain that has the same or very similar
physiological and
biochemical characteristics. Strains that are biotypes of the bacterium
deposited under accession
number NCIMB 43456 and that are suitable for use in the invention may be
identified by
sequencing other nucleotide sequences for the bacterium deposited under
accession number
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NCIMB 43456. For example, substantially the whole genome may be sequenced and
a biotype
strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%,
99.5%, 99.6%,
99.7%, 99.8% or 99.9% sequence identity across at least 80% of its whole
genome (e.g. across
at least 85%, 90%, 95% or 99%, or across its whole genome). For example, in
some
5 embodiments, a biotype strain has at least 98% sequence identity across
at least 98% of its
genome or at least 99% sequence identity across 99% of its genome. Other
suitable sequences
for use in identifying biotype strains may include hsp60 or repetitive
sequences such as BOX,
ERIC, (GTG)5, or REP [28]. Biotype strains may have sequences with at least
95%, 96%, 97%,
98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the
corresponding
10 sequence of the bacterium deposited under accession number NCIMB 43456.
In some
embodiments, a biotype strain has a sequence with at least 95%, 96%, 97%, 98%,
99%, 99.5%,
99.6%, 99.7%, 99.8% or 99.9% sequence identity to the 16S rRNA sequence of SEQ
ID NO: 2.
Alternatively, strains that are biotypes of the bacterium deposited under
accession number
NCIMB 43456 and that are suitable for use in the invention may be identified
by using the
15 accession number NCIMB 43456 deposit and restriction fragment analysis
and/or PCR analysis,
for example by using fluorescent amplified fragment length polymorphism
(FAFLP) and
repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or
partial 16S or 23s
rDNA sequencing. In preferred embodiments, such techniques may be used to
identify other
Enterococcus durans strains.
In certain embodiments, strains that are biotypes of the bacterium deposited
under accession
number NCIMB 43456 and that are suitable for use in the invention are strains
that provide the
same pattern as the bacterium deposited under accession number NCIMB 43456
when analysed
by amplified ribosomal DNA restriction analysis (ARDRA), for example when
using Sau3AI
restriction enzyme (for exemplary methods and guidance see, for example,
[291). Alternatively,
.. biotype strains are identified as strains that have the same carbohydrate
fermentation patterns as
the bacterium deposited under accession number NCIMB 43456.
In some embodiments, strains that are biotypes of the bacterium deposited
under accession
number NCIMB 43456 and that are suitable for use in the invention are
Enterococcus strains that
do not produce acid from glycerol. In some embodiments, strains that are
biotypes of the
bacterium deposited under accession number NCIMB 43456 and that are suitable
for use in the
invention are Enterococcus strains that do not produce acid from mannitol. In
some
embodiments, strains that are biotypes of the bacterium deposited under
accession number
NCIMB 43456 and that are suitable for use in the invention are Enterococcus
strains that do not
produce acid from sucrose. In some embodiments, whether acid is produced from
glycerol,
mannitol and/or sucrose is determined with the API 50CH system, using
bromocresol purple
(0.002%, wt/vol) as the indicator in 1% (wt/vol) peptone water (see reference
23).
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The ability of enterococci to grow under particular conditions is widely used
for their selective
isolation ([30]). In some embodiments, strains that are biotypes of the
bacterium deposited under
accession number NCIMB 43456 and that are suitable for use in the invention
are Enterococcus
strains that are incapable of metabolising one or more of (e.g. more than 1,
2, 3, 4, 5, 6 of or all
7 of) glycerol, D-mannitol, D-sorbitol, L-arabinose, gluconate, melibiose
and/or sucrose,
preferably one or more of (e.g. more than 1, 2, 3 of or all 4 of) glycerol, D-
mannitol, D-sorbitol
and/or sucrose; such strains are in accordance with the phenotype of
Enterococcus durans
provided in references [30, 31 and 77]).
In some embodiments, strains that are biotypes of the bacterium deposited
under accession
number NCIMB 43456 and that are suitable for use in the invention are
Enterococcus strains that
are incapable of metabolising D-mannitol (for example in accordance with the
phenotype of
Enterococcus durans provided in references [30 and 31]).
In some embodiments, strains that are biotypes of the bacterium deposited
under accession
number NCIMB 43456 and that are suitable for use in the invention are
Enterococcus strains that
grow in tellurite (as is the case for Enterococcus durans, see references [30
and 31]).
In some embodiments, the biotype is not an E. casseliflavus strain. In some
embodiments, the
biotype is not an E. malodoratus strain. In some embodiments, the biotype is
not an E. faecium
strain.
In some embodiments, the biotype is an E. hirae strain.
Other Enterococcus durans strains that are useful in the compositions and
methods of the
invention, such as biotypes of the bacterium deposited under accession number
NCIMB 43456,
may be identified using any appropriate method or strategy, including the
assays described in the
examples. For instance, strains for use in the invention may be identified by
culturing in anaerobic
YCFA and/or administering the bacteria to an autism spectrum disorder mouse
model and then
assessing cytokine levels. In particular, bacterial strains that have similar
growth patterns,
metabolic type and/or surface antigens to the bacterium deposited under
accession number
NCIMB 43456 may be useful in the invention. In particular, a biotype strain
will elicit
comparable effects on the autism spectrum disorder models to the effects shown
in the examples,
which may be identified by using the culturing and administration protocols
described in the
.. examples.
A particularly preferred strain of the invention is the Enterococcus durans
strain deposited under
accession number NCIMB 43456. This is the exemplary Enterococcus durans strain
tested in the
examples and shown to be effective for treating disease. Therefore, the
invention provides a cell,
such as an isolated cell, of the Enterococcus durans strain deposited under
accession number
NCIMB 43456, or a derivative thereof. The invention also provides a
composition comprising a
cell of the Enterococcus durans strain deposited under accession number NCIMB
43456, or a
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17
derivative thereof. The invention also provides a biologically pure culture of
the Enterococcus
durans strain deposited under accession number NCIMB 43456. The invention also
provides a
cell of the Enterococcus durans strain deposited under accession number NCIMB
43456, or a
derivative thereof, for use in therapy, in particular for the diseases
described herein.
A derivative of the strain deposited under accession number NCIMB 43456 may be
a daughter
strain (progeny) or a strain cultured (subcloned) from the original. A
derivative of a strain of the
invention may be modified, for example at the genetic level, without ablating
the biological
activity. In particular, a derivative strain of the invention is
therapeutically active. A derivative
strain will have comparable immune modulatory activity to the original NCIMB
43456 strain. In
particular, a derivative strain will elicit comparable effects on the autism
spectrum disorder
models to the effects shown in the examples, which may be identified by using
the culturing and
administration protocols described in the examples. For example, a derivative
strain will elicit
comparable effects on the BTBR mouse model and/or the maternal immune
activation (MIA)
mouse model, which may be identified by the marble burying test, the elevated
plus maze test,
the three-chamber social interaction test, the novel object recognition test,
the forced swim test,
brainstem monoamine levels and/or amygdalar gene expression as set out in
example 2. A
derivative of the NCIMB 43456 strain will generally be a biotype of the NCIMB
43456 strain.
The bacterial strain may also be a strain that has the same safety and
therapeutic efficacy
characteristics as the strains deposited under accession number NCIMB 43456,
and such cells
are encompassed by the invention.
In some embodiments, the bacterial strain in the compositions of the invention
is an E. hirae
strain.
In preferred embodiments, the bacterial strains in the compositions of the
invention are viable
and capable of partially or totally colonising the intestine.
In certain embodiments, the bacterial strain for use in the invention is
resistant to one of more of
amoxicillin, ampicillin, arbekacin and dibekacin, azlocillin, bacampicillin,
carbenicillin,
ceftobiprole, clarithromycin, doripenem, erythromycin, fusidic acid,
gentamicin, grepafloxacin,
imipenem, josamycin, meropenem, meziocillin, piperacillin, rifampin,
rifaximin, rokitamycin,
rosaramicin, roxithromycin, spiramycin, streptomycin,
sulfamethoxazole/trimethoprim,
telithromycin, ticarcillin, ticarcillin/clavulanate, tosufloxacin,
trimethoprim and virginiamycin.
In certain embodiments, the bacterial strain for use in the invention is
susceptible to Quinopristin-
dalfopristin.
In certain embodiments, the bacterial strain for use in the invention is
resistant to f3-lactam
antibiotics. In certain embodiments, the bacterial strain for use in the
invention is resistant to
vancomycin. In certain embodiments, the bacterial strain for use in the
invention is resistant to
ampicillin.
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18
Therapeutic uses
Modulation of the microbiota-gut-brain axis
Communication between the gut and the brain (the microbiota-gut-brain axis)
occurs via a
bidirectional neurohumoral communication system. Recent evidence shows that
the microbiota
that resides in the gut can modulate brain development and produce behavioural
phenotypes via
the microbiota-gut-brain axis. Indeed, a number of reviews suggest a role of
the microbiota-gut-
brain axis in maintaining central nervous system functionality and implicate
dysfunction of the
microbiota-gut-brain axis in the development of central nervous system
disorders and conditions
181,191,1321.
The bidirectional communication between the brain and the gut (i.e. the-gut-
brain axis) includes
the central nervous system, neuroendocrine and neuroimmune systems, including
the
hypothalamus-pituitary-adrenal (HPA) axis, sympathetic and parasympathetic
aims of the
autonomic nervous system (ANS), including the enteric nervous system (ENS) and
the vagus
nerve, and the gut microbiota.
As demonstrated in the examples, the compositions of the present invention can
modulate the
microbiota-gut-brain axis and reduce the behavioural symptoms associated with
a CNS disorder.
Accordingly, the compositions of the invention may be useful for treating or
preventing disorders
of the central nervous system (CNS), in particular those disorders and
conditions associated with
dysfunction of the microbiota-gut-brain axis. The compositions of the
invention may be useful
for reducing behavioural symptoms associated with a CNS disorder.
The compositions of the invention may also be useful for treating or
preventing
neurodevelopmental disorders and/or neuropsychiatric conditions.
Neurodevelopmental diseases
and neuropsychiatric conditions are often associated with the microbiota-gut-
brain axis. The
compositions of the invention may be useful for treating or preventing
neurodevelopmental
diseases and/or neuropsychiatric conditions mediated by dysfunction of the
microbiota-gut-brain
axis. In further preferred embodiments, the compositions of the invention are
for use in treating
or preventing a neurodevelopmental disorder or a neuropsychiatric condition.
In particular embodiments, the compositions of the invention may be useful for
treating or
preventing a disease or condition selected from the group consisting of:
autism spectrum
disorders (ASDs); child developmental disorder; obsessive compulsive disorder
(OCD); major
depressive disorder; depression; seasonal affective disorder; anxiety
disorders; schizophrenia
spectrum disorders; schizophrenia; bipolar disorder; psychosis; mood disorder;
chronic fatigue
syndrome (myalgic encephalomyelitis); stress disorder; post-traumatic stress
disorder; dementia;
Alzheimer's; Parkinson's disease; epilepsy; chronic pain (for example, central
sensitisation or
fibromyalgia); motor neuron disease; Huntington's disease; Guillain-Barre
syndrome and/or
meningitis.
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The compositions of the invention may be particularly useful for treating or
preventing chronic
disease, treating or preventing disease in patients that have not responded to
other therapies (such
as treatment with anti-psychotics and/or anti-depressants), and/or treating or
preventing the tissue
damage and symptoms associated with dysfunction of the microbiota-gut-brain
axis.
In certain embodiments, the compositions of the invention modulate the CNS. In
some
embodiments, the compositions of the invention modulate the autonomic nervous
system (ANS).
In some embodiments, the compositions of the invention modulate the enteric
nervous system
(ENS). In some embodiments, the compositions of the invention modulate the
hypothalamic,
pituitary, adrenal (HPA) axis. In some embodiments, the compositions of the
invention modulate
the neuroendocrine pathway. In some embodiments, the compositions of the
invention modulate
the neuroimmune pathway. In some embodiments, the compositions of the
invention modulate
the CNS, the ANS, the ENS, the HPA axis and/or the neuroendocrine and
neuroimmune
pathways. In certain embodiments, the compositions of the invention module the
levels of
commensal metabolites and/or the gastrointestinal permeability of a subject.
The signalling of the microbiota-gut-brain axis is modulated by neural
systems. Accordingly, in
some embodiments, the compositions of the invention modulate signalling in
neural systems. In
certain embodiments, the compositions of the invention modulate the signalling
of the central
nervous system. In some embodiments, the compositions of the invention
modulate signalling in
sensory neurons. In other embodiments, the compositions of the invention
modulate signalling
in motor neurons. In some embodiments, the compositions of the invention
modulate the
signalling in the ANS. In some embodiments, the ANS is the parasympathetic
nervous system.
In preferred embodiments, the compositions of the invention modulate the
signalling of the vagus
nerve. In other embodiments, the ANS is the sympathetic nervous system. In
other embodiments,
the compositions of the invention modulate the signalling in the enteric
nervous system. In certain
embodiments, the signalling of ANS and ENS neurons responds directly to
luminal contents of
the gastrointestinal tract. In other embodiments, the signalling of ANS and
ENS neurons responds
indirectly to neurochemicals produced by luminal bacteria. In other
embodiments, the signalling
of ANS and ENS neurons responds to neurochemicals produced by luminal bacteria
or
enteroendocrine cells. In certain preferred embodiments, the neurons of the
ENS activate vagal
afferents that influence the functions of the CNS. In some embodiments, the
compositions of the
invention regulate the activity of enterochromaffin cells.
In certain embodiments, the compositions of the invention modulate fear
conditioning in an
animal model. In certain embodiments, the compositions of the invention can be
used to modulate
the development of fear and/or anxiety, and/or modulate the extent to which
the fear and/or
anxiety becomes extinct in a subject. In certain embodiments, the compositions
of the invention
can be used to modulate the extent of stress-induced hyperthermia in an animal
model. In certain
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embodiments, the compositions of the invention modulate the level of stress
and/or anxiety in a
subject.
Autism spectrum disorder (ASD)
Autism spectrum disorder is a set of heterogeneous neurodevelopmental
conditions, characterised
5 by early-onset difficulties in social interaction, communication and
unusually restricted,
repetitive behaviour and interests. Symptoms can be recognised from a very
early age but ASD
is often diagnosed in more able children starting mainstream education. Autism
represents the
primary type of ASD.
Historically, autism has been diagnosed on the basis of three core domains:
impaired social
10 interaction, abnormal communication, and restricted and repetitive
behaviours and interests. In
the International Classification of Diseases (ICD-10R, WHO 1993) and the
Diagnostic and
Statistical Manual (DSM-IV, American Psychiatric Association, 2000), autism
comes under the
umbrella term of Pervasive Developmental Disorder (PDD), with four possible
diagnostic
subtypes: Asperger Syndrome, Childhood Autism/Autistic Disorder, Atyptical
Autism, and
15 PDD-not otherwise specified. In DMS-5, these diagnostic subtypes are
combined into a single
category of autism spectrum disorder (ASD) and the previous use of three core
domains of
impairment has been reduced to two main areas, namely social communication and
interaction,
and repetitive behaviour, which include sensory integration dysfunctions.
ASD is a 'spectrum disorder' as it affects each person in a variety of
different ways and can range
20 from very mild to severe. The functioning of the affected individual
varies substantially
depending on language abilities, level of intelligence, co-morbidity,
composition of symptoms
and access to services. Cognitive functioning, learning, attention and sensory
processing are
usually impaired.
DSM-IV states that the diagnosis of autism requires the presence of at least
six symptoms,
including a minimum of two measures of qualitative impairment in social
interaction, one
symptom of qualitative impairment in communication, and one symptom of
restricted and
repetitive behaviour. DMS-5 redefines diagnosis of ASD into two symptom
domains: (i) social
interaction and social communication deficits; and (ii) restricted, repetitive
patterns of behaviour,
interests or activities.
Co-morbid medical conditions are highly prevalent in ASD. Co-morbid medical
conditions
include anxiety and depression, seizures, attention deficits, aggressive
behaviours, sleep
problems, gastrointestinal disorders, epilepsy, mental retardation,
intellectual disabilities and/or
feeding difficulties.
The examples demonstrate that the compositions of the invention achieve a
reduction in disease
incidence and disease severity in an animal model of autism spectrum disorder
and that they are
therefore useful in the treatment or prevention of ASD.
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21
ASD is a central nervous system disorder that is partially triggered by
environmental factors.
Therefore, dysfunction of the microbiota-gut-brain axis may be responsible for
development and
persistence of ASDs. Accordingly, in preferred embodiments, the compositions
of the invention
are for use in treating or preventing ASDs. In some embodiments, the
compositions of the
invention are for use in treating or preventing autism. In some embodiments,
the autism is
Pervasive Developmental Disorder (PDD). In another embodiment, the PDD is
Asperger
Syndrome, Childhood Autism/Autistic Disorder, Atyptical Autism and/or PDD-not
otherwise
specified. Accordingly, in some embodiments, the compositions of the invention
are for use in
treating or preventing autism spectrum disorders, autism, pervasive
developmental disorder;
Asperger Syndrome; Childhood Autism/Autistic Disorder, Atypical Autism and/or
PDD-not
otherwise specified.
The compositions of the invention may be useful for modulating the microbiota-
gut-brain axis of
a subject. Accordingly, in preferred embodiments the compositions of the
invention are for use
in preventing ASD in a patient that has been identified as at risk of ASD, or
that has been
diagnosed with ASD at a prenatal or an early developmental stage; in childhood
and/or in
adulthood. The compositions of the invention may be useful for preventing the
development of
ASD.
The compositions of the invention may be useful for managing or alleviating
ASDs. Treatment
or prevention of ASD may refer to, for example, an alleviation of the severity
of symptoms or a
reduction in the frequency of exacerbations or the range of triggers that are
a problem for the
patient.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one core symptom of ASD.
In some embodiments, the compositions of the invention prevent, reduce or
alleviate at least one
of the two symptom domains of ASD classified in the DMS-5. In some
embodiments, the
compositions of the invention prevent, reduce or alleviate social interaction
and/or social
communication deficits. In some embodiments, the compositions of the invention
prevent, reduce
or alleviate restrictive, repetitive patterns of behaviour, interests or
activities. In some
embodiments, the compositions of the invention prevent, reduce or alleviate
social interaction,
social communication deficits and/or restrictive, repetitive patterns of
behaviour, interests or
activities.
In some embodiments, the compositions of the invention prevent, reduce or
alleviate repetitive
behaviour, stereotyped behaviour, compulsive behaviour, routine behaviour,
sameness behaviour
and restricted behaviour. In some embodiments, the compositions of the
invention improve social
awareness, learning, memory function, social infonnation processing, capacity
for social
communication, social anxiety/avoidance, and autistic preoccupations and
traits in a subject with
Date Recue/Date Received 2022-07-25
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22
ASD. In preferred embodiments, the compositions of the invention reduce
feelings of despair
or helplessness in a subject with ASD. In preferred embodiments, the invention
provides a
composition for treating or preventing neophobia and / or sociability deficit
in a subject with
ASD.
In some embodiments, the compositions of the invention prevent, reduce or
alleviate additional
symptoms associated with the core symptoms of ASD. In some embodiments, the
compositions
of the invention prevent, reduce or alleviate irritability (including
aggression, deliberate self-
injury and temper tantrums), agitation, crying, lethargy, social withdrawal,
stereotypic behaviour,
hyperactivity, non-compliance, inappropriate speech, anxiety, depression,
and/or over or under-
controlled behaviour in a subject with ASDs. In some embodiments, the
compositions of the
invention improve cognitive functioning, learning, memory function, attention
and/or sensory
processing in a subject with ASD.
In other embodiments, the compositions of the invention improve secondary
outcome measures
in a subject with ASDs. In some embodiments, the secondary outcome measures
include
additional symptom and/or functional rating scales, behavioural scales and
miscellaneous
measures of interest.
In some embodiments, the compositions of the invention cause in a positive
change in the
diagnostic and/or symptomatic scale for the assessment of core symptoms of a
subject with
ASDs. In some embodiments, the diagnostic and/or symptomatic scale is the
Autism Diagnostic
Interview ¨ Revised (ASI-R). In some embodiments, the diagnostic or
symptomatic scale is the
Autism Diagnostic Observation Schedule-Generic (ADOS-G) now ADOS-2. In other
embodiments, the diagnostic or symptomatic scale is the Autism Diagnostic
Interview Revised
(ADI-R). In other embodiments, the diagnostic or symptomatic scale is the
Diagnostic Interview
for Social and Communication Disorders (DISCO). In yet other embodiments, the
diagnostic or
symptomatic scale is the Childhood Autism Rating Scale (CARS and CARS2).
In some embodiments, the compositions of the invention cause a positive change
in generic
measures of the efficacy endpoints of ASD. In certain embodiments, the generic
measures
include, but are not limited to the Aberrant Behaviour Checklist (ABC), the
Child Behaviour
Checklist (CBCL), the Vineland-II Adaptive Behaviour Scales (VABS), the Social
Responsiveness Scale (SRS), and/or the Repetitive Behaviour Scale ¨ Revised
(RBS-R).
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global
functioning of the subject with ASD.
Date Recue/Date Received 2022-07-25
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23
Additional scales would be known to a person skilled in the art. In some
embodiments, the
compositions of the invention would improve the outcome of diagnostic and/or
symptomatic
scales known to a person skilled in the art.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate the
incidence of comorbidities of ASDs. In some embodiments, the compositions of
the invention
prevent, reduce or alleviate the incidence of anxiety and depression,
seizures, attention deficits,
aggressive behaviours, sleep problems, gastrointestinal disorders (including
irritable bowel
syndrome (IBS)), epilepsy, mental retardation, intellectual disabilities
and/or feeding difficulties.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate
.. gastrointestinal comorbidities, such as abdominal pain, diarrhoea and/or
flatulence.
In some embodiments, the compositions of the invention prevent, reduce or
alleviate the
symptoms of certain psychiatric and behavioural disorders that may present
clinically with
similarities to autism. Accordingly, in some embodiments, the compositions of
the invention,
prevent, reduce or alleviate attention deficit disorder (ADHD);
affective/anxiety disorders;
attachment disorders; oppositional defiant disorder (ODD); obsessive
compulsive disorder
(OCD) and/or psychoses including schizophrenia (cognitive impairment).
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating ASD when used in combination with another therapy for
treating ASD.
Such therapies include anti-psychotic, anti-anxiety and anti-depressant drugs.
Such drugs include
risperidone (Risperda10); olanzapine (Zyprexa0); fluoxetine (Prozac0);
sertraline (Zoloft0);
fluvoxamine (Luvox0); clomipramine (Anafrani10); haloperidol (Haldo10);
thioridazine;
fluphenazine; chlorpromazine; ziprasidone (Geogon0); carbamazepine
(Tegreto10); lamotrigine
(Lamictal0); topiramate (Topomax0); valproic acid (Depakote0); methylphenidate
(Ritalin0);
diazepam (Valium()) and lorazepam (Ativan0).
Obsessive compulsive disorder (OCD)
OCD is a heterogeneous, chronic and disabling disorder belonging to the
anxiety disorders.
According to the DSM-IV definition, the essential features of OCD are
recurrent obsessions
and/or compulsions (criterion A) that are severe and time consuming (more than
one hour a day)
or cause marked distress or significantly interfere with the subject's noimal
routine, occupational
functioning, usual social activities or relationships (criterion C). As some
point during the course
of the disorder, the person has recognised that the obsessions or compulsions
are excessive or
unreasonable (criterion B).
Obsessions are defined as recurrent and persistent thoughts, impulses or
images that are
experienced as intrusive and inappropriate and cause marked anxiety or
distress. The thoughts,
.. impulses or images are not simply excessive worries about real-life
problems, they are recognised
by the patient as a product of his own mind (e.g. fear for contamination,
symmetry obsession).
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24
The person attempts to ignore, suppress or neutralise the obsessions with some
other thoughts or
actions.
Compulsions are defined as repetitive behaviours (e.g. hand washing, ordering,
hoarding,
checking) or mental acts (e.g. praying, counting, repeating words silently)
that the person feels
driven to perfonn in response to an obsession or according to rules that must
be applied rigidly.
OCD is often associated with co-morbidity rates of other psychiatric diseases
including major
depressive disorder, other anxiety disorders (generalised anxiety disorder,
social anxiety disorder,
panic disorder), substance abuse and eating disorders (anorexia and bulimia).
OCD is a psychiatric disorder that may develop or persist due to dysfunction
of the microbiota-
gut-brain axis. Accordingly, in preferred embodiments, the compositions of the
invention are for
use in treating or preventing OCD in a subject.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate the
essential symptomatic features of OCD. In certain embodiments, the
compositions of the
invention prevent, reduce or alleviate recurrent obsessions and/or compulsions
in a subject. In
certain embodiments, the obsessions are recurrent or persistent thoughts,
impulses or images that
are experienced as intrusive and inappropriate and cause marked anxiety or
distress. In certain
embodiments, the compulsions are repetitive behaviours that the subject feels
driven to perfonn
in response to an obsession or according to rules that must be applied
rigidly.
In certain embodiments, the compositions of the invention improve symptoms of
OCD in a
subject accordingly to the Y-BOCS and/or the NIMH-OC diagnostic and/or
symptomatic scales.
In some embodiments, the Y-BOCS scale is used to monitor improvement of
primary endpoints.
In some embodiments, the NIMH-OC scale is used to monitor improvement of
secondary
parameters.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social
functioning (relationships, work, etc.) of the subject with ASDs. In some
embodiments, the global
scale is the Sheehan disability scale.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of OCD. The comorbidities of OCD include major depressive
disorder, other
anxiety disorders (generalised anxiety disorder, social anxiety disorder,
panic disorder),
substance abuse and eating disorders (anorexia and bulimia) Gilles de la
Tourette syndrome,
ADHD (Attention-Deficit/Hyperactivity Disorder) and developmental disorders.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating OCD when used in combination with another therapy for
treating OCD.
Date Recue/Date Received 2022-07-25
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Such therapies include serotonin and dopamine reuptake inhibitors;
clomipramine and anti-
psychotics.
Major depressive disorder (MDD)
MDD is associated with substantial psychosocial dysfunction and high
individual mental strain
5 as well as with excess morbidity and mortality (the risk of suicide is
considerable). The tenn
major depressive disorder encompasses clinical depression, major depression,
unipolar
depression, unipolar disorder, recurrent depression and simply depression. The
temi major
depressive disorder covers mood disorders; dysthymia; chronic depression;
seasonal affective
disorder and borderline personality disorder.
10 According to the DMS-5 criteria, MDD symptoms include a depressed mood,
or loss of interest
or pleasure in daily activities for more than two weeks; and impaired social,
occupational and
educational function. Specific symptoms, at least five of the following nine,
present nearly every
day: depressed mood or irritable most of the day; decreased interest or
pleasure in most activities,
most of each day; significant weight change or change in appetite; change in
sleep (insomnia or
15 hypersomnia); change in activity (psychomotor agitation or retardation);
fatigue or loss of
energy; guilt or worthlessness (feelings of worthlessness or excessive or
inappropriate guilt);
reduced concentration (diminished ability to think or concentrate, or more
indecisiveness; and
suicidality (thoughts of death or suicide, or subject has a suicide plan). In
addition, MDD is
associated with anxiety symptoms including irrational worry; preoccupation
with unpleasant
20 worries; trouble relaxing and/or feeling tense. MDD episodes can be
mild, moderate or severe.
MDD episodes are often associated with comorbidity with other psychiatric
disorders or with
somatic disorders like Parkinson's disease, Alzheimer's disease,
cerebrovascular disorders,
cancer and chronic pain syndromes. MDD is frequently associated with a wide
spectrum of other
mental disorders as comorbidities including generalised anxiety disorder;
anxiety disorder;
25 substance use disorders; post-traumatic stress disorder (PTSD);
personality disorders; pain;
stress; irritable bowel syndrome; insomnia; headaches and interpersonal
problems.
Major depressive disorder is a psychiatric disorder that may develop or
persist due to dysfunction
of the microbiota-gut-brain axis. Accordingly, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing MDD in a subject.
In certain embodiments, the compositions of the invention are for use in
treating or preventing
acute major depressive episodes and/or the prevention of new episodes
(recurrence prevention).
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate the
occurrence of mild, moderate or severe MDD episodes.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of MDD as classified by the DMS-5 criteria listed herein.
In a preferred
embodiment, the compositions of the invention prevent, reduce or alleviate a
depressed mood in
Date Recue/Date Received 2022-07-25
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26
a subject. In a preferred embodiment, the compositions of the invention
prevent, reduce or
alleviate a decreased interest or pleasure in most activities in a subject. In
some embodiments,
the compositions of the invention reduce the occurrence of symptoms of MDD
within a 2-week
period.
In some embodiments, the compositions of the invention improve the symptoms of
MDD
according to a symptomatic or diagnostic scale. Such scales for assessing
symptomatic
improvement include the Hamilton Rating Scale of Depression (HAMD) and the
Montgomery
Asberg Depression Rating Scale. In addition, the Zung Self-Rating Depression
Scale (SDS) and
Zung Self-Rating Anxiety Scale (SAS) are also suitable symptomatic improvement
scales.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social
and occupational functioning of the subject with MDD.
In certain embodiments, the compositions of the invention are for use in
treating or preventing
treatment resistant MDD.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of MDD. The comorbidities of MDD include generalised anxiety
disorder;
anxiety disorder; substance use disorders; post-traumatic stress disorder
(PTSD); personality
disorders; pain; stress; IBS; insomnia, and headaches.
.. In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating MDD when used in combination with another therapy for
treating MDD.
Such therapies include antidepressants, augmentation strategies (e.g.
combination therapy,
lithium and other mood stabilizers, thyroid homiones and atypical
antipsychotics) or even second
generation antipsychotics.
Anxiety disorders
Anxiety disorders are psychiatric disorders that may develop or persist due to
dysfunction of the
microbiota-gut-brain axis. Accordingly, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing anxiety disorders in a
subject. In certain
embodiments, the anxiety disorder is generalised anxiety disorder (GAD);
specific phobia; social
anxiety disorder; separation anxiety disorder; agoraphobia; panic disorder and
selective mutism.
GAD is diagnosed according to DMS-5 in six criteria. The first criterion is
too much anxiety or
worry over more than six months wherein the anxiety or worry is present most
of the time in
regards to many activities. The second criterion is that the subject is unable
to manage the
symptoms of the first criterion. The third criterion is that at least three
(one in children) of the
following occurs: restlessness; tires easily; problems concentrating;
irritability; muscle tension
Date Recue/Date Received 2022-07-25
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27
and problems with sleep. The final three criterion are that the symptoms
results in significant
social, occupational and functional impairment; the symptoms are not due to
medications, drugs,
or other physical health problems; and the symptoms do not fit better with
another psychiatric
problem such as panic disorder. All other anxiety disorders may be considered
as differential
diagnoses of GAD.
GAD is frequently associated with a wide spectrum of other mental disorders as
comorbidities
including depression; substance use disorders; stress; IBS; insomnia;
headaches; pain; cardiac
events; interpersonal problems and ADHD.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate one or
.. more of the symptoms of GAD in a subject as classified by the DMS-5
criteria listed herein.
According to DMS-5, the same symptoms are associated with other anxiety
disorders. Therefore,
in certain embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of anxiety disorders in a subject. In preferred
embodiments, the
compositions of the invention prevent, reduce or alleviate the anxiety or
worry of the subject. In
certain embodiments, the compositions of the invention reduce the occurrence
of symptoms
within a six month period. In certain embodiments, the composition of the
invention prevents,
reduces or alleviates restlessness; fatigue; loss of concentration;
irritability; muscle tension;
and/or problems with sleep. In some embodiments, the compositions of the
invention prevent,
reduce or alleviate social, occupational and functional impahment associated
with anxiety
.. disorders.
In some embodiments, the compositions of the invention improve the symptoms of
anxiety
disorders according to a symptomatic or diagnostic scale. In certain
embodiments, the scale for
assessing symptomatic improvement includes the Hamilton Anxiety Rating Scale
(HAM-A). In
some embodiments, the HAM-A total scale is used to assess primary endpoint. In
other
embodiments, the HAM-A psychic anxiety factor may be useful as a secondary
endpoint.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social,
occupational and functional impahment of the subject with anxiety disorder. In
some
.. embodiments, the global scale is the Sheehan disability scale.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of GAD and anxiety disorders. The comorbidities of GAD include
depression;
substance use disorders; stress; IBS; insomnia; headaches; pain; cardiac
events, and ADHD.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating anxiety disorders when used in combination with
another therapy for
treating anxiety disorders. Such therapies include selective serotonin
reuptake inhibitors
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28
(venlafaxine, duloxetine, escitalopram and paroxetine); benzodiazepines
(alprazolam, lorazepam
and clonazepam); pregabalin (Lyrica0) and gabapentin (Neurontin C)); serotonin
receptor partial
agonists (buspirone and tandospirone); atypical serotonergic antidepressants
(such as imipramine
and clomipramine); monoamine oxidase inhibitors (MAOIs) (such as moclobemide
and
phenelzine); hydroxyzine; propranolol; clonidine; guanfacine and prazosin.
Post-traumatic stress disorder (PTSD)
PTSD is a severe and disabling disorder, an essential feature of which is the
inclusion of a
traumatic event as a precipitating factor of this disorder.
The symptoms of PTSD are grouped into four main clusters according to the DMS-
V criteria:
(i) intrusion: examples include nightmares, unwanted thoughts of the traumatic
events,
flashbacks, and reacting to traumatic reminders with emotional distress or
physiological
reactivity; (ii) avoidance: examples include avoiding triggers for traumatic
memories including
places, conversations, or other reminders; (iii) negative alterations in
cognitions and mood:
examples include distorted blame of self or others for the traumatic event,
negative beliefs about
oneself or the world, persistent negative emotions (e.g., fear, guilt, shame),
feeling alienated, and
constricted affect (e.g., inability to experience positive emotions); (iv)
alterations in arousal and
reactivity: examples include angry, reckless, or self-destructive behaviour,
sleep problems,
concentration problems, increased startle response, and hypervigilance.
Symptoms that resolve within 4 weeks of the traumatic event meet the criteria
for an Acute Stress
Disorder. The DSM distinguishes between acute (duration of symptoms for less
than three
months) and chronic PTSD (duration of symptoms longer than 3 months). If the
symptoms begin
more than 6 months after the stressor, the disorder is defined as delayed
onset PTSD.
PTSD carries high comorbidities with major depressive disorder and substance
use disorders.
PTSD is a psychiatric disorder that may develop or persist due to dysfunction
of the
microbiota-gut-brain axis. Accordingly, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing PTSD in a subject. According
to a similar
pathogenesis, in certain embodiments, the compositions of the invention are
for use in treating or
preventing stress disorders. In certain embodiments, the compositions of the
invention treat acute
stress disorder. In some embodiments, the compositions of the invention treat
acute and/or
chronic PTSD. In some embodiments, the compositions of the invention treat
delayed onset
PTSD.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of PTSD (or stress disorder) in a subject as classified
by the DMS-5 criteria
listed herein. In preferred embodiments, the compositions of the invention
prevent, reduce or
alleviate intrusive thoughts in a subject with PTSD. In preferred embodiments,
the compositions
of the invention prevent, reduce or alleviate avoidance behaviour in a subject
with PTSD. In
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29
preferred embodiments, the compositions of the invention prevent, reduce or
alleviate negative
alterations in cognitions and mood in a subject with PTSD. In preferred
embodiments, the
compositions of the invention prevent alterations in arousal and reactivity in
a subject with PTSD.
In some embodiments, the compositions of the invention improve the symptoms of
PTSD and
stress disorders according to a symptomatic or diagnostic scale. In certain
embodiments, the scale
for assessing symptomatic improvement is the Clinical-Administered PTSD (CAPS)
scale.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social,
occupational and functional impaiiment of the subject with PTSD and stress
disorders. In some
embodiments, the global scale is the Sheehan disability scale.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of PTSD and stress disorders. The comorbidities of PTSD and
stress disorders
include MDD, substance use disorders; stress and anxiety.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating PTSD and stress disorders when used in combination
with another therapy
for treating PTSD and stress disorders. Such therapies include serotoninergic
agents, tricyclic
antidepressants, mood stabilisers, adrenergic inhibiting agents,
antipsychotics, benzodiazepines,
sertraline (Zoloft0), fluoxetine (Prozac0) and/or paroxetine (Paxi10).
Schizophrenia spectrum and psychotic disorders
These diseases affect a subject's ability to think clearly, make good
judgements, respond
emotionally, communicate effectively, understand reality, and behave
appropriately. Psychotic
diseases include schizophrenia (symptoms listed below); schizoaffective
disorder (the subject has
symptoms of both schizophrenia and a mood disorder, such as depression or
bipolar disorder);
________ schizophrenifoi in disorder (displays the symptoms of
schizophrenia, but the symptoms last for a
shorter time: between 1 and 6 months); brief psychotic disorder (subjects
display a sudden, short
period of psychotic behaviour, often in response to a very stressful event,
such as a death in the
family - recovery is usually less than a month); delusional disorder
(delusions last for at least 1
month); shared psychotic disorder; substance-induced psychotic disorder;
psychotic disorder due
to another medical condition; paraphrenia (displaying symptoms similar to
schizophrenia and
starting late in life, when people are elderly). The most well-known psychotic
disorder is
schizophrenia and the majority of psychotic disorders display similar symptoms
to schizophrenia.
Schizophrenia is a severe psychiatric disease with a heterogeneous course and
symptom profile.
Schizophrenia presents clinically with so-called positive and negative
symptoms. The positive
symptoms include delusions, hallucinations, disorganised speech, and
disorganised or catatonic
behaviours. Negative symptoms include affective flattening, restriction in the
fluency and
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productivity of thought and speech and in the initiation of goal directed
behaviour. The positive
symptoms appear to reflect an excess or distortion of normal functions,
whereas negative
symptoms appear to reflect a diminution or loss of normal function. In
addition, cognitive deficits
(defects of working memory, information processing, attention/vigilance,
learning, reasoning and
5 social cognition) are common. Cognitive deficits generally show poor
improvement with current
antipsychotic treatment. Schizophrenic patients also suffer from mood
symptoms. Besides these
predominant symptoms, schizophrenia is associated with a comorbidity with
other psychiatric
symptoms such as manic and depressive symptoms, anxiety or obsessive-
compulsive symptoms,
substance abuse and dependence, and personality disorder.
10 According to the DMS-5, for the diagnosis of schizophrenia, a subject
must have at least two of
the following symptoms: delusions; hallucinations; disorganised speech;
disorganised or
catatonic behaviour and negative symptoms. At least one of the symptoms must
be the presence
of delusions, hallucinations or disorganised speech. Continuous signs of
disturbance must persist
for at least 6 months, during which the subject must experience at least 1
month of active
15 symptoms, with social or occupational deterioration problems occurring
over a significant
amount of time.
Schizophrenia spectrum and psychotic disorders are psychiatric disorders that
may develop or
persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in
preferred embodiments,
the compositions of the invention are for use in treating or preventing
schizophrenia spectrum
20 and/or psychotic disorders in a subject. In certain embodiments, the
schizophrenia spectrum and
psychotic disorder is selected from schizophrenia; schizoaffective disorder;
schizophreniform
disorder; brief psychotic disorder; delusional disorder; shared psychotic
disorder;
substance-induced psychotic disorder; psychotic disorder due to another
medical condition and
paraphrenia. In preferred embodiments, the compositions of the invention are
for use in treating
25 or preventing schizophrenia. In certain embodiments, the schizophrenia
is selected from
paranoid, disorganised, catatonic, undifferentiated and residual
schizophrenia.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of schizophrenia in a subject as classified by the DMS-5
criteria listed
herein. These embodiments apply to the prevention, reduction or alleviation of
symptoms of other
30 schizophrenia spectrum and psychotic disorders. In certain embodiments,
the compositions of the
invention prevent, reduce or alleviate negative symptoms of schizophrenia. In
certain
embodiments, the compositions of the invention prevent, reduce or alleviate
positive symptoms
of schizophrenia. Positive symptoms are those that most people do not normally
experience, but
are present in people with schizophrenia, such as delusions, hallucinations,
disordered speech. In
certain embodiments, the compositions of the invention prevent, reduce or
alleviate negative and
positive symptoms of schizophrenia. Negative symptoms are deficits of normal
emotional
responses or of other thought processes, such as affective flattening and
catatonic behaviour. In
Date Recue/Date Received 2022-07-25
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31
preferred embodiments, the compositions of the invention prevent, reduce or
alleviate delusions,
hallucinations, disorganised speech, and disorganised or catatonic behaviours
in a subject with
schizophrenia. In preferred embodiments, the compositions of the invention
prevent, reduce or
alleviate affective flattening, restriction in the fluency and productivity of
thought and speech
and in the initiation of goal directed behaviour in a subject with
schizophrenia. In preferred
embodiments, the compositions of the invention prevent, reduce or alleviate
the cognitive defects
and/or mood disorders in a subject with schizophrenia.
In certain embodiments, the compositions of the invention reduce the
occurrence of positive
and/or negative symptoms of schizophrenia in a subject within a 6 month
period. In certain
embodiments, the compositions of the invention improve social and/or
occupational functionality
in a subject with schizophrenia spectrum or psychotic disorder.
In some embodiments, the compositions of the invention improve the symptoms of
schizophrenia
spectrum or psychotic disorders according to a symptomatic or diagnostic
scale. In certain
embodiments, the scale for assessing symptomatic improvement is the Positive
and Negative
Symptom Scale (PANSS) and Brief Psychiatric Rating Scale (BPRS). In certain
embodiments,
the Scale for Assessment of Negative Symptoms (SANS) is used.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social
and occupational impairment of the subject with schizophrenia spectrum or
psychotic disorders.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of schizophrenia spectrum or psychotic disorder. In certain
embodiments, the
comorbidity is/are: manic and/or depressive symptoms, anxiety or obsessive-
compulsive
symptoms, substance abuse and dependence, and personality disorder.
In certain embodiments, the compositions of the invention are for use in
treating or preventing
treatment resistant of refractory schizophrenia.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating schizophrenia spectrum or psychotic disorders when
used in combination
with another therapy for treating PTSD and stress disorders. In certain
embodiments, such
therapies include first generation antipsychotics including chlorpromazine,
fluphenazine,
haloperidol and/or perphenazine. In certain embodiments, such therapies
include second
generation therapies including aripiprazole (Abilify0); asenapine (Saphris0);
brexpiprazole
(Rexulti0); cariprazine (Vraylar0); clozapine (Clozari10); iloperidone
(Fanapt0); lurasidone
(Latuda0); olanzapine (Zyprexa0); paliperidone (Invega); quetiapine
(Seroque10); risperidone
(Risperda10); ziprasidone (Geodon0).
Date Recue/Date Received 2022-07-25
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32
Bipolar disorder
Bipolar disorder in general is a chronic disease. Mania is the cardinal
symptom of bipolar
disorder. There are several types of bipolar disorder based upon the specific
duration and pattern
of manic and depressive episodes. In DMS-5, a distinction is made between
bipolar I disorder,
bipolar II disorder, cyclothymic disorder, rapid-cycling bipolar disorder and
bipolar disorder
NOS.
According to the DSM, mania is a distinct period of abnormally and
persistently elevated,
expansive, or irritable mood. The episode must last a week, and the mood must
have at least three
of the following symptoms: high self-esteem; reduced need for sleep; increase
rate of speech;
rapid jumping of ideas; easily distracted; an increased interest in goals or
activities; psychomotor
agitation; increased pursuit of activities with a high risk of danger.
Bipolar I disorder involves one or more manic or mixed (mania and depression)
episodes and at
least one major depressive episode (see above for symptoms of MDD episodes).
Bipolar II
disorder has one or more major depressive episodes accompanied by at least one
hypomanic
episode. There are no manic or mixed episodes. Hypomania is a lesser form of
mania. The
symptoms are responsible for significant social, occupational and functional
impairments.
Cyclothymia is characterized by changing low-level depression along with
periods of hypomania.
The symptoms must be present for at least two years in adults or one year in
children before a
diagnosis can be made. Symptom free periods in adults and children last no
longer than two
months or one month, respectively. Rapid cycling bipolar disorder is a severe
form of bipolar
disorder. It occurs when a person has at least four episodes of major
depression, mania,
hypomania, or mixed states within a year. Not-otherwise specified (NOS)
bipolar disorder
classified bipolar symptoms that do not clearly fit into other types. NOS is
diagnosed when
multiple bipolar symptoms are present but not enough to meet the label for any
of the other
subtypes.
Bipolar disorder is associated with the following comorbidities: ADHD; anxiety
disorders;
substance disorders; obesity and metabolic syndrome.
Bipolar disorder is a psychiatric disorder that may develop or persist due to
dysfunction of the
microbiota-gut-brain axis. Therefore, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing bipolar disorder in a subject.
In certain
embodiments, the bipolar disorder is bipolar I disorder. In certain
embodiments, the bipolar
disorder is bipolar II disorder. In certain embodiments, the bipolar disorder
is cyclothymic
disorder. In certain embodiments, the bipolar disorder is rapid-cycling
bipolar disorder. In certain
embodiments, the bipolar disorder is bipolar disorder NOS.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of bipolar disorder in a subject. In certain embodiments,
the compositions
Date Recue/Date Received 2022-07-25
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33
of the invention prevent, reduce or alleviate the occurrence of manic episodes
in a subject. In
certain embodiments, the compositions of the invention prevent, reduce or
alleviate the
occurrence of an abnormally and persistently elevated, expansive, or irritable
mood. In certain
embodiments, the compositions of the invention prevent, reduce or alleviate
one or more of the
following symptoms: high self-esteem; reduced need for sleep; increase rate of
speech; rapid
jumping of ideas; easily distracted; an increased interest in goals or
activities; psychomotor
agitation; increased pursuit of activities with a high risk of danger. In
certain embodiments, the
compositions of the invention prevent, reduce or alleviate the occurrence of
one or more manic
or mixed episodes in a subject. In certain embodiments, the compositions of
the invention reduce
the occurrence of at least one major depressive episode in a subject. In
certain embodiments, the
compositions of the invention prevent, reduce or alleviate the occurrence of
at least one major
depressive episode accompanied by at least one hypomanic episode.
In preferred embodiments, the compositions of the invention treat the acute
phase of bipolar
disorder and/or prevent the occurrence of further episodes. In certain
embodiments, the
compositions of the invention treat the acute phase of manic/depressive
episodes in a subject with
bipolar disorder and prevent occurrence of further manic/depressive episodes.
In some embodiments, the compositions of the invention improve the symptoms of
bipolar
disorder according to a symptomatic or diagnostic scale. In certain
embodiments, the scale for
assessing symptomatic improvement of manic episodes is the Manic State Rating
Scale and the
Young Mania Rating Scale. In certain embodiments, the scale is the Bech-
Rafaelsen Mania Scale
(BRMAS). In certain embodiments, scales for assessing symptomatic improvement
of the switch
from manic to depressive episodes include the Hamilton Depression Rating
Scale, the
Montgomery-Asberg Rating Scale, and the Bech-Rafaelsen Depression Scale.
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social,
occupational and functional impairments of the subject with bipolar disorder.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one comorbidity of bipolar disorder. In certain embodiments, the comorbidity
is selected from
ADHD, anxiety disorders, substance disorder, obesity and metabolic syndrome.
In certain embodiments, the compositions of the invention are for use in
treating or preventing
manic-depressive illness and bipolar disorder unresponsive to lithium and
divalproex.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating bipolar disorder when used in combination with another
therapy for
treating bipolar disorder. In certain embodiments, such therapies include
lithium carbonate,
Date Recue/Date Received 2022-07-25
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34
anticonvulsant drugs (including valproate, divalproex, carbamazepine and
lamotrigine) and
antipsychotic drugs (including aripiprazole, olanzapine, quetiapine and
risperidone).
Neurocognitive disorders and Alzheimer's disease
In DSM-5, the term dementia was replaced with the terms major neurocognitive
disorder and
mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class
of psychiatric
diseases. The most common neurocognitive disorder is Alzheimer's disease,
followed by
vascular dementias or mixed forms of the two. Other forms of neurodegenerative
disorders (e.g.
Lewy body disease, frontotemporal dementia, Parkinson's dementia, Creutzfeldt-
Jakob disease,
Huntington's disease, and Wemicke-Korsakoff syndrome) are accompanied by
dementia.
The symptomatic criteria for dementia under DSM-5 are evidence of significant
cognitive decline
from a previous level of performance in one or more cognitive domains selected
from: learning
and memory; language; executive function; complex attention; perceptual-motor
and social
cognition. The cognitive deficits must interfere with independence in everyday
activities. In
addition, the cognitive deficits do not occur exclusively in the context of a
delirium and are not
better explained by another mental disorder (for example MDD or
schizophrenia).
In addition to the primary symptom, subjects with neurocognitive disorders
display behavioural
and psychiatric symptoms including agitation, aggression, depression, anxiety,
apathy, psychosis
and sleep-wake cycle disturbances.
Neurocognitive disorders are psychiatric disorders that may develop or persist
due to dysfunction
of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing neurocognitive disorders in a
subject. In preferred
embodiments, the neurocognitive disorder is Alzheimer's disease. In other
embodiments, the
neurocognitive disorder is selected from vascular dementias; mixed form
Alzheimer's disease
and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson's
dementia;
Creutzfeldt-Jakob disease; Huntington's disease; and Wemicke-Korsakoff
syndrome.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of neurocognitive disorders in a subject. In certain
embodiments, the
compositions of the invention prevent, reduce or alleviate the occurrence of
cognitive decline in
a subject. In certain embodiments, the compositions of the invention improve
the level of
performance of a subject with neurocognitive disorders in one or more
cognitive domains
selected from: learning and memory; language; executive function; complex
attention;
perceptual-motor and social cognition. In some embodiments, the compositions
of the invention
prevent, reduce or alleviate the occurrence of one or more behavioural and
psychiatric symptoms
associated with neurocognitive disorders selected from agitation, aggression,
depression, anxiety,
apathy, psychosis and sleep-wake cycle disturbances.
Date Recue/Date Received 2022-07-25
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In certain embodiments, the compositions of the invention prevent, reduce or
alleviate
symptomatic disease by intervention in suspected pathogenic mechanisms at a
preclinical stage.
In certain embodiments, the compositions of the invention improve disease
modification, with
slowing or arrest of symptom progression. In some embodiments, the slowing or
arrest of
5 symptom progression correlates with evidence in delaying the underlying
neuropathological
process. In preferred embodiments, the compositions of the invention improve
symptoms of
neurocognitive disorders comprising enhanced cognitive and functional
improvement. In
preferred embodiments, the compositions of the invention improve the
behavioural and
psychiatric symptoms of dementia (BPSD). In preferred embodiments, the
compositions of the
10 invention improve the ability of a subject with neurocognitive disorder
to undertake everyday
activities.
In preferred embodiments, the compositions of the invention improve both
cognition and
functioning in a subject with Alzheimer's disease. In some embodiments, the
composition of the
invention improve the cognitive endpoint in a subject with Alzheimer's
disease. In some
15 embodiments, the compositions of the invention improve the functional
endpoint in a subject
with Alzheimer's disease. In preferred embodiments, the compositions of the
invention improve
the cognitive and functional endpoint in a subject with Alzheimer's disease.
In yet further
preferred embodiments, the compositions of the invention improve the overall
clinical response
(the global endpoint) in a subject with Alzheimer's disease.
20 In some embodiments, the compositions of the invention improve the
symptoms of
neurocognitive disorders according to a symptomatic or diagnostic test. In
certain embodiments,
the tests for assessing symptomatic improvement of Alzheimer's disease (and
other
neurocognitive disorders) are selected from objective cognitive, activities of
daily living, global
assessment of change, health related quality of life tests and tests assessing
behavioural and
25 psychiatric symptoms of neurocognitive disorders.
In certain embodiments, the objective cognitive tests for assessment of
symptomatic
improvement use the Alzheimer's disease Assessment Scale cognitive subscale
(ADAS-cog) and
the classic ADAS scale. In certain embodiments, symptomatic improvement of
cognition is
assessed using the Neurophysiological Test Battery for Use in Alzheimer's
Disease (NTB).
30 In some embodiments, the global assessment of change test uses the
Clinical Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the global scale is the Clinician's Interview Based
Impression of Change plus
(CIBIC-plus). In some embodiments, the global scale is the Alzheimer's Disease
Cooperative
Study Unit Clinician's Global Impression of Change (ADCS-CGIC).
35 In certain embodiments, the health related quality of life measures are
the Alzheimer's Disease-
Related QOL (ADRQL) and the QOL-Alzheimer's Disease (QOL-AD).
Date Recue/Date Received 2022-07-25
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36
In certain embodiments, the tests assessing behavioural and psychiatric
symptoms of
neurocognitive disorders are selected from the Behavioural pathology in
Alzheimer's Disease
Rating Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD);
the
Neuropsychiatric Inventory (NPI); and the Cohen-Mansfield Agitation Inventory
(CMAI).
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating neurocognitive disorders when used in combination with
another therapy
for treating neurocognitive disorders. In certain embodiments, such therapies
include
acetylcholinesterase inhibitors including donepezil (Aricept0), galantamine
(Razadyne0) and
rivastigmine (Exelon C)), and memantine.
Parkinson's disease
Parkinson's disease is a common neurodegenerative disease neuropathologically
characterised
by degeneration of heterogeneous populations of neural cells (dopamine-
producing cells). The
clinical diagnosis of Parkinson's disease requires bradykinesia and at least
one of the following
core symptoms: resting tremor; muscle rigidity and postural reflex impahment.
Other signs and
symptoms that may be present or develop during the progression of the disease
are autonomic
disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances,
sexual functioning,
orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances
in the sense of smell
or sense of temperature. Depressive symptoms and cognitive dysfunction
comorbidities develop
in many Parkinson's disease patients, as well as neurocognitive disorders
related to Lewy Bodies.
Parkinson's disease is a psychiatric disorder that may develop or persist due
to dysfunction of the
microbiota-gut-brain axis. Therefore, in preferred embodiments, the
compositions of the
invention are for use in treating or preventing Parkinson's disease in a
subject.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the symptoms of Parkinson's disease in a subject. In preferred
embodiments, the
compositions of the invention prevent, reduce or alleviate one or more core
symptoms of
Parkinson's disease in a subject. In certain embodiments, the compositions of
the invention
prevent, reduce or alleviate bradykinesia in a subject. In certain
embodiments, the compositions
of the invention prevent, reduce or alleviate resting tremor; muscle rigidity
and/or postural reflex
impahment in a subject. In certain embodiments, the compositions of the
invention prevent,
reduce or alleviate one or more symptoms associated with Parkinson's disease
progression
selected from autonomic disturbances (sialorrhoea, seborrhoea, constipation,
micturition
disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis),
sleep disturbances and
disturbances in the sense of smell or sense of temperature.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate
depressive symptoms comorbid with Parkinson's disease. In certain embodiments,
the
compositions of the invention improve verbal memory and/or executive
functions. In certain
Date Recue/Date Received 2022-07-25
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37
embodiments, the compositions of the invention improve attention, working
memory, verbal
fluency and/or anxiety.
In other preferred embodiments, the compositions of the invention prevent,
reduce or alleviate
cognitive dysfunctions comorbid with Parkinson's disease.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate
Parkinson's disease progression. In certain embodiments, the compositions of
the invention
prevent, reduce or alleviate later motor complications. In certain
embodiments, the compositions
of the invention prevent, reduce or alleviate late motor fluctuations. In
certain embodiments, the
compositions of the invention prevent, reduce or alleviate neuronal loss. In
certain embodiments,
the compositions of the invention improve symptoms of Parkinson's disease
dementia (PDD). In
certain embodiments, the compositions of the invention prevent, reduce or
alleviate impaiiment
of executive function, attention and/or working memory. In certain
embodiments, the
compositions of the invention improve dopaminergic neurotransmission. In
certain
embodiments, the compositions of the invention prevent, reduce or alleviate
impaired
dopaminergic neurotransmission.
In some embodiments, the compositions of the invention improve the symptoms of
Parkinson's
disease according to a symptomatic or diagnostic scale. In certain
embodiments, the tests for
assessing symptomatic improvement of motor function in Parkinson's disease is
the Unified
Parkinson's Disease Rating Scale. In particular, UPDRS II considers the
activity of daily life and
UPDRS III considers motor-examination.
In some embodiments, the compositions of the invention improve the symptoms
associated the
PDD according to a symptomatic or diagnostic test and/or scale. In certain
embodiments, the test
or scale is selected from the Hopkins Verbal Learning Test ¨ Revised (HVLT-R);
the Delis-
Kaplan Executive Function System (D-KEFS) Color-Word Interference Test; the
Hamilton
Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating
Scale (HAM-
A; anxiety) and the Unified Parkinson's Disease Rating Scale (UPDRS; PD
symptom severity).
In some embodiments, the compositions of the invention improve the Clinical
Global Impression
¨ Global Improvement (CGI-I) scale for assessing psychiatric and neurological
disorders. In
some embodiments, the compositions of the invention display a positive effect
on global social
and occupational impaiiment of the subject with Parkinson's disease.
In some embodiments, the compositions of the invention are particularly
effective at preventing,
reducing or alleviating neurocognitive disorders when used in combination with
another therapy
for treating neurocognitive disorders. In certain embodiments, such therapies
include dopamine
agonists (including L-Dopa+); monoamine oxidase inhibitors, catecholamine-O-
methyl
transferase inhibitors; anticholinergics and glutamate modulators.
Date Recue/Date Received 2022-07-25
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38
Other central nervous system disorders
In preferred embodiments, the compositions of the invention are for use in
treating or preventing
a central nervous system disorder associated with dysfunction of the
microbiota-gut-brain axis.
In addition to the embodiments above, the compositions of the invention are
for use in treating
or preventing psychosis; chronic fatigue syndrome (myalgic encephalomyelitis)
and/or chronic
pain. In further embodiments, the compositions of the invention may be useful
for treating or
preventing motor neuron disease; Huntington's disease; Guillain-Barre syndrome
and/or
meningitis.
Neurochemical factors, neuropeptides and neurotransmitters and the microbiota-
gut-brain axis
As outlined above, the microbiota-gut-brain axis is modulated by a number of
different
physiological systems. The microbiota-gut-brain axis is modulated by a number
of signalling
molecules. Alterations in the levels of these signalling molecules results in
defects in central
nervous system development and/or functionality. Indeed, many of the molecules
disclosed in
this section have been implicated in the functionality of the microbiota-gut-
brain axis and the
pathogenesis of central nervous system disorders or conditions ([331, [9],
[32], [341). The
experiments perfonned by the inventors indicate that behavioural changes can
be triggered by
administration of Enterococcus durans. This effect may be mediated by an
effect on levels of the
signalling molecules, in particular those listed in this section. These
alterations may be
responsible for the therapeutic benefits associated with Enterococcus durans.
Accordingly, due
to the fact that the central nervous system disorders and conditions disclosed
herein display a
similar fundamental biochemical and physiological pathogenesis (i.e. via the
microbiota-gut-
brain axis), a similar therapeutic benefit of Enterococcus durans may be also
achieved for these
disorders and conditions.
The signalling of the microbiota-gut-brain axis is modulated by levels of
neurochemical factors,
neuropeptides and neurotransmitters. Accordingly, in certain embodiments, the
compositions of
the invention modulates levels of neurochemical factors, neuropeptides and
neurotransmitters.
Accordingly, in certain preferred embodiments, the compositions of the
invention directly alter
CNS biochemistry. In preferred embodiments, the compositions of the invention
modulate the
levels of brain-derived neurotrophic factor (BDNF). In certain embodiments,
the compositions
of the invention modulate the levels of monoamines. In certain embodiments,
the monoamines
are serotonin (5-hydroxytryptamine (5-HT)), dopamine, norepinephrine and/or
epinephrine. In
certain embodiments, the monoamines are catecholamines. In certain
embodiments, the
catecholamines are dopamine, norepinephrine and epinephrine. In certain
embodiments, the
monoamines are tryptamines. In certain embodiments, the tryptamines are
serotonin and
melatonin. In certain embodiments, the compositions of the invention modulate
the levels of
acetylcholine.
Date Recue/Date Received 2022-07-25
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39
In certain preferred embodiments, the compositions of the invention modulate
the levels of
oxytoxin. Oxytocin is associated with emotional, social, cognitive and
neuroendocrine
physiologies as well as autoregulation. In particular, oxytocin release is
involved in anxiolysis;
positive mood; maternal behaviour, pair bonding; sexual behaviour; social
memory; olfactory
memory; anorexiant effects; attenuation of the HPA axis response to stress;
autoexcitation during
birth and suckling as well as other physiological and psychological processes.
In certain
embodiments, the compositions of the invention increase the levels of
oxytocin. In certain
embodiments, the compositions of the invention decrease the levels of
oxytocin. In certain
embodiments, the compositions of the invention increase or decrease oxytocin
signalling. In
certain embodiments, the compositions of the invention modulate the levels of
oxytocin
receptors. In certain embodiments, the compositions of the invention modulate
the flux of
calcium ions into or out of neuronal, muscle and gastrointestinal cells. In
preferred embodiments,
the compositions of the invention treat and prevent neurodevelopmental and
neuropsychiatric
disorders and diseases associated with the microbiota-gut-brain axis by
modulating the levels of
oxytocin.
In certain embodiments, the compositions of the invention modulate the levels
of brain
monoamines and metabolites thereof. In preferred embodiments, the monoamine is
serotonin. In
certain embodiments, the compositions of the invention modulate the
serotonergic and/or
kynurenine routes of tryptophan metabolism. In certain embodiments, the
compositions of the
invention modulate the levels of serotonin metabolites, such as 5-
Hydroxyindoleacetic acid (5-
HIAA). In certain embodiments, the compositions of the invention modulate the
levels of
dopamine metabolites, such as Homovanillic acid (HVA). Modulation of these
neurotransmitters
and neurochemical factors is useful for treating stress, depression and
anxiety-related disorders.
The signalling of the microbiota-gut-brain axis is modulated by levels of y-
aminobutyric acid
(GABA). Accordingly, in preferred embodiments, the compositions of the
invention modulate
the levels of GABA. GABA is an inhibitory neurotransmitter that reduces
neuronal excitability.
In certain embodiments, the compositions of the invention increase the levels
of GABA. In
certain embodiments, the compositions of the invention decrease the levels of
GABA. In certain
embodiments, the compositions of the invention alter GABAergic
neurotransmission. In certain
.. embodiments, the compositions of the invention modulate the level of GABA
transcription in
different regions of the central nervous system. In certain embodiments, the
commensal derived
GABA crosses the blood-brain barrier and affects neurotransmission directly.
In certain
embodiments, the compositions of the invention lead to a reduction of GABA in
the
hippocampus, amygdala and/or locus coeruleus. In certain embodiments, the
compositions of the
invention lead to an increase of GABA in cortical regions.
Compositions which modulate the levels of GABA have been found to be useful in
the treatment
of epilepsy. Consequently, in some embodiments, the compositions of the
invention are for use
Date Recue/Date Received 2022-07-25
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in the treatment of epilepsy. In some embodiments, the treatment reduces the
frequency and/or
the intensity of epileptic seizures in a subject. The frequency and/or
intensity of epileptic seizures
can vary greatly in different subjects. In some embodiments, the treatment
comprises reducing
the frequency and/or intensity of seizures by at least 20%, at least 30%, at
least 40%, at least
5 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, or at least
99%.
Modes for measuring the reduction in frequency and/or intensity of epileptic
seizures are known
in the art. For example, the intensity of a seizure may be assessed using the
Chalfont Seizure
Severity Scale.
10 In some subjects it may be appropriate to measure the reduction in
frequency and/or intensity
over the course of one day. In some subjects it may be appropriate to measure
the reduction in
frequency and/or intensity over either a day or a longer period, e.g. two
days, a week, a month,
six months, a year or longer. Thus, in some embodiments, the percentage
reduction in the
frequency of the seizures is measured over a period of one day, two days,
three days, a week, two
15 weeks, a month, three months, six months, a year, two years or longer
than two years. In some
embodiments, the treatment comprises reducing the frequency and/or intensity
of epileptic
seizures whilst the subject is awake. In some embodiments, the treatment
comprises reducing
the frequency and/or intensity of epileptic seizures whilst the subject is
asleep. In some
embodiments, the treatment comprises reducing the frequency and/or intensity
of epileptic
20 seizures whilst the subject is awake but not whilst the subject is
asleep, or whilst the subject is
asleep but not whilst the subject is awake. In some embodiments, the treatment
comprises
eliminating epileptic seizures. In some embodiments, the frequency of
epileptic seizures is
reduced to once a day or less, once every two days or less, once every week or
less, once every
two weeks or less, once a month or less, once every three months or less, once
every six months
25 .. or less, once a year or less, once every two years or less, or once
every three years or less. In
some embodiments, the treatment results in the subject experiencing an
epileptic seizure free
period of at least a day, at least two days, at least a week, at least a
month, at least three months,
at least six months, at least a year, at least two years, or at least three
years.
In some embodiments, the compositions of the invention are particularly
effective at treating
30 epilepsy when used in combination with another therapy for treating
epilepsy. In some
embodiments, the other therapy for use in combination with the composition of
the invention is
selected from one or more of: Acetazolamide, Brivaracetam, Carbamazepine,
Clobazam,
Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Gabapentin,
Lacosamide,
Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital,
Phenytoin, Piracetam,
35 Pregabalin, Primidone, Rufinamide, Sodium valproate, Stiripentol,
Tiagabine, Topiramate,
Valproic acid, Vigabatrin, and Zonisamide.
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41
In some embodiments, the treatment results in the subject being able to reduce
the dose or stop
taking one or more other anti-epileptic drugs without seeing a deterioration
in symptoms. For
example, in some embodiments, the dose of a GABA-mediator anti-epileptic drug
may be
reduced or the GABA-mediator anti-epileptic drug may no longer be taken. In
some
embodiments, the GABA-mediator anti-epileptic drug is selected from valproic
acid, sodium
valproate and pregabalin.
The levels of neuroactive molecules, such as serotonin, melatonin, GABA,
histamines and
acetylcholine are linked to the pathophysiology of central nervous system
diseases such as
dementia, Alzheimer's disease, Huntington's disease and epilepsy.
The signalling of the microbiota-gut-brain axis is modulated by levels of
histamines.
Accordingly, in certain embodiments, the compositions of the invention
modulate the levels of
histamines. In certain embodiments, the histamines has an immunoregulatory
effect. In certain
embodiments, histamine levels enable translocation of bacteria from the lumen
into systemic
circulation. Therefore, in some embodiments, the compositions of the invention
alter
gastrointestinal tract peimeability and/or barrier function. In certain other
embodiments, the
histamine acts as a neurotransmitter linked to central processes.
The signalling of the microbiota-gut-brain axis is modulated by the HPA axis.
Accordingly, in
certain embodiments, the compositions of the invention modulate HPA activity.
In certain
embodiments, the compositions of the invention attenuate the HPA stress
response. In certain
preferred embodiments, the compositions of the invention modulate inflammatory
responses
associated with HPA activity. In certain embodiments, the compositions of the
invention
modulate the levels of glucocorticoids. In certain preferred embodiments, the
compositions of
the invention modulate the levels of corticosterone and adrenaline. In certain
embodiments, the
compositions of the invention modulate the levels of corticotrophin-releasing
factor and/or
vasopressin. In certain embodiments, the compositions of the invention
modulate the levels of
vasopressin and/or other neurohypophysial or antidiuretic hoimones.
Alterations in HPA axis
activity are associated with anxiety and stress disorders.
The signalling of the microbiota-gut-brain axis is modulated by alterations in
the immune
response and inflammatory factors and markers. Accordingly, in certain
embodiments, the
compositions of the invention may modulate the immune response. In certain
embodiments, the
compositions of the invention modulate the systemic levels of circulating
neuroimmune
signalling molecules. In certain preferred embodiments, the compositions of
the invention
modulate pro-inflammatory cytokine production and inflammation. In certain
embodiments, the
compositions of the invention modulate the inflammatory state. In certain
embodiments, the
compositions of the invention modulate the splenocyte proliferative response.
In certain
embodiments, the compositions of the invention modulate the systemic and/or
plasma levels of
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42
C-reactive protein; IL-1 family cytokines; IL-1f3; IL-2; IL-4; IL-6; IL-8; IL-
10; IL-12p40; IL-17;
IL-17A; IL-21; IL-23; TNF- a and IFN-y. In some embodiments the compositions
of the
invention module the levels of anti-inflammatory cytokines, for example IL-10.
In preferred
embodiments, the compositions of the invention increase the levels of IL-10.
In some
embodiments, the compositions of the invention modulate the levels of TNF-a.
In preferred
embodiments, the compositions of the invention modulate the levels of IFN-y.
In some
embodiments, the compositions of the invention modulate the IFN-y:IL-10 ratio.
In certain
preferred embodiments, the compositions of the invention decrease the IFN-y:IL-
10 ratio. In
preferred embodiments, the compositions of the invention decrease the levels
of the pro-
inflammatory cytokines TNF-a and IFN-y. Increased circulating levels of
cytokines are closely
associated with various neuropsychiatric disorders, including depression,
anxiety, schizophrenia
and ASD. Evidence of inflammatory state alteration is highlighted in disorders
such as
schizophrenia, major depressive disorder and bipolar disorder.
In certain embodiments, the compositions of the invention modulates the levels
of tolerance-
mediating dendritic cells and reciprocally regulate pro and anti-inflammatory
cytokine responses.
In certain embodiments, the compositions of the invention decrease the
systemic level of
myeloperoxidase (a marker for inflammation and oxidation).Therapeutic
modulators of the
immune system and of inflammatory responses are useful for treating autism
spectrum disorders
and mood disorders.
In certain embodiments, the compositions of the invention modulate the immune
response to an
infection or vaccination. In certain embodiments, the compositions of the
invention modulate the
level of inflammation in response to infection or vaccination. In certain
preferred embodiments,
the compositions of the invention modulate maternal immune activation in
response to an
infection or vaccination during pregnancy. Accordingly, the compositions of
the invention can
.. be administered during pregnancy in order to treat or prevent a central
nervous system disorder
in the offspring.
The signalling of the microbiota-gut-brain axis is modulated by levels
commensal metabolites.
Accordingly, in certain embodiments, the compositions of the invention
modulate the systemic
levels of microbiota metabolites. In certain preferred embodiments, the
compositions of the
invention modulate the level of short chain fatty acids (SCFAs). In certain
embodiments the level
of SCFAs is increased or decreased. In some embodiments, the SCFA is butyric
acid (BA) (or
butyrate). In some embodiments, the SCFA is propionic acid (PPA). In some
embodiments, the
SCFA is acetic acid. In certain embodiments, the compositions of the invention
modulate the
ability of SCFAs to cross the blood-brain barrier. In certain embodiments, the
compositions of
the invention modulate the level of Polysaccharide A (PSA). In certain
embodiments, the
compositions of the invention modulate the levels of the potent pro-
inflammatory endotoxin
lipopolysaccharide (LPS). LPS leads to the production of inflammatory
cytokines that alter
Date Recue/Date Received 2022-07-25
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43
physiological brain activity and modulate neuropeptide synthesis. LPS has an
important influence
on the modulation of the CNS, increasing the activity of areas devoted to the
control of emotions
(e.g. the amygdala). In certain embodiments, the compositions of the invention
modulate the level
of tryptophan and/or its metabolites. In certain embodiments, the compositions
of the invention
modulate the levels of 4-ethylphenylsulphate (4EPS; a uremic toxic associated
with ASD-related
behavioural abnounalities). In preferred embodiments, the compositions of the
invention
decrease the levels of 4-ethylphenylsulphate in a subject. The signals
generated by the stimulation
of neuronal signalling pathways caused by intraluminal gut stimuli strongly
modulate brain
activity, including pain perception, immune-response modulation, emotional
control and other
homeostatic functions. Accordingly, a composition able to modulate levels of
these factors would
have broad therapeutic applications for treating or preventing CNS disorders.
The signalling of the microbiota-gut-brain axis is modulated by levels
gastrointestinal
permeability. Accordingly, in some embodiments, the compositions of the
invention alter the
integrity of the gastrointestinal tract epithelium. In certain embodiments,
the compositions of the
invention modulate the penneability of the gastrointestinal tract. In certain
embodiments, the
compositions of the invention modulate the barrier function and integrity of
the gastrointestinal
tract. In certain embodiments, the compositions of the invention modulate
gastrointestinal tract
motility. In certain embodiments, the compositions of the invention modulate
the translocation
of commensal metabolites and inflammatory signalling molecules into the
bloodstream from the
gastrointestinal tract lumen.
The signalling of the microbiota-gut-brain axis is modulated by microbiome
composition in the
gastrointestinal tract. Accordingly, in certain embodiments, the compositions
of the invention
modulates the microbiome composition of the gastrointestinal tract. In certain
embodiments, the
compositions of the invention prevents microbiome dysbiosis and associated
increases in toxic
metabolites (e.g. LPS). In certain embodiments, the compositions of the
invention modulate the
levels of Clostridium in the gastrointestinal tract. In preferred embodiments,
the compositions of
the invention reduce the level of Clostridium in the gastrointestinal tract.
In certain embodiments,
the compositions of the invention reduce the levels of Campylobacter jejuni.
In certain
embodiments, the compositions of the invention modulate the proliferation of
hannful anaerobic
bacteria and the production of neurotoxins produced by these bacteria. In
certain embodiments,
the compositions of the invention modulate the microbiome levels of
Lactobacillus and/or
Bifidobacterium. In certain embodiments, the compositions of the invention
modulate the
microbiome levels of Sutterella, Prevotella, Ruminoccucs genera and/or the
Alcaligenaceae
family. In certain embodiments, the compositions of the invention increase the
level of
Lactobacillus plantarum and/or Saccharomyces boulardii.
In certain embodiments, the compositions of the invention prevent the
dysregulation of the
composition of the microbiome by extensive antibiotic use. In certain
preferred embodiments,
Date Recue/Date Received 2022-07-25
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44
the compositions of the invention maintain a functional maternal microbiome
composition upon
administration of antibiotics during pregnancy. Accordingly, the compositions
of the invention
can be administered during pregnancy in order to treat or prevent a central
nervous system
disorder in the offspring.
Modulation of the microbiome has been shown to be effective at improving
psychiatric disorder-
related behaviours, including anxiety, depression, autism spectrum disorder,
obsessive-
compulsive disorder and memory abilities (including spatial and non-spatial
memory), as well as
other CNS-related disorders including Parkinson's disease. Certain studies
have suggested that
probiotics can reduce psychological stress, somatisation, depression and anger-
hostility. The
levels of Lactobacillus are associated with depression and have been
implicated in pain signalling
associated with gastrointestinal discomfort.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate at least
one of the behavioural symptoms associated with a central nervous system
disorder described
herein. In preferred embodiments, the compositions of the invention improve
the overall clinical
.. response in a subject.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate
stereotyped, repetitive behaviour in a subject. In preferred embodiments, the
compositions of the
invention prevent, reduce or alleviate the occurrence of unusually restrictive
behaviours and/or
interests. In certain embodiments, the compositions of the invention prevent,
reduce or alleviate
recurrent obsessions and/or compulsions in a subject. In preferred
embodiments, the
compositions of the invention prevent, reduce or alleviate deficits in social
behaviour in a subject.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate
avoidance behaviour in a subject. In preferred embodiments, the compositions
of the invention
prevent, reduce or alleviate deficits in communication behaviour in a subject.
In preferred
embodiments, the compositions of the invention prevent, reduce or alleviate
neophobia and / or
sociability deficit.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate negative
alterations in cognitions and mood in a subject. In preferred embodiments, the
compositions of
the invention prevent, reduce or alleviate anxiety-related behaviour in a
subject. In preferred
embodiments, the compositions of the invention prevent, reduce or alleviate
stress-related
behaviour in a subject. In preferred embodiments, the compositions of the
invention prevent,
reduce or alleviate depression-related behaviour in a subject. In preferred
embodiments, the
compositions of the invention prevent, reduce or alleviate aggressive
behaviour in a subject. In
preferred embodiments, the compositions of the invention prevent, reduce or
alleviate the
occurrence of an abnomially and persistently elevated, expansive, or irritable
mood.
Date Recue/Date Received 2022-07-25
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In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate intrusive
thoughts in a subject. In preferred embodiments, the compositions of the
invention prevent
alterations in arousal and reactivity in a subject. In preferred embodiments,
the compositions of
the invention prevent, reduce or alleviate delusions, hallucinations,
disorganised speech, and
5 disorganised or catatonic behaviours in a subject. In preferred
embodiments, the compositions of
the invention prevent, reduce or alleviate affective flattening, restriction
in the fluency and
productivity of thought and speech and in the initiation of goal directed
behaviour in a subject.
In preferred embodiments, the compositions of the invention prevent, reduce or
alleviate one or
more of the following symptoms: despair, helplessness, high self-esteem;
reduced need for sleep;
10 increase rate of speech; rapid jumping of ideas; easily distracted; an
increased interest in goals or
activities; psychomotor agitation; increased pursuit of activities with a high
risk of danger.
In preferred embodiments, the compositions of the invention improve memory
function, for
example spatial and/or non-spatial memory deficits in a subject. In preferred
embodiments, the
compositions of the invention improve learning in a subject. In preferred
embodiments, the
15 compositions of the invention improve both cognition and functioning in
a subject. In preferred
embodiments, the compositions of the invention improve locomotor activity in a
subject. In
preferred embodiments, the compositions of the invention prevent, reduce or
alleviate
bradykinesia in a subject. In preferred embodiments, the compositions of the
invention prevent,
reduce or alleviate resting tremor; muscle rigidity and/or postural reflex
impairment in a subject.
20 In preferred embodiments, the compositions of the invention prevent,
reduce or alleviate at least
one comorbidity associated with a CNS disorder disclosed herein.
In preferred embodiments, the compositions of the invention improve the scores
of a subject on
at least one of the symptomatic and/or diagnostic scales for CNS disorders
described herein. In
certain other embodiments, the symptomatic and/or diagnostic scale is selected
from the General
25 Health Questionnaire (GHQ); the Depression Anxiety and Stress Scale (DAS
S); the Leiden Index
of Depression Sensitivity-Revised (LEIDS-r); the Positive and Negative Symptom
Scale
(PANSS); the State-Trait Anxiety Inventory (STAI); the Development Behavior
Checklist
(DBC); the Beck Depression Inventory (BDI); the Beck Anxiety Inventory (BAT);
the Hopkins
Symptom Checklist (HSCL-90); the Hospital Anxiety and Depression Scale (HADS);
the
30 Perceived Stress Scale (PSS); the Coping Checklist (CCL) (also used to
counter the stress of
daily life); and the questionnaire-based Profile of Mood State (POMS).
In certain embodiments, the compositions of the invention may improve the
symptomatic and/or
diagnostic scale when assessing therapeutic efficacy in other animal models of
CNS disorders
known to a person skilled in the art. In addition to the behavioural assays
disclosed in the
35 examples, the compositions of the invention may improve reciprocal
social interactions; olfactory
communication; ultrasonic vocalisation; motor stereotypes (such as circling
and vertical
Date Recue/Date Received 2022-07-25
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46
jumping), repetitive behaviour such as self-grooming and diffing; and
perseverance in spatial
tasks.
In addition, the compositions of the invention will be useful in treating
and/or preventing CNS
disorders in other animal models of CNS disorders. Other mouse models include
inbred mice
strains (including BALB/cJ and C58/J) and also genetically modified mice
strains (including
NEUREXIN1, NEUROLIGIN3, NEUROLIGIN4, SHANK2, SHANK3, CNTNAP2, Tsc1/2 and
Fmrl gene mutant mice strains).
In certain embodiments, the compositions of the invention improve social
behaviour of a subject.
In preferred embodiments, the compositions of the invention improve the
recognition of social
novelty in a subject. In preferred embodiments, the compositions of the
invention improve
sociability deficit. In preferred embodiments, the compositions of the
invention improve the
ability to discriminate between familiar and novel objects and familiar and
novel subjects. In
preferred embodiments, the composition of the invention improve ability to
recognise other
subjects.
In certain embodiments, the compositions of the invention regulate plasma
levels of amino acids.
In certain embodiments, the compositions of the invention regulate the
biosynthesis or catabolism
of amino acids. In preferred embodiments, the compositions of the invention
regulate plasma
levels of proline. In preferred embodiments, the compositions of the invention
reduce the plasma
levels of proline. Elevated proline is known to negatively affect brain
function by an increase in
dopamine in the prefrontal cortex [35]. In addition, proline is considered to
be a neurotransmitter
that modulates glutamatergic neurotransmission in the hippocampus, and
neurotransmission
elsewhere in the brain. Accordingly, proline has been implicated in CNS
disorders and
psychiatric disorders, in particular psychosis. In preferred embodiments, the
reduction in plasma
levels of proline treats or prevents CNS disorders, in particular, ADHD, OCD,
mood disorders,
autism spectrum disorder, psychosis and schizophrenia.
In certain embodiments, the compositions of the invention prevent, reduce or
alleviate the
symptoms of psychiatric disorders, for example schizophrenia and bipolar
disorder, associated
with 22q11.2 deletion syndrome (22q1 IDS) [35]. In certain embodiments, the
compositions of
the invention improve the social behavioural and social cognitive problems in
subjects with
22q1 IDS. In preferred embodiments, the compositions of the invention modulate
the associated
cognitive and behavioural outcomes in 22q11DS subjects. In preferred
embodiments, the
modulation of these outcomes is a consequence of reduced plasma levels of
proline. In certain
embodiments, the compositions of the invention modulate the activity of
proline hydrogenase.
In certain embodiments, the compositions of the invention modulate the levels
of NMDA
receptors and/or the subunits thereof. In preferred embodiments, the
compositions of the
invention modulate the levels of the NMDA receptor 2B. In certain embodiments,
the
Date Recue/Date Received 2022-07-25
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47
compositions of the invention increase the levels of the NMDA receptor 2B. In
preferred
embodiments, the compositions of the invention decrease the levels of the NMDA
receptor 2B.
Dysregulation of NMDA receptors have been associated with CNS disorders, in
particular ASD
and schizophrenia. There have been suggestions that NMDA receptor antagonists
may be
effective in treating ASD [36]. In addition, suppression of NMDA receptor
function has been
demonstrated to improve social deficits and reduce repetitive behaviour in
valproic acid induced
models of ASD [37]. In certain embodiments, the compositions of the invention
cause
hypofunction of the NMDA receptor 2B. In certain embodiments, the compositions
of the
invention cause hyperfunction of the NMDA receptor 2B. In certain embodiments,
the
compositions of the invention prevent, reduce or alleviate the symptoms of CNS
disorders, for
example ASD or schizophrenia as a consequence of the modulation of NMDA
receptor 2B
activity. In preferred embodiments, the compositions of the inventions
suppress NMDA receptor
activity and reduce social deficits and stereotypical behaviour in subjects
with CNS disorders.
In certain embodiments, the compositions of the invention modulate the levels
of BDNF. In
preferred embodiments, the compositions of the invention reduce the levels of
BDNF. In certain
embodiments, the reduction in BDNF is localised to the amygdala. Meta-analyses
of ASD
populations have shown that higher levels of BDNF are detected in ASD subjects
compared to
controls [38]. In preferred embodiments, the compositions of the invention
prevent, reduce or
alleviate the symptoms of CNS disorders, in particular ASD, as a consequence
of the reduction
in levels of BDNF. Altered levels of BDNF have been associated with a number
of
neurodevelopmental disorders, as well as psychosis and schizophrenia. In
certain embodiments,
the compositions of the invention modulate levels of BDNF in order to prevent,
reduce or
alleviate the symptoms of neurodevelopmental and psychiatric disorders.
In certain embodiments, the compositions of the invention modulate the levels
of inflammatory
markers produced in response to an antigen challenge. In preferred
embodiments, the
compositions of the invention increase the levels of IL-1f3 in response to a
viral antigen challenge.
In certain embodiments, the compositions of the invention modulate the innate
immune response.
In certain embodiments, the compositions of the invention modulate the
adaptive immune
response. In certain embodiments, the compositions of the invention modulate
the inflammatory
response.
Modes of administration
Preferably, the compositions disclosed herein are to be administered to the
gastrointestinal tract
in order to enable delivery to and / or partial or total colonisation of the
intestine with the bacterial
strain of the invention. In other words, the bacteria may have colonised some
or all of the
gastrointestinal tract and / or such colonisation may be transient or
peimanent.
Date Recue/Date Received 2022-07-25
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48
More specifically, in some embodiments, the "total colonisation of the
intestine" means that
bacteria have colonised all parts of the intestine (i.e. the small intestine,
large intestine and
rectum). Additionally or alternatively, the tenn "total colonisation" means
that the bacteria
engraft permanently in the some or all parts of the intestine.
In some embodiments, "partial colonisation of the intestine" means that
bacteria have colonised
some but not all parts of the intestine. Additionally or alternatively, the
term "partial
colonisation" means that the bacteria engraft transiently in some or all parts
of the intestine.
The transience of engraftment can be detennined by assessing (e.g. in a fecal
sample) the
abundance of the bacterial strain of the invention periodically (e.g. daily)
following the end of a
dosing interval to detennine the washout period, i.e. the period between
conclusion of the dosing
interval and there being no detectable levels of the bacterial strain of the
invention present. In
embodiments of the invention, the washout period is 14 days or less, 12 days
or less, 10 days or
less, 7 days or less, 4 days or less, 3 days or less, 2 days or less or 1 day
or less.
In embodiments of the invention, the bacteria of the present invention engraft
transiently in the
large intestine.
Generally, the compositions of the invention are administered orally, but they
may be
administered rectally, intranasally, or via buccal or sublingual routes.
In some embodiments, the compositions of the invention are administered
orally. Oral
administration may involve swallowing, so that the compound enters the
gastrointestinal tract.
Phannaceutical founulations suitable for oral administration include solid
plugs, solid
microparticulates, semi-solid and liquid (including multiple phases or
dispersed systems) such as
tablets; soft or hard capsules containing multi- or nano-particulates, liquids
(e.g. aqueous
solutions), emulsions or powders; lozenges (including liquid-filled); chews;
gels; fast dispersing
dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
In certain embodiments, the compositions of the invention may be administered
as a foam, as a
spray or a gel.
In certain embodiments, the compositions of the invention may be administered
as a suppository,
such as a rectal suppository, for example in the foun of a theobroma oil
(cocoa butter), synthetic
hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or
soap glycerin
composition.
In certain embodiments, the composition of the invention is administered to
the gastrointestinal
tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube,
jejunostomy tube (J tube),
percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall
port that provides
access to the stomach, jejunum and other suitable access ports.
Date Recue/Date Received 2022-07-25
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49
The compositions of the invention may be administered once, or they may be
administered
sequentially as part of a treatment regimen. In certain embodiments, the
compositions of the
invention are to be administered daily (either once or several times).
In certain embodiments, the compositions of the invention are administered
regularly, such as
daily, every two days, or weekly, for an extended period of time, such as for
at least one week,
two weeks, one month, two months, six months, or one year.
In some embodiments the compositions of the invention are administered for 7
days, 14 days, 16
days, 21 days or 28 days or no more than 7 days, 14 days, 16 days, 21 days or
28 days. For
example, in some embodiments the compositions of the invention are
administered for 16 days.
In certain embodiments of the invention, treatment according to the invention
is accompanied by
assessment of the patient's gut microbiota. Treatment may be repeated if
delivery of and / or
partial or total colonisation with the bacterial strain is not achieved such
that efficacy is not
observed, or treatment may be ceased if delivery and / or partial or total
colonisation is successful
and efficacy is observed.
In certain embodiments, the composition of the invention may be administered
to a pregnant
animal, for example a mammal such as a human in order to prevent an
inflammatory or
autoimmune disease developing in her child in utero and / or after it is born.
The compositions of the invention may be administered to a patient that has
been diagnosed with
a central nervous system disorder or condition, in particular a central
nervous system disorder or
condition mediated by the microbiota-gut-brain axis, or that has been
identified as being at risk
of a central nervous system disorder or condition, in particular central
nervous system disorder
or condition mediated by the microbiota-gut-brain axis. The compositions may
also be
administered as a prophylactic measure to prevent the development of central
nervous system
disorders or conditions, in particular central nervous system disorders or
conditions mediated by
the microbiota-gut-brain axis in a healthy patient.
The compositions of the invention may be administered to a patient that has
been identified as
having an abnoimal gut microbiota. For example, the patient may have reduced
or absent
colonisation by Enterococcus durans.
The compositions of the invention may be administered as a food product, such
as a nutritional
supplement.
Generally, the compositions of the invention are for the treatment or
prevention of disease in
humans, although they may be used to treat or prevent disease in animals
including monogastric
mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions
of the invention
may be useful for enhancing the growth and perfoimance of animals. If
administered to animals,
oral gavage may be used.
Date Recue/Date Received 2022-07-25
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In some embodiments, the subject to whom the composition is to be administered
is a human.
The human may be an adult, a child or an infant.
Compositions
The compositions of the invention comprise bacteria. In preferred embodiments
of the invention,
5 the composition is formulated in freeze-dried fonn. The composition of
the invention may
comprise granules or gelatin capsules, for example hard gelatin capsules,
comprising a bacterial
strain of the invention.
Preferably, the composition of the invention comprises lyophilised bacteria.
Lyophilisation of
bacteria is a well-established procedure and relevant guidance is available
in, for example,
10 references [39-41].
Alternatively, the composition of the invention may comprise a live, active
bacterial culture.
In some embodiments, the bacterial strain in the composition of the invention
has not been
inactivated, for example, has not been heat-inactivated. In some embodiments,
the bacterial
strain in the composition of the invention has not been killed, for example,
has not been heat-
15 killed. In some embodiments, the bacterial strain in the composition of
the invention has not
been attenuated, for example, has not been heat-attenuated. For example, in
some embodiments,
the bacterial strain in the composition of the invention has not been killed,
inactivated and/or
attenuated. For example, in some embodiments, the bacterial strain in the
composition of the
invention is live. For example, in some embodiments, the bacterial strain in
the composition of
20 the invention is viable. For example, in some embodiments, the bacterial
strain in the composition
of the invention is capable of partially or totally colonising the intestine.
For example, in some
embodiments, the bacterial strain in the composition of the invention is
viable and capable of
partially or totally colonising the intestine.
In some embodiments, the composition comprises a mixture of live bacterial
strains and bacterial
25 strains that have been killed.
In preferred embodiments, the composition of the invention is encapsulated to
enable delivery of
the bacterial strain to the intestine. Encapsulation protects the composition
from degradation until
delivery at the target location through, for example, rupturing with chemical
or physical stimuli
such as pressure, enzymatic activity, or physical disintegration, which may be
triggered by
30 changes in pH. Any appropriate encapsulation method may be used.
Exemplary encapsulation
techniques include entrapment within a porous matrix, attachment or adsorption
on solid carrier
surfaces, self-aggregation by flocculation or with cross-linking agents, and
mechanical
containment behind a microporous membrane or a microcapsule. Guidance on
encapsulation that
may be useful for preparing compositions of the invention is available in, for
example, references
35 [42-43].
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51
The composition may be administered orally and may be in the form of a tablet,
capsule or
powder. Encapsulated products are preferred.
A composition of the invention includes a therapeutically effective amount of
a bacterial strain
of the invention. A therapeutically effective amount of a bacterial strain is
sufficient to exert a
beneficial effect upon a patient. A therapeutically effective amount of a
bacterial strain may be
sufficient to result in delivery to and / or partial or total colonisation of
the patient's intestine.
A suitable daily dose of the bacteria, for example for an adult human, may be
from about 1 x 103
to about 1 x 10" colony forming units (CFU); for example, from about 1 x 10 to
about 1 x 101
CFU; in another example from about 1 x 106 to about 1 x 101 CFU; in another
example from
about 1 x 10' to about 1 x 10" CFU; in another example from about 1 x 108 to
about 1 x 101
CFU; in another example from about 1 x 108 to about 1 x 10" CFU.
In certain embodiments, the dose of the bacteria is at least 109 cells per
day, such as at least 1010
,
at least 1011, or at least 1012 cells per day.
A dose of the composition may comprise the bacterial strain from about 1 x 106
to about 1 x 10"
colony forming units (CFU) /g, respect to the weight of the composition. The
dose may be
suitable for an adult human. For example, the composition may comprise the
bacterial strain from
about 1 x 103 to about 1 x 10" CFU/g; for example, from about 1 x 10' to about
1 x 101 CFU/g;
in another example from about 1 x 106 to about 1 x 101 CFU/g; in another
example from about
1 x 10' to about 1 x 10" CFU/g; in another example from about 1 x 108 to about
1 x 101 CFU/g;
in another example from about 1 x 108 to about 1 x 10" CFU/g, from about 1 x
108 to about 1 x
101 CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.
The composition may be formulated as a probiotic. A probiotic is defined by
the FAO/WHO as
a live microorganism that, when administered in adequate amounts, confers a
health benefit on
the host.
Typically, a probiotic, such as the composition of the invention, is
optionally combined with at
least one suitable prebiotic compound. In certain embodiments, the probiotic
composition of the
present invention includes a prebiotic compound in an amount of from about 1
to about 30% by
weight, respect to the total weight composition, (e.g. from 5 to 20% by
weight). Known prebiotics
include commercial products such as inulin and transgalacto-oligosaccharides.
A prebiotic compound is usually a non-digestible carbohydrate such as an oligo-
or
polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the
upper digestive tract.
The carbohydrates may be selected from the group consisting of: fructo-
oligosaccharides (or
FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides,
pectins, xylo-
oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans,
arable gum
modified and resistant starches, polydextrose, D-tagatose, acacia fibers,
carob, oats, and citrus
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52
fibers. In one aspect, the prebiotics are the short-chain fructo-
oligosaccharides (for simplicity
shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible
carbohydrates, generally
obtained by the conversion of the beet sugar and including a saccharose
molecule to which three
glucose molecules are bonded.
Other prebiotic compounds (such as vitamin C, for example), may be included as
oxygen
scavengers and to improve the delivery and / or partial or total colonisation
and survival in vivo.
Alternatively, the probiotic composition of the invention may be administered
orally as a food or
nutritional product, such as milk or whey based fermented dairy product, or as
a pharmaceutical
product.
In certain embodiments, the compositions of the invention are used in
combination with another
therapeutic compound for treating or preventing the central nervous system
disorder. In some
embodiments, the compositions of the invention are administered with
nutritional supplements
that modulate central neurotransmitters and neuropeptides. In preferred
embodiments, the
nutritional supplements comprise or consist of nutritional vitamins. In
certain embodiments, the
vitamins are vitamin B6, magnesium, dimethylglycine (vitamin B16) and vitamin
C. In certain
embodiments, the compositions of the invention are administered in combination
with another
probiotic. In certain preferred embodiments, the probiotic comprises or
consists of Trichuris suis
ova.
The compositions of the invention may comprise pharmaceutically acceptable
excipients or
carriers. Examples of such suitable excipients may be found in reference [44].
Acceptable carriers
or diluents for therapeutic use are well known in the pharmaceutical art and
are described, for
example, in reference [45]. Examples of suitable carriers include lactose,
starch, glucose, methyl
cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of
suitable diluents
include ethanol, glycerol and water. The choice of pharmaceutical carrier,
excipient or diluent
can be selected with regard to the intended route of administration and
standard pharmaceutical
practice. The pharmaceutical compositions may comprise as, or in addition to,
the carrier,
excipient or diluent any suitable binder(s), lubricant(s), suspending
agent(s), coating agent(s),
solubilising agent(s). Examples of suitable binders include starch, gelatin,
natural sugars such as
glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners,
natural and synthetic
gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose
and polyethylene
glycol. Examples of suitable lubricants include sodium oleate, sodium
stearate, magnesium
stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives,
stabilizers, dyes and even flavouring agents may be provided in the
pharmaceutical composition.
Examples of preservatives include sodium benzoate, sorbic acid and esters of p-
hydroxybenzoic
acid. Antioxidants and suspending agents may be also used. A further example
of a suitable
carrier is saccharose. A further example of a preservative is cysteine.
Date Recue/Date Received 2022-07-25
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53
The compositions of the invention may be formulated as a food product. For
example, a food
product may provide nutritional benefit in addition to the therapeutic effect
of the invention, such
as in a nutritional supplement. Similarly, a food product may be formulated to
enhance the taste
of the composition of the invention or to make the composition more attractive
to consume by
being more similar to a common food item, rather than to a pharmaceutical
composition. In
certain embodiments, the composition of the invention is formulated as a milk-
based product.
The tei __ in "milk based product" means any liquid or semi-solid milk- or
whey- based product
having a varying fat content. The milk-based product can be, e.g., cow's milk,
goat's milk, sheep's
milk, skimmed milk, whole milk, milk recombined from powdered milk and whey
without any
.. processing, or a processed product, such as yoghurt, curdled milk, curd,
sour milk, sour whole
milk, butter milk and other sour milk products. Another important group
includes milk beverages,
such as whey beverages, fermented milks, condensed milks, infant or baby
milks, flavoured
milks, ice cream, milk-containing food such as sweets.
In some embodiments, the compositions of the invention comprise one or more
bacterial strains
of the species Enterococcus durans and do not contain bacteria from any other
species, or which
comprise only de minimis or biologically irrelevant amounts of bacteria from
another species.
Thus, in some embodiments, the invention provides a composition comprising one
or more
bacterial strains of the species Enterococcus durans, which does not contain
bacteria from any
other species or which comprises only de minimis or biologically irrelevant
amounts of bacteria
from another species, for use in therapy.
In certain embodiments, the compositions of the invention contain a single
bacterial species and
do not contain any other bacterial species. In certain embodiments, the
compositions of the
invention contain a single bacterial strain and do not contain any other
bacterial strains. Such
compositions may comprise only de minimis or biologically irrelevant amounts
of other bacterial
.. strains or species. Such compositions may be a culture that is
substantially free from other species
of organism. In some embodiments, such compositions may be a lyophilisate that
is substantially
free from other species of organism.
In some embodiments, the invention provides a composition comprising a single
bacterial strain
of the species Enterococcus durans, which does not contain bacteria from any
other strains or
which comprises only de minimis or biologically irrelevant amounts of bacteria
from another
strain for use in therapy.
In embodiments of the invention, the compositions of the invention do not
contain bacteria from
the genus Bacteroides or comprise only de minimis or biologically irrelevant
amounts of bacteria
from the genus Bacteroides for use in therapy.
In some embodiments, the compositions of the invention comprise more than one
bacterial strain
or species. For example, in some embodiments, the compositions of the
invention comprise more
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54
than one strain from within the same species (e.g. more than 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20,
25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteria from
any other species. In
some embodiments, the compositions of the invention comprise less than 50
strains from within
the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7,
6, 5, 4 or 3 strains), and,
optionally, do not contain bacteria from any other species. In some
embodiments, the
compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-
10, 1-9, 1-8, 1-7, 1-
6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-
25, or 31-50 strains
from within the same species and, optionally, do not contain bacteria from any
other species. In
some embodiments, the compositions of the invention comprise more than one
species from
within the same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, 23, 25, 30, 35 or
40 species), and, optionally, do not contain bacteria from any other genus. In
some embodiments,
the compositions of the invention comprise less than 50 species from within
the same genus (e.g.
less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species),
and, optionally, do not
contain bacteria from any other genus. In some embodiments, the compositions
of the invention
.. comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4,
1-3, 1-2, 2-50, 2-40, 2-
30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species from within the
same genus and,
optionally, do not contain bacteria from any other genus. In some embodiments,
the composition
comprises fewer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5
or 4 bacterial strains.
The invention comprises any combination of the foregoing.
In some embodiments, the composition comprises a microbial consortium. For
example, in some
embodiments, the composition comprises the Enterococcus durans bacterial
strain as part of a
microbial consortium. For example, in some embodiments, the Enterococcus
durans bacterial
strain is present in combination with one or more (e.g. at least 2, 3, 4, 5,
10, 15 or 20) other
bacterial strains from the genus Enterococcus and/or other genera with which
it can live
symbiotically in vivo in the intestine. For example, in some embodiments, the
composition
comprises a bacterial strain of Enterococcus durans in combination with a
bacterial strain from
a different genus. In another example, the composition comprises a bacterial
strain of
Enterococcus durans in combination with a bacterial strain from the genus
Enterococcus or the
composition comprises a bacterial strain of Enterococcus in combination with a
bacterial strain
from the genus Enterococcus and a bacterial strain from a different genus. In
some embodiments,
the microbial consortium comprises two or more bacterial strains obtained from
a faeces sample
of a single organism, e.g. a human. In some embodiments, the microbial
consortium is not found
together in nature. For example, in some embodiments, the microbial consortium
comprises
bacterial strains obtained from faeces samples of at least two different
organisms. In some
embodiments, the two different organisms are from the same species, e.g. two
different humans.
In some embodiments, the two different organisms are an infant human and an
adult human. In
some embodiments, the two different organisms are a human and a non-human
mammal.
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In some embodiments, the composition of the invention additionally comprises a
bacterial strain
that has the same safety and therapeutic efficacy characteristics as strain
NCIMB 43456, but
which is not strain NCIMB 43456, or which is not Enterococcus durans.
In some embodiments, the composition of the invention does not comprise a
bacterial strain of
5 the genus Bacillus. In some embodiments, the composition of the invention
does not comprise
Bacillus subtilis and/or does not comprise Bacillus coagulans. In some
embodiments, the CNS
disorder to be treated by the composition of the invention is not bipolar
disorder. In some
embodiments, the patient to be treated by the composition of the invention
does not have a fungal
infection. In some embodiments, the patient to be treated by the composition
of the invention
10 does not suffer from candidiasis. In some embodiments, the patient to be
treated by the
composition of the invention has not been diagnosed as having a fungal
infection and/or has not
been diagnosed as suffering from candidiasis. In preferred such embodiments,
the patient to be
treated by the composition of the invention has never been diagnosed as having
a fungal infection
and/or has never been diagnosed as suffering from candidiasis.
15 In some embodiments, the composition of the invention does not comprise
a bacterial strain of
the species Enterococcus faecium and/or of the species Enterococcus faecalis.
In some embodiments in which the composition of the invention comprises more
than one
bacterial strain, species or genus, the individual bacterial strains, species
or genera may be for
separate, simultaneous or sequential administration. For example, the
composition may comprise
20 all of the more than one bacterial strain, species or genera, or the
bacterial strains, species or
genera may be stored separately and be administered separately, simultaneously
or sequentially.
In some embodiments, the more than one bacterial strains, species or genera
are stored separately
but are mixed together prior to use.
In some embodiments, the bacterial strain for use in the invention is obtained
from human adult
25 faeces. In some embodiments in which the composition of the invention
comprises more than
one bacterial strain, all of the bacterial strains are obtained from human
adult faeces or if other
bacterial strains are present they are present only in de minimis amounts. The
bacteria may have
been cultured subsequent to being obtained from the human adult faeces and
being used in a
composition of the invention.
30 In some embodiments, the one or more Enterococcus durans bacterial
strains is/are the only
therapeutically active agent(s) in a composition of the invention. In some
embodiments, the
bacterial strain(s) in the composition is/are the only therapeutically active
agent(s) in a
composition of the invention.
The compositions for use in accordance with the invention may or may not
require marketing
35 approval.
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56
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
said bacterial strain is lyophilised. In certain embodiments, the invention
provides the above
pharmaceutical composition, wherein said bacterial strain is spray dried. In
certain embodiments,
the invention provides the above pharmaceutical composition, wherein the
bacterial strain is
lyophilised or spray dried and wherein it is live. In certain embodiments, the
invention provides
the above pharmaceutical composition, wherein the bacterial strain is
lyophilised or spray dried
and wherein it is viable. In certain embodiments, the invention provides the
above
pharmaceutical composition, wherein the bacterial strain is lyophilised or
spray dried and
wherein it is capable of partially or totally colonising the intestine. In
certain embodiments, the
invention provides the above pharmaceutical composition, wherein the bacterial
strain is
lyophilised or spray dried and wherein it is viable and capable of partially
or totally colonising
the intestine.
In some cases, the lyophilised or spray dried bacterial strain is
reconstituted prior to
administration. In some cases, the reconstitution is by use of a diluent
described herein.
The compositions of the invention can comprise pharmaceutically acceptable
excipients, diluents
or carriers.
In certain embodiments, the invention provides a pharmaceutical composition
comprising: a
bacterial strain as discussed earlier; and a pharmaceutically acceptable
excipient, carrier or
diluent; wherein the bacterial strain is in an amount sufficient to treat a
disorder when
administered to a subject in need thereof; and wherein the disorder is
selected from the group
consisting of: autism spectrum disorders (ASDs); child developmental disorder;
obsessive
compulsive disorder (OCD); major depressive disorder; depression; seasonal
affective disorder;
anxiety disorders; schizophrenia spectrum disorders; schizophrenia; bipolar
disorder; psychosis;
mood disorder; chronic fatigue syndrome (myalgic encephalomyelitis); stress
disorder; post-
traumatic stress disorder; dementia; Alzheimer's; Parkinson's disease;
epilepsy; chronic pain (for
example, central sensitisation or fibromyalgia); motor neuron disease;
Huntington's disease;
Guillain-Barre syndrome and/or meningitis.
In certain embodiments, the invention provides pharmaceutical composition
comprising: a
bacterial strain as used in the invention; and a pharmaceutically acceptable
excipient, carrier or
diluent; wherein the bacterial strain is in an amount sufficient to treat or
prevent a central nervous
system disorder or condition, in particular central nervous system disorder or
condition mediated
by the microbiota-gut-brain axis. In preferred embodiments, said disease or
condition is selected
from the group consisting of: autism spectrum disorders (ASDs); child
developmental disorder;
obsessive compulsive disorder (OCD); major depressive disorder; depression;
seasonal affective
disorder; anxiety disorders; schizophrenia spectrum disorders; schizophrenia;
bipolar disorder;
psychosis; mood disorder; chronic fatigue syndrome (myalgic
encephalomyelitis); stress
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57
disorder; post-traumatic stress disorder; dementia; Alzheimer's; Parkinson's
disease; epilepsy;
chronic pain (for example, central sensitisation or fibromyalgia); motor
neuron disease;
Huntington's disease; Guillain-Barre syndrome and/or meningitis.
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
the amount of the bacterial strain is from about 1 x 103 to about 1 x 10"
colony forming units
per gram with respect to a weight of the composition.
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
the composition is administered by a method selected from the group consisting
of oral, rectal,
subcutaneous, nasal, buccal, and sublingual.
In certain embodiments, the invention provides the above pharmaceutical
composition,
comprising a carrier selected from the group consisting of lactose, starch,
glucose, methyl
cellulose, magnesium stearate, mannitol and sorbitol.
In certain embodiments, the invention provides the above pharmaceutical
composition,
comprising a diluent selected from the group consisting of ethanol, glycerol
and water.
In certain embodiments, the invention provides the above pharmaceutical
composition,
comprising an excipient selected from the group consisting of starch, gelatin,
glucose, anhydrous
lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth,
sodium alginate,
carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate,
magnesium
stearate, sodium benzoate, sodium acetate and sodium chloride.
In certain embodiments, the invention provides the above pharmaceutical
composition, further
comprising at least one of a preservative, an antioxidant and a stabilizer.
In certain embodiments, the invention provides the above pharmaceutical
composition,
comprising a preservative selected from the group consisting of sodium
benzoate, sorbic acid and
esters of p-hydroxybenzoic acid.
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
said bacterial strain is lyophilised.
In certain embodiments, the invention provides the above pharmaceutical
composition, wherein
when the composition is stored in a sealed container at about 4 C or about 25
C and the container
is placed in an atmosphere having 50% relative humidity, at least 80% of the
bacterial strain as
measured in colony forming units, remains after a period of at least about: 1
month, 3 months, 6
months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.
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58
In some embodiments, the composition of the invention is provided in a sealed
container
comprising a composition as described herein. In some embodiments, the sealed
container is a
sachet or bottle. In some embodiments, the composition of the invention is
provided in a syringe
comprising a composition as described herein.
The composition of the present invention may, in some embodiments, be provided
as a
pharmaceutical fonnulation. For example, the composition may be provided as a
tablet or
capsule. In some embodiments, the capsule is a gelatine capsule ("gel-cap").
The capsule can be
a hard or a soft capsule. In some embodiments, the foimulation is a soft
capsule. Soft capsules
are capsules which may, owing to additions of softeners, such as, for example,
glycerol, sorbitol,
maltitol and polyethylene glycols, present in the capsule shell, have a
certain elasticity and
softness. Soft capsules can be produced, for example, on the basis of gelatine
or starch. Gelatine-
based soft capsules are commercially available from various suppliers.
Depending on the method
of administration, such as, for example, orally or rectally, soft capsules can
have various shapes,
they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules
can be produced
by conventional processes, such as, for example, by the Scherer process, the
Accogel process or
the droplet or blowing process.
In some embodiments the phannaceutical formulation is an enteric foimulation,
i.e. a gastro-
resistant foimulation (for example, resistant to gastric pH) that is suitable
for delivery of the
composition of the invention to the intestine by oral administration. Enteric
foimulations may
be particularly useful when the bacteria or another component of the
composition is acid-
sensitive, e.g. prone to degradation under gastric conditions.
In some embodiments, the enteric foimulation comprises an enteric coating. In
some
embodiments, the foimulation is an enteric-coated dosage fonn. For example,
the fonnulation
may be an enteric-coated tablet or an enteric-coated capsule, or the like. The
enteric coating may
be a conventional enteric coating, for example, a conventional coating for a
tablet, capsule, or the
like for oral delivery. The formulation may comprise a film coating, for
example, a thin film layer
of an enteric polymer, e.g. an acid-insoluble polymer.
In some embodiments, the enteric fonnulation is intrinsically enteric, for
example, gastro-
resistant without the need for an enteric coating. Thus, in some embodiments,
the fonnulation is
an enteric foimulation that does not comprise an enteric coating. In some
embodiments, the
formulation is a capsule made from a thennogelling material. In some
embodiments, the
thennogelling material is a cellulosic material, such as methylcellulose,
hydroxymethylcellulose
or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule
comprises a shell
that does not contain any film fonning polymer. In some embodiments, the
capsule comprises a
shell and the shell comprises hydroxypropylmethylcellulose and does not
comprise any film
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59
forming polymer (e.g. see [461). In some embodiments, the foimulation is an
intrinsically enteric
capsule (for example, Vcaps0 from Capsugel).
Culturing methods
The bacterial strains for use in the present invention can be cultured using
standard microbiology
techniques as detailed in, for example, references [47]-[49].
The solid or liquid medium used for culture may be YCFA agar or YCFA medium.
YCFA
medium may include (per 100m1, approximate values): Casitone (1.0 g), yeast
extract (0.25 g),
NaHCO3 (0.4 g), cysteine (0.1 g), K2HPO4 (0.045 g), KH2PO4 (0.045 g), NaCl
(0.09 g),
(NH4)2SO4 (0.09 g), MgSO4 - 7H20 (0.009 g), CaCl2 (0.009 g), resazurin (0.1
mg), hemin (1 mg),
biotin (1 lag), cobalamin (1 lag), p-aminobenzoic acid (3 lag), folic acid (5
lag), and pyridoxamine
(15 lag).
Bacterial strains for use in vaccine compositions
The inventors have identified that the bacterial strains of the invention are
useful for treating or
preventing central nervous system disorders or conditions, in particular
central nervous system
disorders or conditions mediated by the microbiota-gut-brain axis. This is
likely to be a result of
the effect that the bacterial strains of the invention have on the host
central, autonomic and/or
enteric nervous system; the activity of the HPA pathway; the neuroimmune and
neuroendocrine
pathways; and the level of commensal metabolites in the host gastrointestinal
tract and/or
gastrointestinal peimeability of the host. Therefore, the compositions of the
invention may also
be useful for preventing central nervous system disorders or conditions, in
particular central
nervous system disorders or conditions mediated by the microbiota-gut-brain
axis, when
administered as vaccine compositions. In certain such embodiments, the
bacterial strains of the
invention are viable. In certain such embodiments, the bacterial strains of
the invention are
capable of partially or totally colonising the intestine. In certain such
embodiments, the bacterial
strains of the invention are viable and capable of partially or totally
colonising the intestine. In
other certain such embodiments, the bacterial strains of the invention may be
killed, inactivated
or attenuated. In certain such embodiments, the compositions may comprise a
vaccine adjuvant.
In certain embodiments, the compositions are for administration via injection,
such as via
subcutaneous injection.
General
The practice of the present invention will employ, unless otherwise indicated,
conventional
methods of chemistry, biochemistry, molecular biology, immunology and
phaimacology, within
the skill of the art. Such techniques are explained fully in the literature.
See, e.g., references [50]
and [511457], etc.
The teim "comprising" encompasses "including" as well as "consisting" e.g. a
composition
"comprising" X may consist exclusively of X or may include something
additional e.g. X + Y.
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The tenn "about" in relation to a numerical value x is optional and means, for
example, x+10%.
In certain embodiments the tenn "modulate" means increase or activate. In
alternative
embodiments, the term "modulate" means decrease or suppress.
The word "substantially" does not exclude "completely" e.g. a composition
which is
5 "substantially free" from Y may be completely free from Y. Where
necessary, the word
"substantially" may be omitted from the definition of the invention.
References to a percentage sequence identity between two nucleotide sequences
means that,
when aligned, that percentage of nucleotides are the same in comparing the two
sequences. This
alignment and the percent homology or sequence identity can be detennined
using software
10 programs known in the art, for example those described in section 7.7.18
of ref. [58]. A preferred
alignment is detennined by the Smith-Waterman homology search algorithm using
an affine gap
search with a gap open penalty of 5 and a gap extension penalty of 2, BLOSUM
matrix of 62.
The Smith-Watennan homology search algorithm is disclosed in ref. [59].
Treatment or prevention may refer to, for example, an alleviation of the
severity of symptoms or
15 a reduction in the frequency of exacerbations or the range of triggers
that are a problem for the
subject or a prevention of relapse.
Unless specifically stated, a process or method comprising numerous steps may
comprise
additional steps at the beginning or end of the method, or may comprise
additional intervening
steps. Also, steps may be combined, omitted or perfonned in an alternative
order, if appropriate.
20 Various embodiments of the invention are described herein. It will be
appreciated that the features
specified in each embodiment may be combined with other specified features, to
provide further
embodiments. In particular, embodiments highlighted herein as being suitable,
typical or
preferred may be combined with each other (except when they are mutually
exclusive).
All patent and literature references cited in the present specification are
hereby incorporated by
25 reference in their entirety.
Any reference to a method for treatment comprising administering an agent to a
patient, also
covers that agent for use in said method for treatment, as well as the use of
the agent in said
method for treatment, and the use of the agent in the manufacture of a
medicament.
The following examples are offered for illustrative purposes only, and are not
intended to limit
30 the scope of the present invention in any way.
MODES FOR CARRYING OUT THE INVENTION
Example 1- ex vivo screening for effects on the microbiota gut-brain axis
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised
by deficits in
social behaviour, increased repetitive behaviour, anxiety and gastrointestinal
symptoms. The
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61
aetiology of autism is complex and most likely involves both genetic and
environmental
factors[60]. Recent evidence suggests that the microorganisms resident to the
gastrointestinal
system play a fundamental role in regulating brain health status, behaviour
and mood via the
microbiota-gut-brain axis [61]. Emerging preclinical and clinical studies have
documented a
potential role for gut bacteria in ASD, and consequently, the microbiota
represents a potential
target in the development of novel therapeutics for this neurodevelopmental
disorder.
Additional behavioural complications include depression, anxiety, cognitive
impairments and
seizures in autistic individuals. Moreover, non-neurological symptoms such as
gastrointestinal
dysfunction are becoming increasingly recognised as a major comorbidity of
this
neurodevelopmental disorder. For instance, several studies have documented
pronounced
gastrointestinal distress such as abdominal pain, gaseousness, diarrhoea,
flatulence and
constipation in patients with ASD [62,63,64,65] . These gastrointestinal
disturbances correspond
with reports of alterations to the gut microbiota of individuals with ASD. For
instance, several
studies have documented an increased abundance of potentially pathogenic
strains, such as
Clostridia and Desulfovibrio, along with a reduction in the presence of
beneficial genera such as
Bifidobacteria [66,67,68,69] ;. In light of these pronounced alterations to
the microbiota in ASD,
emerging clinical evidence has shown that targeting the microbiota through
prebiotics or
microbiota transfer therapy can improve both the gastrointestinal and
behavioural symptoms of
children with autism [70,71]. These preliminary studies suggest that targeting
the microbiota may
represent a novel therapeutic strategy for the treatment of autism.
BALB/c mice display an inherent anxiety-like phenotype and are thus a useful
model for
screening the beneficial effects of live biotherapeutic strains on the gut-
brain axis.
Example la ¨ Materials and methods
Animals
BALBc (Envigo, UK) adult male mice were group housed under a 12 h light-dark
cycle; standard
rodent chow and water were available ad libitum. All experiments were
performed in accordance
with European guidelines following approval by University College Cork Animal
Ethics
Experimentation Committee. Animals were 8 weeks old at the start of the
experiment.
Dosing and strain
The strain used was Enterococcus durans bacterium deposited under accession
number NCIMB
43456.
Biotherapeutic was provided in glycerol stock. Live biotherapeutics were grown
in the facility in
anaerobic conditions.
Animals were allowed to habituate to their holding room for one week after
arrival into the animal
unit. They received oral gavage (2004 dose) of strain NCIMB 43456 at a dose of
1 X 109 CFU
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62
or PBS (vehicle control) for 6 consecutive days between 15:00 and 17:00. On
day 7, the animals
are decapitated and tissues are harvested for experimentation.
Tissue collection
Animals were sacrificed in a random fashion regarding treatment and testing
condition; sampling
occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in
potassium EDTA
(Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g.
Plasma was isolated
and stored at ¨80 C for further analysis. The brain was quickly excised,
dissected and each brain
region was snap-frozen on dry ice and stored at ¨80 C for further analysis.
Spleen was removed,
collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758
Sigma + 10
% FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately
after culls
for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of ileum and
colon closest to
the caecum were excised, and the furthest lcm of tissue from the caecum were
used) were
mounted into the Ussing chambers for intestinal permeability assay. A further
lcm of ileum and
colon tissue was taken for tight junction gene expression analysis. The caecum
was removed,
weighed and stored at ¨80 C for SCFAs analysis.
Statistical Analysis
Normally distributed data are presented as mean SEM; non-parametric datasets
are presented
as median with inter-quartile range. Unpaired two-tailed t-test were applied
to analyse parametric
data and Mann-Whitney test was used for non-parametric. Spearman's rank
correlation
coefficient was employed for the correlation analysis in the pooled datasets.
A p value <0.05
was deemed significant in all cases.
Example lb ¨ Tight junction protein mRNA expression
Method and rationale
Total RNA was extracted using the mirVanaTM miRNA Isolation kit (Ambion/Llife
technologies,
Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies)
according to the
manufacturers' recommendations. RNA was quantified using NanoDropTM
spectrophotometer
(Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the
manufacturer's
instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent,
Stockport, UK)
according to the manufacturer's procedure and an RNA integrity number (RIN)
was calculated.
RNA with RIN value >7 was used for subsequent experiments. RNA was reverse
transcribed to
cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems,
Warrington,
UK) according to manufacturer's instructions. Briefly, Multiscribe Reverse
Transcriptase (50
U/nL) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25 C for
10 min, 37 C
for 2 h, 85 C for 5 min and stored at 4 C. Quantitative PCR was carried out
using probes (6
carboxy fluorescein - FAM) designed by Applied Biosystems to mouse specific
targeted genes,
while using f3-actin as an endogenous control. Amplification reactions
contained 1 ptl cDNA, 5
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63
IA of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to
a total of 10
IA by the addition of RNase-free water. All reactions were performed in
triplicate using 96-well
plates on the LightCycler0480 System. Thermal cycling conditions were as
recommended by
the manufacturer (Roche) for 55 cycles. To check for amplicon contamination,
each run
contained no template controls in triplicate for each probe used. Cycle
threshold (Ct) values were
recorded. Data was normalized using f3-actin and transformed using the 2¨AACT
method and
presented as a fold change vs. control group.
Tight junction proteins including TJP1 and occludin help regulate the
permeability of the gut
epithelium. An increase in these genes would be a desirable trait. Indoleamine
2,3 dioxygenase-1
(ID01) is an enzyme that initiates tryptophan catabolism along a pathway that
generates several
bioactive kynurenine-based metabolites. Tryptophan hydroxylase-1 (TPH-1)
catalyze the formation
of 5-hydroxy-L-tryptophan (5-HTP) from L-tryptophan, the first and rate-
limiting step in the
biosynthesis of 5-HT. Thus the relationship between these genes is important
in the regulation of
tryptophan/kynurenine system.
Results
No difference was observed between strain NCIMB 43456-treated and vehicle-
treated mice for
occludin mRNA expression in the ileum or colon (Figure 2).
IDO1 mRNA expression in the colon was increased in the strain NCIMB 43456-
treated mice
relative to the vehicle-treated mice (P=0.062) (Figure 3A). No difference was
observed between
strain NCIMB 43456-treated and vehicle-treated mice for IDO1 mRNA expression
in the ileum
(Figure 3B).
TJP1 mRNA expression in the ileum was increased in the strain NCIMB 43456-
treated mice
relative to the vehicle-treated mice (Figure 4A). No difference was observed
between strain
NCIMB 43456-treated and vehicle-treated mice for TJP1 mRNA expression in the
colon (Figure
4B).
Tphl mRNA expression in the ileum was increased in the strain NCIMB 43456-
treated mice
relative to the vehicle-treated mice (Figure 5A). No difference was observed
between strain
NCIMB 43456-treated and vehicle-treated mice for Tphl mRNA expression in the
colon (Figure
5B).
Conclusions
Daily treatment with strain NCIMB 43456 leads to an increase in TJP1 and Tphl
mRNA
expression in the ileum, and IDO1 mRNA expression in the colon. Chronic
treatment with strain
NCIMB 43456 can therefore be expected to improve regulation of permeability of
the gut
epithelium.
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Example lc ¨ Short chain fatty acid production
Method and rationale
Caecum content was mixed and vortexed with MilliQ water and incubated at room
temperature
for 10 min. Supernatants were obtained by centrifugation (10000 g, 5 min, 4
C) to pellet bacteria
and other solids and filtration by 0.2 m. It was transferred to a clear GC
vial and 2-Ethylbutyric
acid (Sigma) was used as the internal standard. The concentration of short-
chain fatty acids (SCFAs)
was analyzed using a Varian 3500 GC flame-ionization system, fitted with a
with a ZB-FFAP column
(30 m x 0.32 mm x 0.25 mm; Phenomenex). A standard curve was built with
different concentrations
of a standard mix containing acetate, propionate, iso-butyrate, n-butyrate,
isovalerate and valerate
(Sigma). Peaks were integrated by using the Varian Star Chromatography
Workstation version 6.0
software. All SCFA data are expressed as gmoUg.
SCFAs are microbial byproducts of dietary fibre. An increase in acetate,
propionate, valerate,
butyrate, isobutyrate or isovalerate suggest an increase in productivity of
the microbiota and a
desirable trait.
Results
Mice treated with strain NCIMB 43456 had significantly increased caecum levels
of acetate and
butyrate relative to vehicle-treated mice (Figure 6).
Conclusions
Treatment with strain NCIMB 43456 results in increased levels of acetate and
butyrate in the
caecum, suggesting increased productivity of the microbiota.
Example id ¨ Screening for effects of administration of live biotherapeutics
on central gene
expression
Methods and rationale
The mRNA levels for markers for the oxytocinergic system (oxytocin receptor
and vasopressin
receptor), endocrine system (mineralocorticoid (Nr3c1); glucocorticoid
receptor (Nr3c2);
corticosterone releasing hounone (CRH) and receptors; Brain derived
neurotrophic factor
(BDNF)), immune system (I1-6); and neurotransmitter systems (NMDA receptor 2A
(Grin2A);
NMDA receptor 2B (Grin2B); GABAA receptor subunit A2; GABAB receptor subunit B
1;
serotonin 2C) were assessed in the amygdala, hippocampus and prefrontal cortex
¨ key brain
regions of the limbic system involved in emotional response.
RNA was extracted and quantified as described in example lb above.
Results
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Expression of oxytocin receptor mRNA, CRH mRNA, IL-6 mRNA, Grin2a mRNA and
GABA
B1R in the hippocampus was significantly increased in strain NCIMB 43456-
treated mice
relative to vehicle-treated mice (Figure 7).
Expression of oxytocin receptor mRNA in the amygdala was also significantly
increased in strain
5 NCIMB 43456-treated mice relative to vehicle-treated mice (Figure 8).
Expression of BDNF mRNA in the prefrontal cortex was significantly increased
in strain NCIMB
43456-treated mice relative to vehicle-treated mice (Figure 9).
Conclusions
Treatment with strain NCIMB 43456 results in increased mRNA expression for
stress markers
10 (CRH), immune proteins (IL-6), markers of the oxytocinergic system
(oxytocin receptor) and
neurotransmitters (Grin2A, GABA B1R, BDNF) in regions of the brain involved in
emotional
response.
These data suggest that treatment strain NCIMB 43456 can be used to treat
diseases and
conditions of the brain, for example, those which have a gastrointestinal
component.
15 Overall conclusions for ex vivo screening experiments
The ex vivo screening data show that daily treatment with strain NCIMB 43456
led to an increase
in productivity of the microbiota, demonstrated by increased production of
SCFA microbial by-
products of dietary fibre. The treatment with strain NCIMB 43456 led to
increased expression in
the tight junction protein TJP1 in the ileum and a concomitant improvement in
ileal peimeability
20 (reduced leakiness). Expression of catabolic enzymes IDO1 and Tphl was
increased in the colon
and ileum respectively.
These changes in the gut microbiota were accompanied by increased mRNA
expression for
various neurological markers, suggesting that treatment with strain NCIMB
43456 will be
effective for the treatment of CNS disorders with a gastrointestinal
component, such as autism
25 and Parkinson's disease.
Example 2 ¨ Assessing anxiety, depression, cognitive and social domains of
behaviour in
mouse models of autism spectrum disorders
The present study aimed to assess the effect of strain NCIMB 43456 treatment
on psychiatric and
neurological disorders in two different mouse models that display behavioural
characteristics
30 associated with neurodevelopmental and psychiatric disorders. In
particular, the study focused
on autistic-related behaviour in (i) C57BL/6 wt mouse model, (ii) a BTBR
inbred, genetically
modified mouse model and (iii) a maternal immune activation (MIA) mouse model.
The effects
of chronic strain NCIMB 43456 versus vehicle treatment across anxiety,
depression, and
cognitive and social domains of behaviour in the three mouse models were
investigated. In
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addition, physiological and anatomical analyses were performed as well as
analysis of
neurotransmitter concentrations in brainstem tissue.
The BTBR mouse model uses inbred, genetically modified mice that display a
robust autistic-
like phenotype. Deficits in social behaviours, increased repetitive behaviours
and increased
anxiety-related behaviours have been reported in this strain [72]. Due to this
robust behavioural
phenotype, the BTBR mouse is an ideal animal model to assess the efficacy of
novel therapeutic
agents for the treatment of autistic-related behaviours. Alleviation of such
symptoms by a live
biotherapeutic can also be indicative of efficacy of the biotherapeutic in the
treatment of other
psychiatric or neurological diseases.
The MIA mouse model uses an environmental immune challenge in pregnant mice in
order to
trigger the core symptoms of autism spectrum disorder in the offspring. MIA
mice typically
display stereotyped behaviour (as shown by the grooming and marble burying
tests) and deficits
in social communication (as shown by the social play, 3-chamber social
interaction, and social
transmission of food preference tests). The offspring display the three core
symptoms of autism
.. (reduced communication; reduced sociability; and increased repetitive or
stereotyped behaviour)
and therefore provide a suitable model in which to determine whether
administration of a
therapeutic can alleviate the behavioural phenotypes associated with autistic
spectrum disorders
and indeed in a number of other neurological disorders. It is well established
that alteration of
behavioural phenotypes in animal models is indicative of a potentially
clinically relevant
intervention, irrespective of an understanding of the underlying biological or
physiological
mechanism [73].
The EMA Guidelines on the clinical development of medicinal products for the
treatment of
autism spectrum disorder state that, due to the heterogeneity of the diseases,
it may not be possible
to achieve a significant effect on all core symptoms with a single compound,
and so short term
efficacy has to be demonstrated on at least one core symptom. The strain NCIMB
43456 live
biotherapeutic tested in the Examples has shown effective treatment of at
least one core symptom
of autistic spectrum disorder, so this strain and related Enterococcus strains
are expected to be
effective against human disease. Similarly, other central nervous system
disorders or conditions
display complex pathology with multiple different symptoms, and have few
approved treatments.
Therefore, it is understood that an effective treatment does not need to treat
all symptoms of a
central nervous system disorder or condition. A treatment would be considered
therapeutically
useful if it treated one of the symptoms associated with a central nervous
system disorder or
condition. However, as is demonstrated in the examples that follow,
Enterococcus durans was
advantageously able to elicit improvements in a range of symptoms.
Example 2a ¨ Materials and methods
Mice and dosing
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BTBR animals were bred in house with brother-sister mating. The male offspring
from these animals
were separated from their mothers at 3 weeks old and daily administration of
the live biotherapeutic
or control commenced at 8 weeks of age. Behaviour assessment started at 11
weeks old. A control
age-matched C57BL/6 group was included as a reference control group.
For the maternal immune activation studies, female C57B/6 mice (8 weeks old)
and age matched
males were purchased from Harlan UK. After 1-week habituation these animals
were mated. At
embryonic day 12.5, females received either an injection of the viral mimetic
poly-IC to activate the
maternal immune system, or a saline vehicle injection. The male offspring from
these animals were
separated from their mothers at 3 weeks old and daily administration of the
live biotherapeutic or
control commenced at 8 weeks of age. Behaviour started at 11 weeks old.
For dosing, animals received daily oral gavage of PBS or the live
biotherapeutics prepared to 109
cfu/mL in PBS. Dosing continued daily throughout the behavioural paradigm.
All experiments were conducted in accordance with the European Directive
2010/63/EEC, the
requirements of the S.I No 543 of 2012, and approved by the Animal
Experimentation Ethics
Committee of University College Cork.
Strain
The strain used was Enterococcus durans bacterium deposited under accession
number NCIMB
43456.
Biotherapeutic was provided in glycerol stock. Live biotherapeutics were grown
in the facility in
anaerobic conditions.
Administration schedule
The treatment groups for the study are shown below. The vehicle for oral
administration is PBS.
Daily oral administration occurs via oral gavage.
Group Treatment BTBR MIA
1 Control 11 10
2 Vehicle 11 11
3 NCIMB 43456 11 11
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For the MIA and B 1BR animals, once they reached 8 weeks they started getting
a daily gavage
of the live biotherapeutic of interest. This they got from 8 weeks old until
they were culled. At
week 11 (3 weeks later) the animals began undergoing behaviour testing. There
were 10-12
animals per group. Both the MIA and BTBR were run over 2 cohorts each ¨ each
animal within
each cohort underwent the exact same procedures in the exact same order for
the same length of
time. Animals were always randomised such that not all one group was run on
one individual
day.
Experimental design and methods
As outlined above, dosing with strain NCIMB 43456 commenced when the mice were
8 weeks
old. The initial dosing took place for 3 weeks before the behavioural
experiments. The
behavioural battery occurred in the following order: marble burying and
grooming tests at week
4; the elevated plus maze and three chamber tests at week 5; the open field
and novel object
recognition tests, and the in vivo intestinal motility assay at week 6; and
the forced swim test at
week 7. Finally, in week 8, the mice were culled and dissected for brain,
proximal and distal
colon, and spleen regions, along with plasma samples.
Graphical design and statistical analysis
Normally distributed data are presented as mean SEM; non-parametric datasets
are presented as
median with inter-quartile range. Unpaired two-tailed t-test were applied to
analyse parametric data
and Mann-Whitney test was used for non-parametric. Spearman's rank correlation
coefficient was
employed for the correlation analysis in the pooled datasets. A p value < 0.05
was deemed significant
in all cases.
Example 2b ¨ Assessing stereotype-related behaviour ¨ the marble bulying test
Rationale
The marble burying test is a useful model of neophobia, anxiety and obsessive
compulsive
behaviour. It is also used to test novel antidepressants, anxiolytics and
antipsychotics. Mice
pre-treated with pharmacological agents such as anxiolytics show decreased
marble burying
behaviour, compared to the control mice. A higher number of marbles buried
represents a
heightened state of stereotype behaviour or higher levels of anxiety
(neophobia).
Methods
Mice were individually placed in a novel plexiglas cage (35 x 28 x 18.5 cm,
LxWx H), filled up
with sawdust (5-10 cm) and 20 marbles on top of it (five rows or marbles
regularly spaced 2 cm away
from the walls and 2 cm apart). Thirty minutes later, the number of marbles
buried for more than 2/3
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of their surface was counted.
Results
The number of marbles buried during the test was reduced for strain NCIMB
43456-treated
BTBR mice and strain NCIMB 43456-treated MIA mice relative to the respective
vehicle-treated
controls (Figure 10).
Conclusions
Chronic treatment with strain NCIMB 43456 reduces anxiety-like behaviour in
BTBR and MIA
mice in the marble burying test.
Example 2c ¨ Assessing stereotype-related behaviour ¨ the grooming test
Rationale
This test is used as an index for stereotyped and repetitive behaviour. An
increase in time spent
grooming is indicative of increased stereotyped or repetitive behaviour.
Methods
Self-grooming is evaluated in a 6.5 cm diameter >< 10 cm tall, clear glass
beaker covered with a filter
.. top. Experimental animals will be brought to the test room to habituate up
to 1 hour prior to the test.
The test is approximately 20 minutes in duration. The investigator records the
cumulative time spent
by the test animal grooming. Between tests the beakers are cleaned thoroughly
for next use.
Results
Treatment with strain NCIMB 43456 did not have a significant effect on the
time spent grooming
.. by BTBR or MIA mice (Figure 11).
Conclusions
Chronic treatment with strain NCIMB 43456 did not observably alter repetitive
behaviours in
BTBR or MIA mice in the grooming test.
Example 2d ¨ Assessment of anxiety-like behaviours ¨ the elevated plus maze
Rationale
The elevated plus maze (EPM) is a widely used test to assess anxiety-like
behaviours in rodents.
The EPM assesses general anxiety behaviour, with less anxious mice spending
more time in the
open aims of the maze. An increase in time or number of entries into an open
aim is an index of
reduced anxiety.
Methods
The set up was made of a grey plastic cross-shaped maze 1 m elevated from the
floor, comprising
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two open (fearful) and two closed (safe) arms (50 x 5 x 15 cm walls or 1 cm no
wall). Experiments
occurred under red light (-5 lux). Mice were individually placed into the
center of the maze
facing an open arm (to avoid direct entrance into a closed one) and were
allowed 5-min free
exploration. Experiments were videotaped using a ceiling camera for further
parameters analysis
5 using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham,
UK). The time spent in
the open aims and the number of entries in each aim were measured (entrance in
an arm was
defined as all four paws inside the arm).
Results
Treatment with strain NCIMB 43456 led to a significant increase in the number
of entries into
10 the open aims of the maze (OA entries) for MIA mice relative to vehicle-
treated mice (Figure
12). This increase corresponds to a full recovery of the deficit in OA entries
for the vehicle-
treated MIA mice relative to the wild type controls. Treatment with strain
NCIMB 43456 did not
have a significant effect on the time spent by MIA mice in the open arms of
the maze (OA
duration), nor did it have a significant effect on the OA entries or OA
duration for BTBR mice
15 (Figure 12).
Conclusions
Chronic treatment with strain NCIMB 43456 significantly reduces anxiety-like
behaviour in MIA
mice in the elevated plus maze. Chronic treatment with strain NCIMB 43456 has
no effect on
anxiety-like behaviour in BTBR mice in the elevated plus maze.
20 Example 2e ¨ Assessment of anxiety-like behaviours ¨ the open field
arena
Rationale
The open field arena is used to assess the response of exposure to a novel
stressful environment
and locomotor activity. Naïve mice naturally spend most of their time
alongside the walls of the
arena, as it is less exposed than the centre of the arena. An increase in
duration of time spent in
25 the centre represents a decrease in anxiety-like behaviour.
Methods
To assess the response to a novel stressful environment and locomotor
activity, mice were placed
into open arena (40 x 32 x 23 cm, Lxwx h) with ¨60 lux lighting and allowed to
explore for
10-mins. Experiments were videotaped using a ceiling camera for further
parameter analysis
30 using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham,
UK). The distance
travelled and the latency to enter a virtual central zone (defined at 50% away
from the edges) was
scored.
Results
There was no significant difference in the total distance travelled or time
spent in the centre of
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the area between strain NCIMB 43456-treated mice and the vehicle treated
controls (Figure 13).
Conclusions
Chronic treatment with strain NCIMB 43456 did not observably alter anxiety-
like behaviour in
BTBR or MIA mice in the open field arena test.
Example 2f¨ Assessment of social behaviours ¨ the three-chamber social
interaction test
Rationale
The 3-Chamber Social Interaction Test (3-CSIT) is a well validated
ethologically relevant model
that assesses social interaction between sex-matched conspecifics and allows
for readouts of
social novelty and social preference in mice. The test allows mice to freely
explore between an
inanimate object or sex-matched conspecific mice. Control animals naturally
are more interested
in a conspecific mouse more than an inanimate object (sociability). Similarly,
control animals
spend more time interacting with a novel unfamiliar mouse then one they have
already had
interactions with.
Methods
The social testing apparatus was a rectangle, three-chambered box. Each
chamber was 20 cm L
x 40 cm W x 22 cm H. Dividing walls were made with small circular openings (5
cm in diameter)
allowing access into each chamber. Two identical wire cup-like cages, with a
bottom diameter of
10 cm, 13 cm in height and bars spaced 1.2 cm, allowing nose contact between
the bars, but
prevented fighting, were placed inside each side chamber in bilaterally
symmetric positions. The
test has three phases of 10 mm each: 1) habitation 2) mouse versus object 3)
novel mouse versus
familiar mouse. Experiments were videotaped using a ceiling camera for further
parameters
analysis using Ethovision software (3.1 version, Noldus, TrackSys, Nottingham,
UK). For the
first phase the test mouse was placed into the middle chamber and allowed to
explore the entire
box with empty small wire cages inside for a 10-min habituation session. After
the habituation
period, the test mouse is removed from the testing box for a short interval
while an object is
placed in one side chamber and an unfamiliar conspecific male mouse (no prior
contact with the
test subject) in the other side chamber, both enclosed in a wire cup-like
cage. During phase two,
the test mouse is placed in the middle chamber and allowed to explore the
entire box for 10 min.
The amount of time spent exploring the object or mouse in each chamber and the
number of
entries into each chamber were evaluated. The location of the unfamiliar mouse
in the left vs
right side chamber was systematically alternated between trials. An entry was
defined as all four
paws in one chamber. During the third phase an object was replaced with an
unfamiliar mouse
serving as a novel mouse and in the other chamber the mouse used in phase two
was kept the
same, now serving as familiar mouse. After every trial, all chambers and cup-
like wire cages
.. were cleaned with 10% ethanol, dried and ventilated for a few minutes to
prevent olfactory cue
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bias and to ensure proper disinfection. Lack of innate side preference was
confirmed during the
initial 10 min of habituation to the entire arena.
Results
For both BTBR and MIA mice, treatment with strain NCIMB 43456 led to an
increase in the
time spent with an unfamiliar mouse relative to an inanimate object, and with
an unfamiliar
mouse relative to a familiar mouse. In each case, treatment with strain NCIMB
43456 led to a
complete recovery of the wild type behavioural phenotype (Figure 14).
Conclusions
Chronic treatment with strain NCIMB 43456 increases social novelty and social
cognition of
BTBR mice and MIA mice in the three chamber test.
Example 2g ¨ Assessment of depression-like behaviour ¨ the novel object
recognition test
Rationale
The novel object recognition test is used to test recognition memory and
learning. Improved
memory is a reflection of reduced depression-like behaviour. Control animals
will discriminate
between an object they have had time to explore and a new object.
Methods
Mice were placed in the middle of a grey plastic rectangular box (40 x 32 x 23
cm, LxWx H)
under a dimly light, 60 lux at the level of the arena, for 10 min. 24 h after
mice were placed in
the box with the two identical objects for a total time of 10 min (acquisition
phase). After a 24 h,
one of the two identical objects were substituted with a novel object and mice
were placed in the
middle of the box at the mid-point of the wall opposite the sample objects for
a total time of 10
min (retention phase). Animals were acclimatized to the testing room for 30
min prior each
experiment. Box and objects were cleaned with alcohol 10% to avoid any cue
smell between each
trial. Experiments were videotaped using a ceiling camera for further
parameter analysis.
Directed contacts with the objects, include any contact with mouth, nose or
paw or minimal
defined distance were counted as an interaction.
Results
Vehicle-treated BTBR mice spent more time interacting with the familiar object
than with the
novel object. Treatment with strain NCIMB 43456 did not have an observable
effect on the time
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spent interacting with a novel object versus time spent interacting with a
familiar object in BTBR
mice (Figure 15A).
Vehicle-treated MIA mice also spent more time interacting with the familiar
object than with the
novel object. However, in strain NCIMB 43456-treated MIA mice this trend was
reversed: strain
NCIMB 43456-treated MIA mice spent more time interacting with the novel object
(Figure 15B).
Conclusions
Chronic treatment with strain NCIMB 43456 led to a reduction of depression-
like behaviour in
MIA mice in the novel object recognition test. Chronic treatment with strain
NCIMB 43456 has
no effect on cognitive behaviour for BTBR mice in the novel object recognition
test.
Example 2h ¨ Assessment of depression-like behaviour ¨ the forced swim test
Rationale
The forced swim test (FST) is the most widely used experimental paradigm to
assess
antidepressant activity. Naïve animals will display escape behaviour in the
foim of swimming,
climbing and diving before adapting an immobile floating posture. The duration
of immobility is
indicative of behavioural despair. An increase in immobility means an increase
in depressive-
like behaviour as the animal has resigned itself to its situation (learned
helplessness).
Antidepressant drugs decrease the time spent immobile in this test.
Methods
Mice were individually placed in a clear glass cylinder (24 x 21 cm diameter),
containing 15-cm-
depth water (25 0.5 C). The water was changed between each animal. The test
lasted 6 min
and experiments were videotaped using a numeric tripod-fixed camera; data were
further scored
twice using the videos (Video Media Player software) and averaged by an
experimenter blind to
conditions. The latency to immobility was scored. The time of immobility (s)
was measured for
the last 4 mm of the test, with immobility being defined as a total absence of
movement except
slight motions to maintain the head above the water.
Results
For both BTBR and MIA mice, treatment with strain NCIMB 43456 led to a
significant decrease
in immobility time relative to vehicle-treated mice (Figure 16).
Conclusions
Chronic treatment with strain NCIMB 43456 results in a reduction in depression-
like behaviour
for both BTBR mice and MIA mice in the forced swimming test.
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Example 2i ¨ in vivo intestinal motility assay
Rationale
The MIA model has been reported to lead to changes in gut barrier function.
Therefore, it was
important to ascertain whether chronic treatment with the biotherapeutic
alters intestinal motility.
Methods
This procedure involves the oral administration of a given amount of a non-
toxic, coloured
marker (Carmin Red) to deteimine motility of the gut. Time to excretion of the
first coloured
faecal bolus is recorded as 'time of whole gut transit' and is used as an
index of peristaltic motility
in the whole intestine. Mice are single housed for 3h prior to the assay to
allow habituation to a
new cage. Carmin red dye (100-200 ul of 6% Caunin red in 0.5% methylcellulose
per mouse) is
given orally by gavage. Each cage is visually inspected every 10 mm. The time
of the first
coloured bolus (red) is recorded. Following the appearance of the first
coloured bolus mice are
returned to noimal housing conditions.
Results
Treatment with strain NCIMB 43456 did not lead to a significant difference in
intestinal motility
time relative to vehicle-treated controls for BTBR or MIA mice (Figure 17).
Conclusions
Chronic treatment with strain NCIMB 43456 did not affect intestinal motility
compared to the
control or vehicle groups for BTBR mice or MIA mice.
Example 2)¨ ex vivo gastrointestinal permeability assay
Rationale
MIA mice exhibit increases in intestinal permeability which corresponded with
reduced
expression of tight junction proteins and inflammation in the colon [74].
Methods
The permeability of the ileum and colon was assessed ex vivo using Ussing
chambers. An increase
in FITC concentration represents an undesired effect as it indicates an
increase in the 'leakiness'
of the intestinal barrier.
Mice were euthanized by cervical dislocation, and the distal ileum and colon
were removed,
placed in chilled Krebs solution opened along the mesenteric line and
carefully rinsed.
Preparations were then placed in Ussing chambers (Harvard Apparatus, Kent, UK,
exposed area
of 0.12 cm2) as described previously [75] with oxygenated (95% 02, 5% CO2)
Krebs buffer
maintained at 37 C. 4 kDa FITC-dextran was added to the mucosal chamber at a
final
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concentration of 2.5 mg/mL; 200 1_, samples were collected from the serosal
chamber every 30
min for the following 3 h.
Results
A significant reduction in in FITC concentration in the serosal chamber was
observed for ileum
5 preparations from strain NCIMB 43456-treated MIA mice relative to ileum
preparations of
vehicle-treated MIA mice. No significant difference was observed for colon
preparations from
strain NCIMB 43456-treated MIA mice versus vehicle-treated MIA mice, and no
significant
difference was observed for colon or ileum preparations from strain NCIMB
43456-treated
BTBR mice versus vehicle-treated BTBR mice (Figure 18).
10 .. Conclusions
Chronic treatment with strain NCIMB 43456 reduced leakiness of the ileal
intestinal barrier in
MIA mice. Chronic treatment with strain NCIMB 43456 did not affect colonic
intestinal barrier
permeability in MIA mice, and did not affect colonic or ileal intestinal
barrier permeability in
BTBR mice.
15 Example 2k ¨ Weight monitoring
Animal body weights were assessed weekly throughout the study to determine
whether chronic
treatment with strain NCIMB 43456 influences this particular parameter.
Conclusions
Chronic treatment with strain NCIMB 43456 does not influence body weight in
BTBR mice or
20 MIA mice (Figure 19).
Example 21¨ Organ weight and colon length
Methods
Animals were sacrificed in a random fashion regarding treatment and testing
condition; sampling
occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in
potassium EDTA
25 (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000
g. Plasma was isolated
and stored at ¨80 C for further analysis. The brain was quickly excised,
dissected and each brain
region was snap-frozen on dry ice and stored at ¨80 C for further analysis.
Spleen was removed,
collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758
Sigma + 10
% FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately
after culls
30 for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of
ileum and colon closest to
the caecum were excised, and the furthest lcm of tissue from the caecum were
used) were
mounted into the Ussing chambers for intestinal permeability assay (see
Example 2j above). A
further lcm of ileum and colon tissue was taken for tight junction gene
expression analysis. The
caecum was removed, weighed and stored at ¨80 C for SCFAs analysis.
Date Recue/Date Received 2022-07-25
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76
Results
For MIA mice, treatment with strain NCIMB 43456 did not result in a
significant difference in
colon length, caecum weight/body weight or spleen weight/body weight relative
to vehicle-
treated controls.
For BTBR mice, treatment with strain NCIMB 43456 did not result in a
significant difference in
colon length or spleen weight/body weight relative to vehicle-treated
controls. Caecum
weight/body weight was increased for strain NCIMB 43456-treated BTBR mice
relative to
vehicle-treated BTBR mice (p=0.06) (Figure 20).
Conclusions
Treatment with strain NCIMB 43456 did not affect colon length or spleen
weight/body weight
for BTBR or MIA mice. Treatment with strain NCIMB 43456 resulted in a non-
significant
increase in caecum weight/body weight in BTBR mice, but did not have an effect
on caecum
weight/body weight in MIA mice.
Example 2m ¨ Brainstem monoamine levels
Rationale
Serotonin and noradrenaline levels, and 5-HIAA/5-HT turnover were assessed in
the brainstem.
The brainstem encompasses a number of cell bodies for all the major
neurotransmitter systems
of the brain including serotonin and noradrenaline. 5-HIAA is the main
metabolite of serotonin
(5HT). Elevated 5HT and 5-HIAA levels and reduced 5-HIAA/5HT turnover were
found in
brainstem samples from suicides relative to those from non-psychiatric, sudden
death controls,
and may be linked to major depressive disorder (MDD) [76]
Methods
Neurotransmitter concentration was analysed by HPLC on samples from the
brainstem. Briefly,
brainstem tissue was sonicated in 500 IA of chilled mobile phase spiked with 4
ng/40 pi of N-
Methyl 5-HT (Sigma Chemical Co., UK) as internal standard. The mobile phase
contained 0.1
M citric acid, 5.6 mM octane- 1-sulphonic acid (Sigma), 0.1 M sodium
dihydrogen phosphate,
0.01 mM EDTA (Alkem/Reagecon, Cork) and 9% (v/v) methanol (Alkem/Reagecon),
and was
adjusted to pH 2.8 using 4 N sodium hydroxide (Alkem/Reagecon). Homogenates
were then
centrifuged for 15 min at 22,000 x g at 4 C and 40 IA of the supernatant
injected onto the HPLC
system which consisted of a SCL 10-Avp system controller, LECD 6A
electrochemical detector
(Shimadzu), a LC-10AS pump, a CTO-10A oven, a SIL-10A autoinjector (with
sample cooler
maintained at 40 C) and an online Gaston Degasser (ISS, UK). A reverse-phase
column (Kinetex
2.6 u C18 100 x 4.6 mm, Phenomenex) maintained at 30 C was employed in the
separation
(Flow rate 0.9 ml/min). The glassy carbon working electrode combined with an
Ag/AgC1
reference electrode (Shimdazu) operated a +0.8 V and the chromatograms
generated were
Date Recue/Date Received 2022-07-25
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77
analyzed using Class-VP 5 software (Shimadzu). The neurotransmitters were
identified by their
characteristic retention times as deteimined by standard injections, which run
at regular intervals
during the sample analysis. The ratios of peak heights of analyte versus
internal standard were
measured and compared with standard injection. Results were expressed as ng of
neurotransmitter per g fresh weight of tissue.
Results
The vehicle-treated BTBR mice had significantly reduced brainstem
noradrenaline levels and 5-
HIAA/5HT turnover relative to wild type C57BL/6 controls. Treatment of BTBR
mice with strain
NCIMB 43456 resulted in a significant increase in 5-HIAA/5HT turnover and a
significant
decrease in serotonin (5HT) in the brainstem relative to vehicle-treated BTBR
mice. Levels of
noradrenaline were also increased in strain NCIMB 43456-treated BTBR mice
relative to
vehicle-treated controls (p=0.061) (Figure 21).
The vehicle-treated MIA mice had significantly elevated brainstem 5HT levels
and significantly
reduced 5-HIAA/5HT turnover relative to wild type C57BL/6 controls. Treatment
of MIA mice
with strain NCIMB 43456 also led to a significant decrease in brainstem 5HT
levels relative to
vehicle-treated controls, but did not affect brainstem noradrenaline levels or
5-HIAA/5HT
turnover (Figure 21).
Conclusions
Chronic treatment with strain NCIMB 43456 resulted in a reduction in 5HT
levels in both BTBR
and MIA mice and an increase in 5-HIAA/5HT turnover, indicating that strain
NCIMB 43456
may be effective in the treatment of MDD. Chronic treatment with strain NCIMB
43456 also led
to an increase in noradrenaline levels in BTBR mice, but did not affect
noradrenaline levels or 5-
HIAA/5HT turnover in MIA mice.
Example 2n ¨ Amygdalar gene expression
Methods
Total RNA was extracted using the mirVanaTM miRNA Isolation kit (Ambion/Llife
technologies,
Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies)
according to the
manufacturers recommendations. RNA was quantified using NanoDropTM
spectrophotometer
(Theimo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the
manufacturer's
instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent,
Stockport, UK)
according to the manufacturer's procedure and an RNA integrity number (RIN)
was calculated.
RNA with RIN value >7 was used for subsequent experiments. RNA was reverse
transcribed to
cDNA using the Applied Biosystems High Capacity cDNA kit (Applied Biosystems,
Warrington,
UK) according to manufacturer's instructions. Briefly, Multiscribe Reverse
Transcriptase (50
U/A) (1)(2)(1)(10) was added as part of RT master mix, incubated for 25 C for
10 min, 37 C
Date Recue/Date Received 2022-07-25
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78
for 2 h, 85 C for 5 min and stored at 4 C. Quantitative PCR was carried out
using probes (6
carboxy fluorescein - FAM) designed by Applied Biosystems to mouse specific
targeted genes,
while using f3-actin as an endogenous control. Amplification reactions
contained 1 IA cDNA, 5
pi of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to
a total of 10
IA by the addition of RNase-free water. All reactions were performed in
triplicate using 96-well
plates on the LightCycler0480 System. Thermal cycling conditions were as
recommended by
the manufacturer (Roche) for 55 cycles. To check for amplicon contamination,
each run
contained no template controls in triplicate for each probe used. Cycle
threshold (Ct) values were
recorded. Data was normalized using f3-actin and transformed using the 2¨AACT
method and
presented as a fold change vs. control group.
mRNA levels for markers for the oxytocinergic system (oxytocin receptor),
endocrine system
(mineralocorticoid (Nr3c1); glucocorticoid receptor (Nr3c2); corticosterone
releasing factor
(CRF) and receptors; Brain derived neurotrophic factor (BDNF)); and
neurotransmitter systems
(NMDA receptor 2A (Grin2A); NMDA receptor 2B (Grin2B); GABAA receptor subunit
A2;
GABAB receptor subunit Bl; serotonin IA receptor (5HT1AR)) were assessed in
the amygdala.
RNA was extracted and quantified as described in example lb above.
Results
In MIA mice, chronic treatment with strain NCIMB 43456 led to a significant
increase in oxtocin
receptor, Grin2A and GABA A2 mRNA expression relative to vehicle-treated
controls. No
significant difference was observed in amygdalar expression for any other of
the mRNAs tested
(Figure 22).
In BTBR mice, chronic treatment with strain NCIMB 43456 led to a significant
decrease in
GABA A2 mRNA expression relative to vehicle-treated controls. No significant
difference was
observed in amygdalar expression for any other of the mRNAs tested (Figure
22).
Conclusions
Treatment with strain NCIMB 43456 resulted in increased amygdala mRNA
expression for
oxytocin receptor and the neurotransmitters, Grin2A GABA A2 for BTBR mice, but
resulted in
decreased amygdala GABA A2 mRNA expression in MIA mice.
Overall conclusions regarding strain NCIMB 43456 in the treatment of autistic
spectrum
disorders
Chronic treatment with strain NCIMB 43456 led to a reduction in the number of
marbles buried
during the marble burying test in BTBR and MIA mice, but did not affect the
time spent
grooming. Treatment with strain NCIMB 43456 also led to a significant increase
in the number
of entries made into the open arms of the maze by MIA mice in the elevated
plus maze test, but
did not have an effect on time spent in the open arms of the maze for MIA
mice, and did not
Date Recue/Date Received 2022-07-25
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79
affect the behaviour of BTBR mice in the elevated plus maze test. The ability
of strain NCIMB
43456 to improve anxiety related behaviours in mouse models of ASD is
promising and indicates
that it may be an effective therapeutic.
In both BTBR and MIA mice, chronic treatment with strain NCIMB 43456 led to a
complete
recovery of wild type social behaviour phenotypes in the three-chamber social
interaction test.
This strongly suggests that strain NCIMB 43456 treatment may be effective in
the treatment of
diseases characterised by impaired social behaviour.
Chronic treatment with strain NCIMB 43456 led to a reduction in depression-
like behaviour for
both BTBR and MIA mice in the forced swim test. Treatment with strain NCIMB
43456 also led
to a reduction of depression-like behaviour in MIA mice in the novel object
recognition test, but
did not significantly affect the behaviour of BTBR mice in this test. This is
concomitant with an
increase in brainstem 5-HIAA/5HT turnover following strain NCIMB 43456
treatment in BTBR
mice, and a decrease in brainstem serotonin levels following strain NCIMB
43456 treatment in
both BTBR and MIA mice, i.e. an improvement in markers that may be linked with
major
depressive disorder. These data strongly suggest that strain NCIMB 43456
treatment may be
effective in the treatment of depression and depressive disorders.
Chronic treatment with strain NCIMB 43456 did not significantly affect the
majority of the
physiological parameters measured. Treatment with strain NCIMB 43456 did not
affect the
weight of BTBR or MIA mice. Intestinal motility was also unaffected, as was
colon length and
spleen weight/body weight. Caecum weight/body weight was unaffected by strain
NCIMB 43456
treatment in MIA mice, but was non-significantly increased in BTBR mice. A
reduction in ileum
permeability (i.e. reduced leakiness) was observed following strain NCIMB
43456 treatment in
MIA mice, but no effect was observed for colon permeability. Treatment with
strain NCIMB
43456 did not affect intestinal barrier permeability in BTBR mice.
The EMA Guidelines on the clinical development of medicinal products for the
treatment of
autism spectrum disorder state that, due to the heterogeneity of the diseases,
it may not be possible
to achieve a significant effect on all core symptoms with a single compound,
and so short term
efficacy has to be demonstrated on at least one core symptom. The strain NCIMB
43456 live
biotherapeutic has shown effective treatment of at least one core symptom or
autistic spectrum
disorder, so it and related Enterococcus durans strains are expected to be
effective against human
disease.
SEQUENCES
SED ID NO:1 ¨ Enterococcus durans type strain 98D, 16S rRNA gene, GenBank
accession
NR 036922
1 tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgta cgcttctttt
Date Recue/Date Received 2022-07-25
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61 tccaccggag cttgctccac cggaaaaaga agagtggcga acgggtgagt aacacgtggg
121 taacctgccc atcagaaggg gataacactt ggaaacaggt gctaataccg tataacaatc
181 gaaaccgcat ggttttgatt tgaaaggcgc tttcgggtgt cgctgatgga tggacccgcg
241 gtgcattagc tagttggtga ggtaacggct caccaaggcc acgatgcata gccgacctga
5 301
gagggtgatc ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt
361 agggaatctt cggcaatgga cgaaagtctg accgagcaac gccgcgtgag tgaagaaggt
421 tttcggatcg taaaactctg ttgttagaga agaacaagga tgagagtaac tgttcatccc
481 ttgacggtat ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt
541 aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg tttcttaagt
10 601
ctgatgtgaa agccgccggc tcaaccgggg agggtcattg gaaactggga gacttgagtg
661 cagaagagga gagtggaatt ccatgtgtag cggtgaaatg cgtagatata tggaggaaca
721 ccagtggcga aggcggctct ctggtctgta actgacgctg aggctcgaaa gcgtggggag
781 caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttggag
841 ggtttccgcc cttcagtgct gcagctaacg cattaagcac tccgcctggg gagtacgacc
15 901
gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt
961 aattcgaagc aacgcgaaga accttaccag gtcttgacat cctttgacca ctctagagat
1021 agagcttccc cttcgggggc aaagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg
1081 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttgttagttg ccatcattta
1141 gttgggcact ctagcaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa
20 1201
tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggaagta caacgagtcg
1261 cgaagtcgcg aggctaagct aatctcttaa agcttctctc agttcggatt gtaggctgca
1321 actcgcctac atgaagccgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac
1381 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg
1441 gtgaggtaac cttttggagc cagccgccta aggtgggata gatgattggg gtgaagtcgt
25 1501 aacaaggtag ccgtatcgga aggtgcggct ggat
SED ID NO:2 ¨ 16srRNA sequence for Enterococcus durans strain NCIMB 43456
assembled
from 2 reads using Geneious
30 AAAAGGCGGCTGGCTCCAAAGGTTACCTCAMCGACTTCGGGTGTTACAAACTCTCG
TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTG
ATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCG
AACTGAGAGAAGCTTTAAGAGATTAGCTTAGCCTCGCGACTTCGCGACTCGTTGTA
CTTCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGT
35 CATCCCCACCTTCC TCCGGTTTGTCAC CGGCAGTCTTGC TAGAGTGCC CAACTRAAT
GATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC
GACACGAGC TGACGACAACCATGCAC CAC CTGTCAC TTTGCCCCCGAAGGGGAAG
CTCTATCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGC
TTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGT
40 TTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCAC
TGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCGTGGACTACCA
GGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACC
AGAGAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACCGCTACA
CATGGAATTCCACTCTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCC
Date Recue/Date Received 2022-07-25
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81
CCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTA
CGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCAC
GTAGTTAGCCGTGGCTTTCTGGTTAGATACCGTCAAGGGATGAACAGTTACTCTCA
TCCTTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGC
GGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCG
TAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGC
TATGCATCGTGGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCACCGCGGG
TCCATCCATCAGCGACACCCGAAAGCGCCTTTCAAATCAAAACCATGCGGTTTCGA
TTGTTATACGGTATTAGCAC CTGTTTCCAAGTGTTATCC CC TTCTGATGGGCAGGTT
AC CCACGTGTTACTCACCCGTTCGC CACTCTTCTTTTTCC GGTGGAGCAAGCTC CGG
TGAAAAAGAAGCGTACGACTTGCATGTAT
Date Recue/Date Received 2022-07-25
CA 03173055 2022-07-25
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