U.S. patent application number 17/530517 was filed with the patent office on 2022-06-16 for compositions comprising bacterial strains.
The applicant listed for this patent is 4D Pharma Research Limited. Invention is credited to John CRYAN, Ted DINAN, Imke Elisabeth MULDER, Helene SAVIGNAC, Alexander James STEVENSON.
Application Number | 20220184145 17/530517 |
Document ID | / |
Family ID | 1000006170065 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220184145 |
Kind Code |
A1 |
SAVIGNAC; Helene ; et
al. |
June 16, 2022 |
COMPOSITIONS COMPRISING BACTERIAL STRAINS
Abstract
The invention provides compositions comprising bacterial strains
for treating and preventing central nervous system disorders and
conditions.
Inventors: |
SAVIGNAC; Helene; (Aberdeen,
GB) ; MULDER; Imke Elisabeth; (Aberdeen, GB) ;
STEVENSON; Alexander James; (Aberdeen, GB) ; DINAN;
Ted; (County Cork, IE) ; CRYAN; John; (County
Cork, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
4D Pharma Research Limited |
Aberdeen |
|
GB |
|
|
Family ID: |
1000006170065 |
Appl. No.: |
17/530517 |
Filed: |
November 19, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16692667 |
Nov 22, 2019 |
|
|
|
17530517 |
|
|
|
|
PCT/GB2018/051389 |
May 22, 2018 |
|
|
|
16692667 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 25/18 20180101; A61K 35/74 20130101; A61P 25/24 20180101; A61K
9/48 20130101; A61K 35/741 20130101; A61P 25/00 20180101; A61K
9/0053 20130101; A61K 9/0031 20130101; A61P 25/22 20180101; A61P
25/28 20180101 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61P 25/28 20060101 A61P025/28; A61P 25/22 20060101
A61P025/22; A61P 25/18 20060101 A61P025/18; A61P 25/24 20060101
A61P025/24; A61P 25/16 20060101 A61P025/16; A61P 25/00 20060101
A61P025/00; A61K 9/00 20060101 A61K009/00; A61K 35/741 20060101
A61K035/741; A61K 9/48 20060101 A61K009/48 |
Foreign Application Data
Date |
Code |
Application Number |
May 22, 2017 |
GB |
1708176.1 |
Sep 6, 2017 |
GB |
1714298.5 |
Sep 6, 2017 |
GB |
1714305.8 |
Sep 6, 2017 |
GB |
1714309.0 |
Oct 9, 2017 |
GB |
1716493.0 |
Nov 9, 2017 |
GB |
1718551.3 |
Claims
1.-81. (canceled)
82. A method of treating a disease or a condition associated with
dysfunction of a microbiota-gut-brain axis in a subject in need
thereof, comprising administering to the subject a pharmaceutical
composition that comprises a therapeutically effective amount of a
bacterial strain of the genus Blautia comprising a 16S rRNA gene
sequence that has at least 95% sequence identity to the
polynucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID
NO:7, wherein the administering is effective to treat the disease
or the condition associated with dysfunction of the
microbiota-gut-brain axis in the subject.
83. The method of claim 82, wherein the disease or the condition
associated with the microbiota-gut-brain axis is a disease or a
condition of the central nervous system (CNS).
84. The method of claim 82, wherein the disease or the condition
associated with the microbiota-gut-brain axis comprises a
neurodevelopmental disorder or a neuropsychiatric condition.
85. The method of claim 82, wherein the disease or the condition
associated with the microbiota-gut-brain axis comprises 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 disease, Parkinson's disease,
chronic pain, motor neuron disease, Huntington's disease,
Guillain-Barre syndrome, or meningitis.
86. The method of claim 82, wherein the treating comprises treating
one or more symptoms, tissue damages, or behaviors associated with
the disease or the condition.
87. The method of claim 82, wherein the bacterial strain modulates
a level of commensal metabolites in the subject.
88. The method of claim 82, wherein the bacterial strain increases
the level of butyrate in the subject.
89. The method of claim 82, wherein the pharmaceutical composition
is formulated for delivery to an intestine of the subject.
90. The method of claim 82, wherein the bacterial strain is live
and capable of at least partially colonizing an intestine of the
subject.
91. The method of claim 82, wherein the pharmaceutical composition
is formulated for oral administration.
92. The method of claim 82, wherein the bacterial strain is
lyophilized.
93. The method of claim 82, wherein the therapeutically effective
amount comprises from about 1.times.10.sup.3 to about
1.times.10.sup.11 colony forming units (CFU).
94. The method of claim 82, wherein the at least 95% sequence
identity is determined by a Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2.
95. The method of claim 82, wherein the pharmaceutical composition
comprises one or more pharmaceutically acceptable excipients or
carriers.
96. The method of claim 82, wherein the bacterial strain is of the
species Blautia stercoris, Blautia wexlerae, or Blautia
hydrogenotrophica.
97. The method of claim 82, wherein the bacterial strain comprises
a 16S rRNA gene sequence that has at least 98% sequence identity to
the polynucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID
NO:7, as determined by a Smith-Waterman homology search algorithm
using an affine gap search with a gap open penalty of 12 and a gap
extension penalty of 2.
98. The method of claim 82, wherein the bacterial strain comprises
a 16S rRNA gene sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID
NO:7.
99. The method of claim 82, wherein the bacterial strain is the
bacterial strain deposited under accession number NCIMB 42381, the
bacterial strain deposited under accession number NCIMB 42486, or
the bacterial strain deposited under accession number DSM
14294.
100. A method of modulating a microbiota-gut-brain axis in a
subject in need thereof, comprising administering to the subject a
pharmaceutical composition that comprises a therapeutically
effective amount of a bacterial strain of the genus Blautia
comprising a 16S rRNA gene sequence that has at least 95% sequence
identity to the polynucleotide sequence of SEQ ID NO:2, SEQ ID
NO:4, or SEQ ID NO:7, as determined by a Smith-Waterman homology
search algorithm using an affine gap search with a gap open penalty
of 12 and a gap extension penalty of 2, wherein the administering
is effective to modulate the microbiota-gut-brain axis in the
subject.
101. The method of claim 100, wherein the bacterial strain
comprises a 16S rRNA gene sequence that has at least 98% sequence
identity to the polynucleotide sequence of SEQ ID NO:2, SEQ ID
NO:4, or SEQ ID NO:7, as determined by a Smith-Waterman homology
search algorithm using an affine gap search with a gap open penalty
of 12 and a gap extension penalty of 2.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/692,667, filed Nov. 22, 2019, which is a continuation of
International Application No. PCT/GB2018/051389, filed May 22,
2018, which claims the benefit of Great Britain Application No.
1708176.1, filed May 22, 2017, Great Britain Application No.
1714309.0, filed Sep. 6, 2017, Great Britain Application No.
1714298.5, filed Sep. 6, 2017, Great Britain Application No.
1714305.8, filed Sep. 6, 2017, Great Britain Application No.
1716493.0, filed Oct. 9, 2017, and Great Britain Application No.
1718551.3, filed Nov. 9, 2017, all of which are hereby incorporated
by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ANSI format and is hereby
incorporated by reference in its entirety. Said ANSI copy, created
on Nov. 17, 2021, is named 56708_723_302_SL and is 4,325,277 bytes
in size.
TECHNICAL FIELD
[0003] 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
[0004] 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 500-1000 different phylotypes
belonging essentially to two major bacterial divisions, the
Bacteroidetes and the Firmicutes [2]. 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. Similarly, the
immunological importance of the gut microbiota is well-recognized
and is exemplified in germfree animals which have an impaired
immune system that is functionally reconstituted following the
introduction of commensal bacteria [3-5].
[0005] The discovery of the size and complexity of the human
microbiome has resulted in an on-going evaluation of many concepts
of health and disease. Certainly, dramatic changes in microbiota
composition have been documented in gastrointestinal disorders such
as inflammatory bowel disease (IBD)[6-9]. More recently, there is
increased interest in the art regarding alternations in the gut
microbiome that may play a pathophysiological role in human brain
diseases [10]. Preclinical and clinical evidence are strongly
suggesting a link between brain development and microbiota
[11].
[0006] 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 [12], and an imbalance of the
Bacteroidetes and Firmicutes phyla has also been implicated in
brain development disorders [13]. 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 [14]. 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.
[0007] 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, [14-17]). Also, certain strains, including mostly
Lactobacillus and Bifidobacterium strains, have been proposed for
use in treating various inflammatory and autoimmune diseases that
are not directly linked to the intestines (see [18] and [19] for
reviews). In addition, a range of probiotics have been investigated
in animal models to determine 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 [20-22]. 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.
[0008] There is a growing body of evidence to suggest that the
microbiota-gut-brain axis is affected in autism spectrum disorders
(ASD) and other neurodevelopmental and neuropsychiatric disorders.
Animal models have provided considerable insight into how the
microbiota may be involved in ASD. Furthermore, 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, including
the maternal immune activation (MIA) mouse model and the black and
tan, brachyuric (BTBR) mouse. The BTBR mouse is a genetically
modified, inbred mouse strain that displays a number of behaviours
associated with ASD such as impaired sociability, repetitive
behaviour and increased anxiety. Moreover, these mice also exhibit
gastrointestinal dysfunctions along with alterations to the
composition of the gut microbiota. Consequently, it represents an
appropriate animal model for investigating the role of the
microbiota-gut-brain axis in ASD.
[0009] Accordingly, there is a requirement in the art for new
methods of treating central nervous system disorders. There is also
a requirement for the potential effects of gut bacteria to be
characterised so that new therapies using gut bacteria can be
developed.
SUMMARY OF THE INVENTION
[0010] The inventors have developed new therapies for treating and
preventing central nervous system disorders. In particular, the
inventors have developed new therapies for treating and preventing
central nervous system disorders and conditions mediated by the
microbiota-gut-brain axis. In particular, the inventors have
identified that bacterial strains of the genus Blautia can be
effective for treating and preventing diseases and conditions
mediated by the microbiota-gut-brain axis. As described in the
examples, oral administration of compositions comprising a Blautia
strain may reduce symptoms associated with dysfunction of the
microbiota-gut-brain axis in a mouse model of autism spectrum
disorders. In addition, as described in the examples, oral
administration of compositions comprising a Blautia strain may
modulate the levels of signalling molecules associated with the
function of the microbiota-gut-brain axis, and neurodevelopmental
and neuropsychiatric disorders.
[0011] Therefore, in a first embodiment, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing a central nervous system
disorder or condition. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia stercoris for use in a method of treating or preventing a
central nervous system disorder or condition. Compositions using
Blautia stercoris may be particularly effective for treating a
central nervous system disorder or condition. In certain
embodiments, the invention provides a composition comprising a
bacterial strain of the species Blautia wexlerae for use in a
method of treating or preventing a central nervous system disorder
or condition. In particular embodiments, the central nervous system
disorder or condition is mediated by the microbiota-gut-brain axis.
In further embodiments, the invention provides a composition
comprising a bacterial strain of the genus Blautia, for use in a
method of treating or preventing a neurodevelopmental disorder or a
neuropsychiatric condition. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia stercoris for use in a method of treating or preventing a
neurodevelopmental disorder or neuropsychiatric condition.
Compositions using Blautia stercoris may be particularly effective
for treating a neurodevelopmental disorder or neuropsychiatric
condition. In certain embodiments, the invention provides a
composition comprising a bacterial strain of the species Blautia
wexlerae for use in a method of treating or preventing a
neurodevelopmental disorder or neuropsychiatric condition. The
inventors have identified that treatment with bacterial strains
from this genus can provide clinical benefits in mouse models of
central nervous system disorders, in particular those mediated by
the microbiota-gut-brain axis. The inventors have identified that
treatment with bacterial strains from this genus 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. Compositions using Blautia stercoris may be particularly
effective at modulating signalling in the central, autonomic and
enteric nervous systems; modulating the activity of the
hypothalamus-pituitary-adrenal (HPA) axis pathway; modulating
neuroendocrine and/or neuroimmune pathways; and/or modulating the
levels of commensal metabolites, inflammatory markers and/or
gastrointestinal permeability of a subject. In certain embodiments
compositions using Blautia wexlerae may also be effective.
[0012] In particular embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of 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; 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; and/or chronic pain. In further embodiments,
the compositions of the invention may be useful for treating or
preventing multiple sclerosis; motor neuron disease; Huntington's
disease; Guillain-Barre syndrome and/or meningitis. The effect
shown for the bacterial strains from the genus Blautia on the
microbiota-gut-brain axis and on diseases mediated by the
microbiota-gut-brain axis may provide 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 genus Blautia, for use in a method of treating comorbidities
associated with diseases and conditions mediated by the
microbiota-gut-brain axis, such as those listed above. In
particularly preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating gastrointestinal comorbidities
associated with diseases and conditions mediated by the
microbiota-gut-brain axis, such as those 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 other central
nervous system disorders including those listed above [23-25].
[0013] In particularly preferred embodiments, the invention
provides a composition comprising a bacterial strain of the genus
Blautia, for use in a method of treating or preventing autism
spectrum disorders, such as autism. The inventors have identified
that treatment with Blautia strains can reduce symptom severity in
a mouse model of autism spectrum disorders and can prevent or
reduce stereotyped, repetitive, compulsive and anxious behaviour.
In preferred embodiments, the invention provides a composition
comprising a bacterial strain of the genus Blautia, for use in the
treatment of autism spectrum disorders. Compositions using Blautia
may be particularly effective for treating autism spectrum
disorders. In preferred embodiments, the invention provides a
composition for use in reducing stereotyped, repetitive, compulsive
or anxious behaviour, in particular in the treatment of autism
spectrum disorders. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the genus
Blautia, for use in the treatment of the behavioural symptoms of
autism spectrum disorders. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the genus
Blautia 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 genus Blautia, for use in the treatment of the behavioural and
gastrointestinal symptoms of autism spectrum disorders. Treatment
with Blautia 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
permeability of a subject, all of which are implicated in the
neuropathology of autism spectrum disorders. In certain
embodiments, treatment with Blautia strains may modulate the levels
of oxytocin and/or vasopressin hormones. In preferred embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia stercoris for use in a method of treating or
preventing autism spectrum disorders. Compositions using Blautia
stercoris may be particularly effective for treating autism
spectrum disorders. In certain embodiments, the invention provides
a composition comprising a bacterial strain of the species Blautia
wexlerae for use in a method of treating or preventing autism
spectrum disorders.
[0014] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, 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 in the treatment of OCD. Treatment with
Blautia 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
and/or gastrointestinal permeability of a subject, all of which are
implicated in the neuropathology of OCD. In preferred embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia stercoris for use in a method of treating or
preventing OCD. Compositions using Blautia stercoris may be
particularly effective for treating OCD. In certain embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia wexlerae for use in a method of treating or
preventing OCD.
[0015] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing major depressive disorder
(MDD). Treatment with Blautia 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 genus Blautia, for use in the treatment of depression.
Compositions using Blautia strains may be particularly effective
for treating depression. In preferred embodiments, the invention
provides a composition for use in reducing stereotyped, repetitive,
compulsive or anxious behaviour in the treatment of depression.
Treatment with Blautia 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 MDD. In certain embodiments, treatment with
Blautia strains may modulate the levels of oxytocin and/or
vasopressin hormones. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia stercoris for use in a method of treating or preventing
MDD. Compositions using Blautia stercoris may be particularly
effective for treating MDD. In certain embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia wexlerae for use in a method of treating or preventing
MDD.
[0016] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing anxiety disorders.
Treatment with Blautia strains reduces disease incidence and
disease severity in a mouse model of anxiety in the examples of
this application. In preferred embodiments, the invention provides
a composition comprising a bacterial strain of the genus Blautia,
for use in the treatment of anxiety disorder. Compositions using
Blautia strains 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 in the treatment of anxiety. In preferred
embodiments, the invention provides a composition comprising a
bacterial strain of the species Blautia stercoris for use in a
method of treating or preventing anxiety disorders. Compositions
using Blautia stercoris may be particularly effective for treating
anxiety disorders. In certain embodiments, the invention provides a
composition comprising a bacterial strain of the species Blautia
wexlerae for use in a method of treating or preventing anxiety
disorders.
[0017] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing stress disorders, such as
post-traumatic stress disorder. Compositions comprising a bacterial
strain of the genus Blautia may reduce stress in mouse models of
stress disorders. Treatment with Blautia 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 stress disorder. In certain
embodiments, treatment with Blautia strains may modulate the levels
of oxytocin and/or vasopressin hormones. In preferred embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia stercoris for use in a method of treating or
preventing stress disorders. Compositions using Blautia stercoris
may be particularly effective for treating stress disorders. In
certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia wexlerae for
use in a method of treating or preventing stress disorders.
[0018] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing schizophrenia spectrum
and psychotic disorders, such as schizophrenia. Compositions
comprising a bacterial strain of the genus Blautia may improve
positive and negative symptoms in mouse models of schizophrenia
spectrum and psychotic disorders. Treatment with Blautia 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 preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia stercoris for use in a method of treating or preventing
schizophrenia spectrum and psychotic disorders. Compositions using
Blautia stercoris may be particularly effective for treating
schizophrenia spectrum and psychotic disorders. In certain
embodiments, the invention provides a composition comprising a
bacterial strain of the species Blautia wexlerae for use in a
method of treating or preventing schizophrenia spectrum and
psychotic disorders.
[0019] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing bipolar disorder.
Compositions comprising a bacterial strain of the genus Blautia may
reduce occasions of mania and/or depression in mouse models of
bipolar disorder. Treatment with Blautia 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 bipolar disorder. In certain
embodiments, treatment with Blautia strains may modulate the levels
of oxytocin and/or vasopressin hormones. In preferred embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia stercoris for use in a method of treating or
preventing bipolar disorder. Compositions using Blautia stercoris
may be particularly effective for treating bipolar disorder. In
certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia wexlerae for
use in a method of treating or preventing bipolar disorder.
[0020] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing neurocognitive disorders,
such as Alzheimer's disease. Compositions comprising a bacterial
strain of the species genus Blautia may improve cognitive and
behavioural functioning in mouse models of neurocognitive
disorders. Treatment with Blautia 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
neurocognitive disorders. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Blautia stercoris for use in a method of treating or preventing
neurocognitive disorders. Compositions using Blautia stercoris may
be particularly effective for treating neurocognitive disorders. In
certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia wexlerae for
use in a method of treating or preventing neurocognitive
disorders.
[0021] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Blautia, for
use in a method of treating or preventing Parkinson's disease.
Compositions comprising a bacterial strain of the genus Blautia may
improve motor and cognitive functions in mouse models of
Parkinson's disease. Treatment with Blautia 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 Blautia strains may modulate the levels
of oxytocin and/or vasopressin hormones. In preferred embodiments,
the invention provides a composition comprising a bacterial strain
of the species Blautia stercoris for use in a method of treating or
preventing Parkinson's disease. Compositions using Blautia
stercoris may be particularly effective for treating Parkinson's
disease. In certain embodiments, the invention provides a
composition comprising a bacterial strain of the species Blautia
wexlerae for use in a method of treating or preventing Parkinson's
disease.
[0022] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing a
central nervous system disorder or condition. Compositions using
Blautia hydrogenotrophica may be particularly effective for
treating a central nervous system disorder or condition.
[0023] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing a
neurodevelopmental disorder or neuropsychiatric condition.
Compositions using Blautia hydrogenotrophica may be particularly
effective for treating a neurodevelopmental disorder or
neuropsychiatric condition.
[0024] Compositions using Blautia hydrogenotrophica may be
particularly effective at modulating signalling in the central,
autonomic and enteric nervous systems; modulating the activity of
the hypothalamus-pituitary-adrenal (HPA) axis pathway; modulating
neuroendocrine and/or neuroimmune pathways; and/or modulating the
levels of commensal metabolites, inflammatory markers and/or
gastrointestinal permeability of a subject. In a particularly
preferred embodiment, Blautia hydrogenotrophica modulates the
levels of butyrate. In certain embodiments, the modulation of the
levels of butyrate treats or prevents a central nervous system
disorder or condition.
[0025] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
autism spectrum disorders. Compositions using Blautia
hydrogenotrophica may be particularly effective for treating autism
spectrum disorders.
[0026] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
OCD. Compositions using Blautia hydrogenotrophica may be
particularly effective for treating OCD.
[0027] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
MDD. Compositions using Blautia hydrogenotrophica may be
particularly effective for treating MDD.
[0028] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
anxiety disorders. Compositions using Blautia hydrogenotrophica may
be particularly effective for treating anxiety disorders.
[0029] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
stress disorders. Compositions using Blautia hydrogenotrophica may
be particularly effective for treating stress disorders.
[0030] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
schizophrenia spectrum and psychotic disorders. Compositions using
Blautia hydrogenotrophica may be particularly effective for
treating schizophrenia spectrum and psychotic disorders.
[0031] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
bipolar disorder. Compositions using Blautia hydrogenotrophica may
be particularly effective for treating bipolar disorder.
[0032] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
neurocognitive disorders. Compositions using Blautia
hydrogenotrophica may be particularly effective for treating
neurocognitive disorders.
[0033] In certain embodiments, the invention provides a composition
comprising a bacterial strain of the species Blautia
hydrogenotrophica for use in a method of treating or preventing
Parkinson's disease. Compositions using Blautia hydrogenotrophica
may be particularly effective for treating Parkinson's disease.
[0034] 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.
[0035] In preferred embodiments of the invention, the bacterial
strain in the composition is of Blautia stercoris. Closely related
strains may also be used, such as bacterial strains that have a 16s
rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or
99.9% identical to the 16s rRNA sequence of a bacterial strain of
Blautia stercoris. Preferably, the bacterial strain has a 16s rRNA
sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% 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.
[0036] In preferred embodiments of the invention, the bacterial
strain in the composition is of Blautia wexlerae. Closely related
strains may also be used, such as bacterial strains that have a 16s
rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or
99.9% identical to the 16s rRNA sequence of a bacterial strain of
Blautia wexlerae. Preferably, the bacterial strain has a 16s rRNA
sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%
identical to SEQ ID NO:3 or 4. Preferably, the sequence identity is
to SEQ ID NO:4. Preferably, the bacterial strain for use in the
invention has the 16s rRNA sequence represented by SEQ ID NO:4.
[0037] In preferred embodiments of the invention, the bacterial
strain in the composition is of Blautia hydrogenotrophica. Closely
related strains may also be used, such as bacterial strains that
have a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial
strain of Blautia hydrogenotrophica. Preferably, the bacterial
strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,
99%, 99.5% or 99.9% identical to SEQ ID NO:7. Most preferably, the
bacterial strain in the composition is the Blautia
hydrogenotrophica strain deposited under accession number DSM
14294.
[0038] In certain embodiments, the composition of the invention is
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 administration is convenient
for patients and practitioners and allows delivery to and/or
partial or total colonisation of the intestine.
[0039] In certain embodiments, the composition of the invention
comprises one or more pharmaceutically acceptable excipients or
carriers.
[0040] In certain embodiments, the composition of the invention
comprises a bacterial strain that has been lyophilised.
Lyophilisation is an effective and convenient technique for
preparing stable compositions that allow delivery of bacteria.
[0041] In certain embodiments, the invention provides a food
product comprising the composition as described above.
[0042] In certain embodiments, the invention provides a vaccine
composition comprising the composition as described above.
[0043] Additionally, the invention provides a method of treating or
preventing a disease or condition mediated by dysfunction of the
microbiota-gut-brain axis, comprising administering a composition
comprising a bacterial strain of the genus Blautia.
[0044] In developing the above invention, the inventors have
identified and characterised a bacterial strain that is
particularly useful for therapy. The Blautia stercoris 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 Blautia
stercoris strain deposited under accession number NCIMB 42381, or a
derivative thereof. The invention also provides compositions
comprising such cells, or biologically pure cultures of such cells.
The invention also provides a cell of the Blautia stercoris strain
deposited under accession number NCIMB 42381, or a derivative
thereof, for use in therapy, in particular for the diseases
described herein.
[0045] In especially preferred embodiments, the invention provides
a composition comprising the strain deposited under accession
number NCIMB 42381, 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 42381,
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 42381, 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 42381, for use in a method of reducing
stereotyped, repetitive, compulsive or anxious behaviour,
especially in the treatment of autism.
[0046] In developing the above invention, the inventors have
identified and characterised a further bacterial strain that is
particularly useful for therapy. The Blautia wexlerae 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 Blautia
wexlerae strain deposited under accession number NCIMB 42486, or a
derivative thereof. The invention also provides compositions
comprising such cells, or biologically pure cultures of such cells.
The invention also provides a cell of the Blautia wexlerae strain
deposited under accession number NCIMB 42486, or a derivative
thereof, for use in therapy, in particular for the diseases
described herein.
[0047] In especially preferred embodiments, the invention provides
a composition comprising the strain deposited under accession
number NCIMB 42486, 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 42486,
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 42486, 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 42486, for use in a method of reducing
stereotyped, repetitive, compulsive or anxious behaviour,
especially in the treatment of autism.
[0048] In developing the above invention, the inventors have
identified and characterised a bacterial strain that is
particularly useful for therapy. The Blautia hydrogenotrophica
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
Blautia hydrogenotrophica strain deposited under accession number
DSM 14294, or a derivative thereof. The invention also provides
compositions comprising such cells, or biologically pure cultures
of such cells. The invention also provides a cell of the Blautia
hydrogenotrophica strain deposited under accession number DSM
14294, or a derivative thereof, for use in therapy, in particular
for the diseases described herein.
[0049] In especially preferred embodiments, the invention provides
a composition comprising the strain deposited under accession
number DSM 14294, 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 DSM 14294
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 DSM 14294, 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 DSM 14294, for use in a method of reducing
stereotyped, repetitive, compulsive or anxious behaviour,
especially in the treatment of autism.
BRIEF DESCRIPTION OF DRAWINGS
[0050] FIGS. 1A-1B: Effect of treatment with MRX006 on C57Bl/6 mice
in the 3-chamber test. *p<0.05 novel versus familiar (FIG. 1A)
and **p<0.01 object versus animal (FIG. 1B).
[0051] FIG. 2: Effect of treatment with MRX006 on C57Bl/6 mice in
the forced swim test. Mrx006 significantly different to the vehicle
group **p<0.01; Vehicle group significantly different to the
naive group ##p<0.01.
[0052] FIG. 3: Effect of treatment with MRX006 on C57Bl/6 mice in
the tail suspension test.
[0053] FIGS. 4A-4C: Effect of treatment with MRX006 on C57Bl/6 mice
in the fear conditioning test. * MRX006 significantly different to
the Vehicle group; # Vehicle significantly different to the Naive
group; *p<0.05, #p<0.05, ##p<0.01, ###p<0.001. FIG. 4A,
Acquisition; FIG. 4B, Retrieval; FIG. 4C, Extinction.
[0054] FIG. 5: Effect of treatment with MRX006 on C57Bl/6 mice in
the novel object recognition test. # Significantly different vs.
familiar object within groups; #p<0.05.
[0055] FIG. 6: Effect of treatment with MRX006 on C57Bl/6 mice in
the marble burying test.
[0056] FIGS. 7A-7D: Effect of treatment with MRX006 on C57Bl/6 mice
in the elevated plus maze test. FIG. 7A, Time spent in closed arms;
FIG. 7B, % Time in closed arms; FIG. 7C, Time spent in open arms;
FIG. 7D, % Time in open arms.
[0057] FIG. 8: Effect of treatment with MRX006 on stress induced
hyperthermia in C57Bl/6 mice. # Vehicle group significantly
different to naive group, #p<0.05.
[0058] FIG. 9: Effect of treatment with MRX006 on circulating
oxytocin levels in C57Bl/6 mice. * Mrx006 significantly different
from vehicle group; *p<0.05.
[0059] FIGS. 10A-10B: Effect of treatment with MRX006 on
corticosterone plasma levels in C57Bl/6 mice. * Significantly
different to naive (FIG. 10A) or vehicle (FIG. 10B) group;
*p<0.05.
[0060] FIG. 11: Effect of treatment with MRX006 on gut permeability
in C57Bl/6 mice.
[0061] FIG. 12: Effect of treatment with MRX006 on organ weight and
colon length in C57Bl/6 mice.
[0062] FIGS. 13A-13F: Effect of treatment with MRX006 on BTBR mice
in the three chamber social interaction test. ##p<0.01 relative
to respective within group. ###p<0.001 relative to respective
within group. *p<0.05 relative to vehicle group. FIG. 13A, Time
in chamber, objective vs conspecific; FIG. 13B, Time in chamber,
familiar vs novel; FIG. 13C, % Time, % Time spent investigating
novel conspecific; FIG. 13D, Interaction time, objective vs
conspecific; FIG. 13E, Interaction time, familiar vs novel; FIG.
13F, % Time interacting, % Time spent investigating novel
conspecific.
[0063] FIG. 14: Effect of treatment with MRX006 on BTBR mice in the
forced intruder test.
[0064] FIG. 15: Effect of treatment with MRX006 on BTBR mice in the
marble burying test. *p<0.05 relative to vehicle group as
determined by a priori comparisons.
[0065] FIGS. 16A-16B: Effect of treatment with MRX006 on BTBR mice
in the grooming test. *p<0.05 relative to vehicle group.
**p<0.01 relative to vehicle group as revealed by a priori
comparisons.
[0066] FIG. 16A, Grooming time, vehicle vs Mrx006; FIG. 16B
Grooming time, grooming.
[0067] FIGS. 17A-17D: Effect of treatment with MRX006 on BTBR mice
in elevated plus maze test. FIG. 17A, % time spent in closed arms;
FIG. 17B, % time spent in open arms; FIG. 17C, No. entries to
closed arm; FIG. 17D, No. entries to open arm.
[0068] FIGS. 18A-18F: Effect of treatment with MRX006 on BTBR mice
in the open field arena. *p<0.05 relative to vehicle group. FIG.
18A, Total distance moved; FIG. 18B, Time spent in outer zone; FIG.
18C, Time spent in inner zone; FIG. 18D, Total distance moved; FIG.
18E, Time spent in outer zone; FIG. 18F, Time spent in inner
zone.
[0069] FIG. 19: Effect of treatment with MRX006 on BTBR mice in the
forced swim test.
[0070] FIGS. 20A-20B: Effect of treatment with MRX006 on BTBR mice
in female urine sniffing test. #p<0.05 relative to water vehicle
group. **p<0.01 relative to vehicle group. FIG. 20A, Time spent
sniffing, Female urine sniffing test; FIG. 20B, Female urine
sniffing test, vehicle vs Mrx006, water vs urine.
[0071] FIGS. 21A-21C: Effect of treatment with MRX006 on BTBR mice
in the novel object recognition test. FIG. 21A, NOR day 1; FIG.
21B, NOR day 2; FIG. 21C, NOR discrimination Index.
[0072] FIGS. 22A-22B: Effect of treatment with MRX006 on ex vivo
gastrointestinal permeability in BTBR mice. FIG. 22A, Ex vivo
colon; FIG. 22B, Ex vivo ileum.
[0073] FIG. 23: Effect of treatment with MRX006 on in vivo
gastrointestinal permeability in BTBR mice.
[0074] FIG. 24: Effect of treatment with MRX006 on in vivo
gastrointestinal motility in BTBR mice. *p<0.05 relative to
vehicle group as revealed by a priori comparisons.
[0075] FIG. 25: Effect of treatment with MRX006 on stress-induced
corticosterone plasma levels in BTBR mice.
[0076] FIGS. 26A-26D: Effect of treatment with MRX006 on organ
weight and colon length in BTBR mice. FIG. 26A, Adrenal weight %
body weight; FIG. 26B, Spleen weight % body weight; FIG. 26C,
Caecum weight % body weight; FIG. 26D, Colon length.
[0077] FIG. 27: Effect of treatment with MRX006 on weight of BTBR
mice over time.
[0078] FIG. 28: Chronic treatment with Mrx006 decreased the number
of marbles buried in a MIA mice model. #p<0.05 relative to
control group; **p<0.01 relative to vehicle MIA group.
[0079] FIGS. 29A-29B: Effect of chronic treatment with MRX006 on
sociability in MIA mice in the social transmission of food
preference test. FIG. 29A, 0 Hour; FIG. 29B, 24 Hour.
[0080] FIGS. 30A-30C: Chronic treatment with MRX006 attenuates
stress-induced locomotor activity caused by exposure to the open
field arena in MIA mice. ##p<0.01 relative to control group,
*p<0.05 relative to vehicle MIA group. FIG. 30A, Total distance
moved; FIG. 30B, Time spent in outer zone; FIG. 30C, Time spent in
inner zone.
[0081] FIG. 31: Effect of chronic treatment with MRX006 on
depressive-like behaviour in MIA mice in the female urine sniffing
test. &p<0.05 relative to respective water group.
[0082] FIG. 32: Effect of treatment with MRX006 on in vivo
gastrointestinal motility in MIA mice.
[0083] FIGS. 33A-33C: Effect of treatment with MRX006 on organ
weight and colon length in MIA mice. FIG. 33A, Colon length; FIG.
33B, Caecum weight % body weight; FIG. 33C, Spleen weight % body
weight.
[0084] FIG. 34: Effect of treatment with MRX006 on circulating
cytokine concentrations in BTBR mice.
[0085] FIGS. 35A-35B: Effect of treatment with MRX008 on MIA mice
in the marble burying test. ##p<0.01 relative to control group.
FIG. 35A, control vs. vehicle vs. Mrx008; FIG. 35B, control vs.
vehicle.
[0086] FIGS. 36A-36B: Effect of chronic treatment with MRX008 on
MIA mice in the social transmission of food preference test. FIG.
36A, T0; FIG. 36B, T24.
[0087] FIG. 37: Effect of chronic treatment with MRX008 on MIA mice
in the forced swimming test.
[0088] FIG. 38: Effect of chronic treatment with MRX008 on
intestinal permeability.
[0089] FIG. 39: Effect of chronic treatment with MRX008 on
intestinal motility. ##p<0.01 relative to control group.
[0090] FIGS. 40A-40B: Effect of chronic treatment with MRX008 on
BTBR mice in the social transmission of food preference test. FIG.
40A, STFP T0; FIG. 40B, STFP 24 HR.
[0091] FIG. 41: Effect of chronic treatment with MRX008 on BTBR
mice in the forced intruder test.
[0092] FIG. 42: Effect of chronic treatment with MRX008 on BTBR
mice in the marble burying test.
[0093] FIGS. 43A-43D: Effect of chronic treatment with MRX008 on
BTBR mice in the elevated plus maze. FIG. 43A, % time spent in
closed arms; FIG. 43B, % time spent in open arms; FIG. 43C, No.
entries to closed arms; FIG. 43D, No. entries to open arms.
[0094] FIGS. 44A-44C: Effect of chronic treatment with MRX008 on
BTBR mice in the open field arena. *p<0.05 relative to vehicle
group as revealed by a priori pairwise comparisons. FIG. 44A,
Distance moved; FIG. 44B, Time spent in outer zone; FIG. 44C, Time
spent inner zone.
[0095] FIG. 45: Effect of chronic treatment with MRX008 on BTBR
mice in the forced swim test.
[0096] FIG. 46: Effect of chronic treatment with MRX008 on
depressive-like behaviour in BTBR mice in the female urine sniffing
test. ##p<0.01 relative to water vehicle group.
[0097] FIG. 47: Effect of chronic treatment with MRX008 on in vivo
intestinal motility in BTBR mice.
[0098] FIGS. 48A-48D: Effect of chronic treatment with MRX008 on
selective anatomical markers in BTBR mice. FIG. 48A, Adrenal weight
% body weight; FIG. 48B, Spleen weight % body weight; FIG. 48C,
Caecum weight % body weight; FIG. 48D, Colon length.
[0099] FIGS. 49A-49D: Effect of chronic treatment with MRX006 on
expression of oxytoxin, vasopressin and their respective receptors
in the hypothalamus of BTBR mice. *p<0.05 relative to the
vehicle group. FIG. 49A, OXTR mRNA; FIG. 49B, AVPR1b mRNA; FIG.
49C, OXT mRNA; FIG. 49D, AV mRNA.
[0100] FIGS. 50A-50D: Effect of chronic treatment with MRX006 on
expression of oxytoxin, vasopressin and their respective receptors
in the amygdala of BTBR mice. *p<0.05 relative to the vehicle
group. FIG. 50A, OXTR mRNA; FIG. 50B, AVPR1b mRNA; FIG. 50C, OXT
mRNA; FIG. 50D, AVP mRNA.
[0101] FIGS. 51A-51H: Effect of chronic treatment with Blautia
hydrogenotrophica and butyrate on BTBR mice in the open field
arena. The data in FIGS. 51B, 51D, 51F and 51H are identical to
that in FIGS. 51A, 51C, 51E and 51G, respectively, except the PBS
and LYO control numbers have been pooled. p.ltoreq.0.05: * vs.
C57BL/6 (same treatment, where applicable); # vs. PBS same
genotype; .sctn. BTBR: But vs. PBS or Bact vs. Lyo. PBS is the
negative control for butyrate administration; LYO is the negative
control for bacterial (Blautia hydrogenotrophica) administration;
BUT is the experimental administration of butyrate; BACT is the
experimental administration of Blautia hydrogenotrophica.
[0102] FIGS. 52A-52B: Effect of chronic treatment with Blautia
hydrogenotrophica and butyrate on BTBR mice in the marble burying
test. The data in FIG. 52B are identical to that in FIG. 52A,
except the PBS and LYO control numbers have been pooled.
p.ltoreq.0.05: * vs. C57BL/6 (same treatment, where applicable); #
vs. PBS same genotype; .sctn. BTBR: But vs. PBS or Bact vs. Lyo.
PBS is the negative control for butyrate administration; LYO is the
negative control for bacterial (Blautia hydrogenotrophica)
administration; BUT is the experimental administration of butyrate;
BACT is the experimental administration of Blautia
hydrogenotrophica.
[0103] FIGS. 53A-53B: Effect of chronic treatment with Blautia
hydrogenotrophica and butyrate on BTBR mice in the digging test.
FIG. 53A shows the time spent digging, while FIG. 53B shows the
number of digging bouts. p<0.05: * vs. C57BL/6 (same treatment,
where applicable); # vs. PBS same genotype; .sctn. BTBR: But vs.
PBS or Bact vs. Lyo. PBS is the negative control for butyrate
administration; LYO is the negative control for bacterial (Blautia
hydrogenotrophica) administration; BUT is the experimental
administration of butyrate; BACT is the experimental administration
of Blautia hydrogenotrophica.
[0104] FIGS. 54A-54F: Effect of chronic treatment with Blautia
hydrogenotrophica and butyrate on BTBR mice in the self-grooming
test. FIG. 54A shows the time spent grooming; FIG. 54C shows the
number of grooming bouts, and FIG. 54E shows the time spent
grooming per bout. The data in FIGS. 54B, 54D and 54F are identical
to FIGS. 54A, 54C, and 54E respectively, except the PBS and LYO
control numbers have been pooled. P<0.05: * vs. C57BL/6 (same
treatment, where applicable); # vs. PBS same genotype; .sctn. BTBR:
But vs. PBS or Bact vs. Lyo. PBS is the negative control for
butyrate administration; LYO is the negative control for bacterial
(Blautia hydrogenotrophica) administration; BUT is the experimental
administration of butyrate; BACT is the experimental administration
of Blautia hydrogenotrophica.
[0105] FIG. 55: Effect of Blautia hydrogenotrophica (10.sup.10/day
for 14 days) on short chain fatty acids production (RMN .sup.1H) in
caecal contents of healthy HIM rats.
[0106] FIG. 56: Qpcr evaluation of B. hydrogenotrophica population
in faecal samples of IBS-HMA rats treated or not with a composition
comprising B. hydrogenotrophica (BlautiX) for 28 days.
[0107] FIGS. 57A-57B: Short chain fatty acids (SCFA) concentrations
in caecal samples of IBS-HMA rats treated or not with B.
hydrogenotrophica (Blautix) for 28 days. FIG. 57A shows
concentration of total SCFA. FIG. 57B shows concentration of Acetic
acid, Propionic acid and Butyric acid.
[0108] FIGS. 58A-58B: Effect of chronic treatment with Blautia
hydrogenotrophica and butyrate on BTBR mice in the three chamber
test. FIG. 58A shows the effect of administration on sociability
(the preference for sniffing an object or another mouse), while
FIG. 58B shows the preference for social novelty (i.e. sniffing a
new mouse vs. a familiar mouse). p<0.05: S vs. 50%; * vs.
C57BL/6 (same treatment, where applicable); # vs. PBS same
genotype; .sctn. BTBR: But vs. PBS or Bact vs. Lyo. PBS is the
negative control for butyrate administration; LYO is the negative
control for bacterial (Blautia hydrogenotrophica) administration;
BUT is the experimental administration of butyrate; BACT is the
experimental administration of Blautia hydrogenotrophica.
[0109] FIG. 59: Effect of chronic treatment with MRX006 on
expression of oxytoxin and oxytoxin receptor in the hypothalamic
cell lines.
[0110] FIG. 60: Effect of chronic treatment with MRX006 on ex vivo
gastrointestinal permeability and tight junction expression in
colon in BALBc mouse model.
[0111] FIG. 61: Effect of chronic treatment with MRX006 on ex vivo
gastrointestinal permeability and tight junction expression in
ileum in BALBc mouse model.
[0112] FIG. 62: Effect of chronic treatment with MRX006 on caecal
short chain fatty acid production in BALBc mouse model.
[0113] FIG. 63: Effect of chronic treatment with MRX006 on cytokine
expression from splenocytes in BALBc mouse model.
[0114] FIG. 64: Effect of chronic treatment with MRX006 on plasma
levels of amino acids in BALBc mouse model.
[0115] FIG. 65: Effect of chronic treatment with MRX006 on
neurotransmitter levels in the brainstem in BALBc mouse model.
[0116] FIG. 66: Effect of chronic treatment with MRX006 on gene
expression of hippocampus neurotransmitter receptors in BALBc mouse
model.
[0117] FIG. 67: Effect of chronic treatment with MRX006 on gene
expression of amygdalar neurotransmitter receptors in BALBc mouse
model.
[0118] FIG. 68: Effect of chronic treatment with MRX006 on gene
expression of prefrontal cortex neurotransmitter receptors in BALBc
mouse model.
[0119] FIG. 69: Effect of chronic treatment with MRX006 on gene
expression of inflammatory markers in the hippocampus in BALBc
mouse model.
[0120] FIG. 70: Effect of chronic treatment with MRX006 on gene
expression of inflammatory markers in the amygdalar in BALBc mouse
model.
[0121] FIG. 71: Effect of chronic treatment with MRX006 on gene
expression of inflammatory markers in the prefrontal cortex in
BALBc mouse model.
[0122] FIG. 72: Effect of chronic treatment with MRX006 on gene
expression of hippocampal endocrine markers in BALBc mouse
model.
[0123] FIG. 73: Effect of chronic treatment with MRX006 on gene
expression of amygdalar endocrine markers in BALBc mouse model.
[0124] FIG. 74: Effect of chronic treatment with MRX006 on gene
expression of prefrontal cortex endocrine markers in BALBc mouse
model.
[0125] FIG. 75: Effect of chronic treatment with MRX006 on in vivo
gastrointestinal permeability in the colon and ileum in MIA mouse
model.
[0126] FIG. 76: Effect of treatment with MRX006 on social novelty
in the three chamber social interaction test in MIA mice.
[0127] FIG. 77: Effect of treatment with MRX006 on social
preference in the three chamber social interaction test in MIA
mice.
[0128] FIG. 78: Effect of treatment with MRX006 on MIA mice in the
grooming test.
[0129] FIG. 79: Effect of treatment with MRX006 on MIA mice in the
elevated plus maze test.
[0130] FIG. 80: Effect of treatment with MRX006 on MIA mice in the
forced swim test.
[0131] FIG. 81: Effect of treatment with MRX006 on stress-induced
corticosterone plasma levels in MIA mice.
DISCLOSURE OF THE INVENTION
[0132] Bacterial Strains
[0133] The compositions of the invention comprise a bacterial
strain of the genus Blautia. The examples demonstrate that bacteria
of this species are useful for treating or preventing autism
spectrum disorders and central nervous system disorders 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 other central nervous system disorders
including those listed above
[0134] The invention provides a composition comprising a bacterial
strain of the genus Blautia 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. In certain
embodiments, the compositions of the invention comprise strains of
the genus Blautia and do not contain any other bacterial genera. In
certain embodiments, the compositions of the invention comprise a
single strain of the genus Blautia and do not contain any other
bacterial strains, genera or species.
[0135] Examples of Blautia strains for use in the invention include
Blautia stercoris, B. faecis, B. coccoides, B. glucerasea, B.
hansenii, B. hydrogenotrophica, B. luti, B. producta, B. schinkii
and B. wexlerae. Preferred species are Blautia stercoris, B.
wexlerae and B. hydrogenotrophica. The Blautia species are
Gram-reaction-positive, non-motile bacteria that may be either
coccoid or oval and all are obligate anaerobes that produce acetic
acid as the major end product of glucose fermentation [26]. Blautia
may be isolated from the human gut, although B. producta was
isolated from a septicaemia sample. The GenBank accession number
for the 16S rRNA gene sequence of Blautia stercoris strain
GAM6-1.sup.T is HM626177 (disclosed herein as SEQ ID NO: 1). An
exemplary Blautia stercoris strain is described in [27]. The type
strain of Blautia wexlerae is WAL 14507=ATCC BAA-1564=DSM 19850
[28]. The GenBank accession number for the 16S rRNA gene sequence
of Blautia wexlerae strain WAL 14507 T is EF036467 (disclosed
herein as SEQ ID NO:3). This exemplary Blautia wexlerae strain is
described in [28].
[0136] The Blautia stercoris bacterium deposited under accession
number NCIMB 42381 was tested in the Examples and is also referred
to herein as MRX006 (strain 830). The terms "MRX006", "MRx0006"
"Mrx006", "Mrx0006" and strain 830 are used interchangeably herein.
A 16S rRNA sequence for MRX006 (830 strain) that was tested is
provided in SEQ ID NO:2. MRX006 (Strain 830) was deposited with the
international depositary authority NCIMB, Ltd. (Ferguson Building,
Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (Life Sciences
Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12 Mar. 2015
as "Blautia stercoris 830" and was assigned accession number NCIMB
42381. GT Biologics Ltd. subsequently changed its name to 4D Pharma
Research Limited.
[0137] The genome of MRX006 (strain 830) comprises a chromosome and
plasmid. A chromosome sequence for MRX006 (strain 830) is provided
in SEQ ID NO:5. A plasmid sequence for MRX006 (strain 830) is
provided in SEQ ID NO:6. These sequences were generated using the
PacBio RS II platform.
[0138] The Blautia wexlerae bacterium deposited under accession
number NCIMB 42486 was tested in the Examples and is also referred
to herein as strain MRX008. The terms "MRX008", "MRx0008" "Mrx008"
and "Mrx0008" are used interchangeably herein. A 16S rRNA sequence
for the MRX008 strain that was tested is provided in SEQ ID NO:4.
Strain MRX008 was deposited with the international depositary
authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA,
Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation
Building, Aberdeen, AB25 2ZS, Scotland) on 16 Nov. 2015 as
"Blautia/Ruminococcus" and was assigned accession number NCIMB
42486.
[0139] A further preferred strain of the invention is the Blautia
hydrogenotrophica bacterium deposited under accession number DSM
14294. This strain was deposited with the Deutsche Sammlung von
Mikroorganismen [German Microorganism Collection] (Mascheroder Weg
1b, 38124 Braunschweig, Germany) under accession number DSM 14294
as "S5a33" on 10 May 2001. The depositor was INRA Laboratoire de
Microbiologie CR de Clermont-Ferrand/Theix 63122 Saint Genes
Champanelle, France. Ownership of the deposits has passed to 4D
Pharma Plc by way of assignment. 4D Pharma Plc has authorised, by
way of an agreement, 4D Pharma Research Limited to refer to the
deposited biological material in the application and has given its
unreserved and irrevocable consent to the deposited material being
made available to the public. The deposit under accession number
DSM 14294 was published on 11 May 2000.
[0140] The Blautia hydrogenotrophica bacterium deposited under
accession number DSM 14294 was tested in the Examples and is a
preferred strain of the invention.
[0141] Bacterial strains closely related to the strain tested in
the examples 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% or 99.9% identical to the 16s rRNA sequence of a
bacterial strain of Blautia stercoris. 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% 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 bacterial strain for use in the invention has a
16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%
or 99.9% identical to the 16s rRNA sequence of a bacterial strain
of Blautia wexlerae. 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% or 99.9% identical to SEQ ID NO:3 or 4.
Preferably, the sequence identity is to SEQ ID NO:4. Preferably,
the bacterial strain for use in the invention has the 16s rRNA
sequence represented by SEQ ID NO:4.
[0142] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5.
In preferred embodiments, the bacterial strain for use in the
invention has a chromosome with at least 90% sequence identity
(e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity) to SEQ ID NO:5 across at least 60% (e.g. at least 65%,
70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID
NO:5. For example, the bacterial strain for use in the invention
may have a chromosome with at least 90% sequence identity to SEQ ID
NO:5 across 70% of SEQ ID NO:5, or at least 90% sequence identity
to SEQ ID NO:5 across 80% of SEQ ID NO:5, or at least 90% sequence
identity to SEQ ID NO:5 across 90% of SEQ ID NO:5, or at least 90%
sequence identity to SEQ ID NO:5 across 100% of SEQ ID NO:5, or at
least 95% sequence identity to SEQ ID NO:5 across 70% of SEQ ID
NO:5, or at least 95% sequence identity to SEQ ID NO:5 across 80%
of SEQ ID NO:5, or at least 95% sequence identity to SEQ ID NO:5
across 90% of SEQ ID NO:5, or at least 95% sequence identity to SEQ
ID NO:5 across 100% of SEQ ID NO:5, or at least 98% sequence
identity to SEQ ID NO:5 across 70% of SEQ ID NO:5, or at least 98%
sequence identity to SEQ ID NO:5 across 80% of SEQ ID NO:5, or at
least 98% sequence identity to SEQ ID NO:5 across 90% of SEQ ID
NO:5, or at least 98% sequence identity to SEQ ID NO:5 across 100%
of SEQ ID NO:5.
[0143] In certain embodiments, the bacterial strain for use in the
invention has a plasmid with sequence identity to SEQ ID NO:6. In
preferred embodiments, the bacterial strain for use in the
invention has a plasmid with at least 90% sequence identity (e.g.
at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity) to SEQ ID NO:6 across at least 60% (e.g. at least 65%,
70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID
NO:6. For example, the bacterial strain for use in the invention
may have a plasmid with at least 90% sequence identity to SEQ ID
NO:6 across 70% of SEQ ID NO:6, or at least 90% sequence identity
to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 90% sequence
identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 90%
sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6, or at
least 95% sequence identity to SEQ ID NO:6 across 70% of SEQ ID
NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 80%
of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6
across 90% of SEQ ID NO:6, or at least 95% sequence identity to SEQ
ID NO:6 across 100% of SEQ ID NO:6, or at least 98% sequence
identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 98%
sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at
least 98% sequence identity to SEQ ID NO:6 across 90% of SEQ ID
NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 100%
of SEQ ID NO:6.
[0144] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5
and a plasmid with sequence identity to SEQ ID NO:6.
[0145] Bacterial strains that are biotypes of the bacterium
deposited under accession number 42381 are also expected to be
effective for treating or preventing autism spectrum disorder and
central nervous system disorders and conditions, in particular
central nervous system disorders and conditions mediated by the
microbiota-gut-brain axis. Bacterial strains that are biotypes of
the bacterium deposited under accession number 42486 are also
expected to be effective for treating or preventing autism spectrum
disorder and central nervous system disorders and conditions, in
particular central nervous system disorders and conditions mediated
by the microbiota-gut-brain axis. A biotype is a closely related
strain that has the same or very similar physiological and
biochemical characteristics.
[0146] Strains that are biotypes of the bacterium deposited under
accession number NCIMB 42381 or 42486 and that are suitable for use
in the invention may be identified by sequencing other nucleotide
sequences for the bacterium deposited under accession number NCIMB
42381 or 42486. 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% 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
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).sub.5, or REP or [29]. Biotype
strains may have sequences with at least 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% sequence identity to the corresponding sequence of
the bacterium deposited under accession number NCIMB 42381 or
42486.
[0147] In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of strain MRX006 deposited as NCIMB
42381 and comprises a 16S rRNA sequence that is at least 99%
identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ
ID NO:2. In some embodiments, a biotype strain has a sequence with
at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity
to the corresponding sequence of strain MRX006 deposited as NCIMB
42381 and has the 16S rRNA sequence of SEQ ID NO:2.
[0148] In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of strain MRX008 deposited as NCIMB
42486 and comprises a 16S rRNA sequence that is at least 99%
identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ
ID NO:4. In some embodiments, a biotype strain has a sequence with
at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity
to the corresponding sequence of strain MRX008 deposited as NCIMB
42486 and has the 16S rRNA sequence of SEQ ID NO:4.
[0149] Alternatively, strains that are biotypes of a bacterium
deposited under accession number NCIMB 42381 or 42486 and that are
suitable for use in the invention may be identified by using the
accession number NCIMB 42381 deposit or the accession number NCIMB
42486 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 Blautia stercoris or Blautia wexlerae
strains.
[0150] In certain embodiments, strains that are biotypes of a
bacterium deposited under accession number NCIMB 42381 or 42486 and
that are suitable for use in the invention are strains that provide
the same pattern as a bacterium deposited under accession number
NCIMB 42381 or 42486 when analysed by amplified ribosomal DNA
restriction analysis (ARDRA), for example when using Sau3AI
restriction enzyme (for exemplary methods and guidance see, for
example, [30]). Alternatively, biotype strains are identified as
strains that have the same carbohydrate fermentation patterns as a
bacterium deposited under accession number NCIMB 42381 or
42486.
[0151] Other Blautia stercoris strains that are useful in the
compositions and methods of the invention, such as biotypes of the
bacterium deposited under accession number NCIMB 42381 or 42486,
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 42381 or 42486 may be useful in the
invention. A useful strain will have comparable immune modulatory
activity to the NCIMB 42381 or 42486 strain. 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.
[0152] A particularly preferred strain of the invention is the
Blautia stercoris strain deposited under accession number NCIMB
42381. This is the exemplary MRX006 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 Blautia
stercoris strain deposited under accession number NCIMB 42381, or a
derivative thereof. The invention also provides a composition
comprising a cell of the Blautia stercoris strain deposited under
accession number NCIMB 42381, or a derivative thereof. The
invention also provides a biologically pure culture of the Blautia
stercoris strain deposited under accession number NCIMB 42381. The
invention also provides a cell of the Blautia stercoris strain
deposited under accession number NCIMB 42381, or a derivative
thereof, for use in therapy, in particular for the diseases
described herein.
[0153] A particularly preferred strain of the invention is the
Blautia wexlerae strain deposited under accession number NCIMB
42486. This is the exemplary MRX008 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 Blautia
wexlerae strain deposited under accession number NCIMB 42486, or a
derivative thereof. The invention also provides a composition
comprising a cell of the Blautia wexlerae strain deposited under
accession number NCIMB 42486, or a derivative thereof. The
invention also provides a biologically pure culture of the Blautia
wexlerae strain deposited under accession number NCIMB 42486. The
invention also provides a cell of the Blautia wexlerae strain
deposited under accession number NCIMB 42486, or a derivative
thereof, for use in therapy, in particular for the diseases
described herein.
[0154] A derivative of the strain deposited under accession number
NCIMB 42381 or 42486 may be a daughter strain (progeny) or a strain
cultured (subcloned) from the original. A derivative of the strain
deposited under accession number NCIMB 42381 or 42486 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 42381
or 42486 strain. In particular, a derivative strain will elicit
comparable effects on the central nervous system disorder or
condition models and comparable effects on cytokine levels to the
effects shown in the Examples, which may be identified by using the
culturing and administration protocols described in the Examples. A
derivative of the NCIMB 42381 strain will generally be a biotype of
the NCIMB 42381 strain. A derivative of the NCIMB 42486 strain will
generally be a biotype of the NCIMB 42486 strain.
[0155] References to cells of the Blautia stercoris strain
deposited under accession number NCIMB 42381 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 42381, and
such cells are encompassed by the invention. Thus, in some
embodiments, reference to cells of the Blautia stercoris strain
deposited under accession number NCIMB 42381 refers only to the
MRX006 strain deposited under NCIMB 42381 and does not refer to a
bacterial strain that was not deposited under NCIMB 42381. In some
embodiments, reference to cells of the Blautia stercoris strain
deposited under accession number NCIMB 42381 refers to cells that
have the same safety and therapeutic efficacy characteristics as
the strains deposited under accession number NCIMB 42381, but which
are not the strain deposited under NCIMB 42381.
[0156] References to cells of the Blautia wexlerae strain deposited
under accession number NCIMB 42486 encompass any cells that have
the same safety and therapeutic efficacy characteristics as the
strains deposited under accession number NCIMB 42486, and such
cells are encompassed by the invention. Thus, in some embodiments,
reference to cells of the Blautia wexlerae strain deposited under
accession number NCIMB 42486 refers only to the strain deposited
under NCIMB 42486 and does not refer to a bacterial strain that was
not deposited under NCIMB 42486. In some embodiments, reference to
cells of the Blautia wexlerae strain deposited under accession
number NCIMB 42486 refers to cells that have the same safety and
therapeutic efficacy characteristics as the strains deposited under
accession number NCIMB 42486, but which are not the strain
deposited under NCIMB 42486.
[0157] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, and a 16S rRNA sequence with
sequence identity to SEQ ID NO:1, 2, 3 or 4, for example as
described above, preferably with a 16s rRNA sequence that is at
least 99% identical to SEQ ID NO: 2 or 4, more preferably which
comprises the 16S rRNA sequence of SEQ ID NO:2 or 4.
[0158] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, and is effective for treating or
preventing central nervous system disorders and conditions, in
particular central nervous system disorders and conditions mediated
by the microbiota-gut-brain axis.
[0159] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, and a 16S rRNA sequence with
sequence identity to SEQ ID NO: 1, 2, 3 or 4, for example as
described above, and is effective for treating or preventing
central nervous system disorders and conditions, in particular
central nervous system disorders and conditions mediated by the
microbiota-gut-brain axis.
[0160] In certain embodiments, the bacterial strain for use in the
invention has a 16s rRNA sequence that is at least 99%, 99.5% or
99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:
2 or 4 (for example, which comprises the 16S rRNA sequence of SEQ
ID NO:2 or 4) and a chromosome with at least 95% sequence identity
to SEQ ID NO:5 across at least 90% of SEQ ID NO:5, and which is
effective for treating or preventing central nervous system
disorders and conditions, in particular central nervous system
disorders and conditions mediated by the microbiota-gut-brain
axis.
[0161] In certain embodiments, the bacterial strain for use in the
invention is a Blautia stercoris and has a 16s rRNA sequence that
is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence
represented by SEQ ID NO: 2 or 4 (for example, which comprises the
16S rRNA sequence of SEQ ID NO:2 or 4) and a chromosome with at
least 98% sequence identity (e.g. at least 99% or at least 99.5%
sequence identity) to SEQ ID NO:5 across at least 98% (e.g. across
at least 99% or at least 99.5%) of SEQ ID NO:5, and which is
effective for treating or preventing central nervous system
disorders and conditions, in particular central nervous system
disorders and conditions mediated by the microbiota-gut-brain
axis.
[0162] In certain embodiments, the bacterial strain for use in the
invention has a plasmid with sequence identity to SEQ ID NO:6, for
example as described above, and a 16S rRNA sequence with sequence
identity to SEQ ID NO:1, 2, 3 or 4, for example as described above,
preferably with a 16s rRNA sequence that is at least 99% identical
to SEQ ID NO: 2 or 4, more preferably which comprises the 16S rRNA
sequence of SEQ ID NO:2 or 4.
[0163] In certain embodiments, the bacterial strain for use in the
invention has a plasmid with sequence identity to SEQ ID NO:6, for
example as described above, and is effective for treating or
preventing central nervous system disorders and conditions, in
particular central nervous system disorders and conditions mediated
by the microbiota-gut-brain axis.
[0164] In certain embodiments, the bacterial strain for use in the
invention has a plasmid with sequence identity to SEQ ID NO:6, for
example as described above, and a 16S rRNA sequence with sequence
identity to SEQ ID NO: 1, 2, 3 or 4, for example as described
above, and is effective for treating or preventing central nervous
system disorders and conditions, in particular central nervous
system disorders and conditions mediated by the
microbiota-gut-brain axis.
[0165] In certain embodiments, the bacterial strain for use in the
invention has a 16s rRNA sequence that is at least 99%, 99.5% or
99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:
2 or 4 (for example, which comprises the 16S rRNA sequence of SEQ
ID NO:2 or 4) and a plasmid with at least 95% sequence identity to
SEQ ID NO:6 across at least 90% of SEQ ID NO:6, and which is
effective for treating or preventing central nervous system
disorders and conditions, in particular central nervous system
disorders and conditions mediated by the microbiota-gut-brain
axis.
[0166] In certain embodiments, the bacterial strain for use in the
invention is a Blautia stercoris and has a 16s rRNA sequence that
is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence
represented by SEQ ID NO: 2 or 4 (for example, which comprises the
16S rRNA sequence of SEQ ID NO:2 or 4) and a plasmid with at least
98% sequence identity (e.g. at least 99% or at least 99.5% sequence
identity) to SEQ ID NO:6 across at least 98% (e.g. across at least
99% or at least 99.5%) of SEQ ID NO:6, and which is effective for
treating or preventing central nervous system disorders and
conditions, in particular central nervous system disorders and
conditions mediated by the microbiota-gut-brain axis.
[0167] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, a plasmid with sequence identity to
SEQ ID NO:6, for example as described above, and a 16S rRNA
sequence with sequence identity to SEQ ID NO:1, 2, 3 or 4, for
example as described above, preferably with a 16s rRNA sequence
that is at least 99% identical to SEQ ID NO: 2 or 4, more
preferably which comprises the 16S rRNA sequence of SEQ ID NO:2 or
4.
[0168] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, and a plasmid with sequence
identity to SEQ ID NO:6, for example as described above, and is
effective for treating or preventing central nervous system
disorders and conditions, in particular central nervous system
disorders and conditions mediated by the microbiota-gut-brain
axis.
[0169] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:5,
for example as described above, a plasmid with sequence identity to
SEQ ID NO:6, for example as described above, and a 16S rRNA
sequence with sequence identity to SEQ ID NO: 1, 2, 3 or 4, for
example as described above, and is effective for treating or
preventing central nervous system disorders and conditions, in
particular central nervous system disorders and conditions mediated
by the microbiota-gut-brain axis.
[0170] In certain embodiments, the bacterial strain for use in the
invention has a 16s rRNA sequence that is at least 99%, 99.5% or
99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:
2 or 4 (for example, which comprises the 16S rRNA sequence of SEQ
ID NO:2 or 4), a chromosome with at least 95% sequence identity to
SEQ ID NO:5 across at least 90% of SEQ ID NO:5, and a plasmid at
least 95% sequence identity to SEQ ID NO:6 across at least 90% of
SEQ ID NO:6, and which is effective for treating or preventing
central nervous system disorders and conditions, in particular
central nervous system disorders and conditions mediated by the
microbiota-gut-brain axis.
[0171] In certain embodiments, the bacterial strain for use in the
invention is a Blautia stercoris and has a 16s rRNA sequence that
is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence
represented by SEQ ID NO: 2 or 4 (for example, which comprises the
16S rRNA sequence of SEQ ID NO:2 or 4), a chromosome with at least
98% sequence identity (e.g. at least 99% or at least 99.5% sequence
identity) to SEQ ID NO:5 across at least 98% (e.g. across at least
99% or at least 99.5%) of SEQ ID NO:5, and a plasmid with at least
98% sequence identity (e.g. at least 99% or at least 99.5% sequence
identity) to SEQ ID NO:6 across at least 98% (e.g. across at least
99% or at least 99.5%) of SEQ ID NO:6, and which is effective for
treating or preventing central nervous system disorders and
conditions, in particular central nervous system disorders and
conditions mediated by the microbiota-gut-brain axis.
[0172] In preferred embodiments, the bacterial strains in the
compositions of the invention are viable and capable of partially
or totally colonising the intestine.
[0173] Blautia hydrogenotrophica (previously known as Ruminococcus
hydrogenotrophicus) has been isolated from the guts of mammals, is
strictly anaerobic, and metabolises H.sub.2/CO.sub.2 to acetate,
which may be important for human nutrition and health. The type
strain of Blautia hydrogenotrophica is S5a33=JCM 14656. The GenBank
accession number for the 16S rRNA gene sequence of Blautia
hydrogenotrophica strain S5a36 is X95624.1 (disclosed herein as SEQ
ID NO:7). This exemplary Blautia hydrogenotrophica strain is
described in [28] and [31]. The S5a33 strain and the S5a36 strain
correspond to two subclones of a strain isolated from a faecal
sample of a healthy subject. They show identical morphology,
physiology and metabolism and have identical 16S rRNA sequences.
Thus, in some embodiments, the Blautia hydrogenotrophica for use in
the invention has the 16S rRNA sequence of SEQ ID NO:7.
[0174] 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% or 99.9% identical to the 16s rRNA sequence of a
bacterial strain of Blautia hydrogenotrophica. 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% or 99.9% identical
to SEQ ID NO:7. Preferably, the bacterial strain for use in the
invention has the 16s rRNA sequence represented by SEQ ID NO:7.
[0175] Bacterial strains that are biotypes of the bacterium
deposited under accession number DSM 14294 are also expected to be
effective for treating or preventing autism spectrum disorder and
central nervous system disorders and conditions, in particular
central nervous system disorders and conditions mediated by the
microbiota-gut-brain axis.
[0176] Strains that are biotypes of the bacterium deposited under
accession number DSM 14294 and that are suitable for use in the
invention may be identified by sequencing other nucleotide
sequences for the bacterium deposited under accession number DSM
14294. 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% 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
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).sub.5, or REP or [29]. Biotype
strains may have sequences with at least 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% sequence identity to the corresponding sequence of
the bacterium deposited under accession number DSM 14294.
[0177] In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of the strain deposited as accession
number DSM 14294 and comprises a 16S rRNA sequence that is at least
99% identical (e.g. at least 99.5% or at least 99.9% identical) to
SEQ ID NO:7. In some embodiments, a biotype strain has a sequence
with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence
identity to the corresponding sequence of the strain deposited as
accession number DSM 14294 and has the 16S rRNA sequence of SEQ ID
NO:7.
[0178] Alternatively, strains that are biotypes of a bacterium
deposited under accession number DSM 14294 and that are suitable
for use in the invention may be identified by using the accession
number DSM 14294 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 Blautia hydrogenotrophica strains.
[0179] In certain embodiments, strains that are biotypes of a
bacterium deposited under accession number NCIMB 42381 or 42486 and
that are suitable for use in the invention are strains that provide
the same pattern as a bacterium deposited under accession number
DSM 14294 when analysed by amplified ribosomal DNA restriction
analysis (ARDRA), for example when using Sau3AI restriction enzyme
(for exemplary methods and guidance see, for example, [30]).
Alternatively, biotype strains are identified as strains that have
the same carbohydrate fermentation patterns as a bacterium
deposited under accession number DSM 14294.
[0180] Other Blautia hydrogenotrophica strains that are useful in
the compositions and methods of the invention, such as biotypes of
the bacterium deposited under accession number DSM 14294, 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 DSM 14294 may be useful in the invention. A useful strain
will have comparable immune modulatory activity to the accession
number DSM 14294 strain. 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.
[0181] A particularly preferred strain of the invention is the
Blautia hydrogenotrophica strain deposited under accession number
DSM 14294. This is the exemplary 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 Blautia
hydrogenotrophica strain deposited under accession number DSM
14294, or a derivative thereof. The invention also provides a
composition comprising a cell of the Blautia hydrogenotrophica
strain deposited under accession number DSM 14294, or a derivative
thereof. The invention also provides a biologically pure culture of
the Blautia hydrogenotrophica strain deposited under accession
number DSM 14294. The invention also provides a cell of the Blautia
hydrogenotrophica strain deposited under accession number DSM
14294, or a derivative thereof, for use in therapy, in particular
for the diseases described herein.
[0182] A derivative of the strain deposited under accession number
DSM 14294 may be a daughter strain (progeny) or a strain cultured
(subcloned) from the original. A derivative of the strain deposited
under accession number DSM 14294 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 strain deposited under accession number
DSM 14294. In particular, a derivative strain will elicit
comparable effects on the central nervous system disorder or
condition models and comparable effects on cytokine levels to the
effects shown in the Examples, which may be identified by using the
culturing and administration protocols described in the Examples. A
derivative of the DSM 14294 strain will generally be a biotype of
the DSM 14294 strain.
[0183] References to cells of the Blautia hydrogenotrophica strain
deposited under accession number DSM 14294 encompass any cells that
have the same safety and therapeutic efficacy characteristics as
the strains deposited under accession number DSM 14294, and such
cells are encompassed by the invention. Thus, in some embodiments,
reference to cells of the Blautia hydrogenotrophica strain
deposited under accession number DSM 14294 refers only to the
strain deposited under DSM 14294 and does not refer to a bacterial
strain that was not deposited under DSM 14294. In some embodiments,
reference to cells of the Blautia hydrogenotrophica strain
deposited under accession number DSM 14294 refers to cells that
have the same safety and therapeutic efficacy characteristics as
the strains deposited under accession number DSM 14294, but which
are not the strain deposited under DSM 14294.
[0184] In preferred embodiments, the bacterial strains in the
compositions of the invention are viable and capable of partially
or totally colonising the intestine.
[0185] Therapeutic Uses
[0186] Modulation of the Microbiota-Gut-Brain Axis
[0187] 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
[10],[13],[14],[32].
[0188] 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 arms of the autonomic nervous system (ANS),
including the enteric nervous system (ENS) and the vagus nerve, and
the gut microbiota.
[0189] As demonstrated in the examples, the compositions of the
present invention can modulate the microbiota-gut-brain axis and
reduce 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.
[0190] 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.
[0191] 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; 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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 embodiments, the compositions of the
invention modulate the level of stress and/or anxiety in a
subject.
[0196] Autism Spectrum Disorder (ASD)
[0197] Autism spectrum disorder is a set of heterogeneous
neurodevelopmental conditions, characterised 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.
[0198] Historically, autism has been diagnosed on the basis of
three core domains: impaired social 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 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.
[0199] ASD is a `spectrum disorder` as it affects each person in a
variety of different ways and can range 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.
[0200] 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.
[0201] Co-morbid medical conditions are highly prevalent in ASDs.
Co-morbid include anxiety and depression, seizures, attention
deficits, aggressive behaviours, sleep problems, gastrointestinal
disorders, epilepsy, mental retardation, intellectual disabilities
and feeding difficulties.
[0202] 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 so they
may be useful in the treatment or prevention of autism spectrum
disorders.
[0203] 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
composition of the invention are for use in treating or preventing
autism spectrum disorders. 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.
[0204] 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 an ASD in a patient that has been identified as
at risk of an ASD, or that has been diagnosed with an 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 ASDs.
[0205] The compositions of the invention may be useful for managing
or alleviating ASDs. Treatment or prevention of ASDs 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.
[0206] In preferred embodiments, the compositions of the invention
prevent, reduce or alleviate at least one core symptom of ASDs.
[0207] 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.
[0208] 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, social
information processing, capacity for social communication, social
anxiety/avoidance, and autistic preoccupations and traits in a
subject with ASDs.
[0209] In some embodiments, the compositions of the invention
prevent, reduce or alleviate additional symptoms associated with
the core symptoms of ASDs. 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, attention and/or sensory
processing in a subject with ASD.
[0210] 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.
[0211] 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).
[0212] In some embodiments, the compositions of the invention cause
a positive change in generic measures of the efficacy endpoints of
ASDs. 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).
[0213] 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 ASDs.
[0214] 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.
[0215] 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 flatulence.
[0216] 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).
[0217] In some embodiments, the compositions of the invention are
particularly effective at preventing, reducing or alleviating ASDs
when used in combination with another therapy for treating ASDs.
Such therapies include anti-psychotic, anti-anxiety and
anti-depressant drugs. Such drugs include risperidone
(Risperdal.RTM.); olanzapine (Zyprexa.RTM.); fluoxetine
(Prozac.RTM.); sertraline (Zoloft.RTM.); fluvoxamine (Luvox.RTM.);
clomipramine (Anafranil.RTM.); haloperidol (Haldol.RTM.);
thioridazine; fluphenazine; chlorpromazine; ziprasidone
(Geogon.RTM.); carbamazepine (Tegretol.RTM.); lamotrigine
(Lamictal.RTM.); topiramate (Topomax.RTM.); valproic acid
(Depakote.RTM.); methylphenidate (Ritalin.RTM.); diazepam
(Valium.RTM.) and lorazepam (Ativan.RTM.).
[0218] 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 live biotherapeutic strains used in the
Examples have shown effective treatment of at least one core
symptom of autistic spectrum disorder, so these strains and related
Blautia strains are expected to be effective against human
disease.
[0219] Obsessive Compulsive Disorder (OCD)
[0220] 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 normal 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).
[0221] 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). The
person attempts to ignore, suppress or neutralise the obsessions
with some other thoughts or actions.
[0222] 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 perform in response to an obsession or according to rules
that must be applied rigidly.
[0223] 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).
[0224] 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.
[0225] 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
experiences as intrusive and inappropriate and cause marked anxiety
or distress. In certain embodiments, the compulsions are repetitive
behaviours that the subject feels driven to perform in response to
an obsession or according to rules that must be applied
rigidly.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
Such therapies include serotonin and dopamine reuptake inhibitors;
clomipramine and anti-psychotics.
[0230] Major Depressive Disorder (MDD)
[0231] MDD is associated with substantial psychosocial dysfunction
and high individual mental strain as well as with excess morbidity
and mortality (the risk of suicide is considerable). The term major
depressive disorder encompasses clinical depression, major
depression, unipolar depression, unipolar disorder, recurrent
depression and simply depression. The term major depressive
disorder covers mood disorders; dysthymia; chronic depression;
seasonal affective disorder and borderline personality
disorder.
[0232] 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 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 worries; trouble relaxing and/or feeling tense. MDD
episodes can be mild, moderate or severe.
[0233] 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; substance use disorders; post-traumatic stress disorder
(PTSD); personality disorders; pain; stress; irritable bowel
syndrome; insomnia; headaches and interpersonal problems.
[0234] 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.
[0235] 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.
[0236] 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 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.
[0237] 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.
[0238] 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.
[0239] In certain embodiments, the compositions of the invention
are for use in treating or preventing treatment resistant MDD.
[0240] 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;
headaches and interpersonal problems.
[0241] 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 hormones and atypical antipsychotics) or even second
generation antipsychotics.
[0242] Anxiety Disorders
[0243] Anxiety disorders are a group of mental disorders
characterised by feelings of anxiety and fear. There are a number
of anxiety disorders including generalised anxiety disorder (GAD);
specific phobia; social anxiety disorder; separation anxiety
disorder; agroraphobia; panic disorder and selective mutism.
[0244] GAD is diagnosed according to DMS-5 in six criterion. 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 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.
[0245] 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.
[0246] 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.
[0247] 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 impairment associated
with anxiety disorders.
[0248] 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.
[0249] 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 impairment of
the subject with anxiety disorder. In some embodiments, the global
scale is the Sheehan disability scale.
[0250] 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; interpersonal problems and ADHD.
[0251] 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 (venlafaxine, duloxetine,
escitalopram and paroxetine); benzodiazepines (alprazolam,
lorazepam and clonazepam); pregabalin (Lyrica.RTM.) and gabapentin
(Neurontin.RTM.); 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.
[0252] Post-Traumatic Stress Disorder (PTSD)
[0253] 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.
[0254] 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.
[0255] 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.
[0256] PTSD carries high comorbidities with major depressive
disorder and substance use disorders.
[0257] 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.
[0258] 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 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.
[0259] 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.
[0260] 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 impairment of
the subject with PTSD and stress disorders. In some embodiments,
the global scale is the Sheehan disability scale.
[0261] 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.
[0262] 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 (Zoloft.RTM.), fluoxetine (Prozac.RTM.) and/or
paroxetine (Paxil.RTM.).
[0263] Schizophrenia Spectrum and Psychotic Disorders
[0264] 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); schizophreniform
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.
[0265] 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
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 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.
[0266] 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 symptoms, with social or occupational
deterioration problems occurring over a significant amount of
time.
[0267] 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 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 or preventing schizophrenia. In certain embodiments, the
schizophrenia is selected from paranoid, disorganised, catatonic,
undifferentiated and residual schizophrenia.
[0268] 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 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. In certain embodiments, the compositions of the
invention prevent, reduce or alleviate negative and positive
symptoms of schizophrenia. In 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.
[0269] 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.
[0270] 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.
[0271] 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.
[0272] 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 as manic and depressive symptoms,
anxiety or obsessive-compulsive symptoms, substance abuse and
dependence, and personality disorder.
[0273] In certain embodiments, the compositions of the invention
are for use in treating or preventing treatment resistant of
refractory schizophrenia.
[0274] 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 (Abilify.RTM.); asenapine (Saphris.RTM.);
brexpiprazole (Rexulti.RTM.); cariprazine (Vraylar.RTM.); clozapine
(Clozaril.RTM.); iloperidone (Fanapt.RTM.); lurasidone
(Latuda.RTM.); olanzapine (Zyprexa.RTM.); paliperidone (Invega);
quetiapine (Seroquel.RTM.); risperidone (Risperdal.RTM.);
ziprasidone (Geodon.RTM.).
[0275] Bipolar Disorder
[0276] 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.
[0277] 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.
[0278] 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.
[0279] Bipolar disorder is associated with the following
comorbidities: ADHD; anxiety disorders; substance disorders;
obesity and metabolic syndrome.
[0280] 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.
[0281] 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 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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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, anticonvulsant drugs (including
valproate, divalproex, carbamazepine and lamotrigine) and
antipsychotic drugs (including aripiprazole, olanzapine, quetiapine
and risperidone).
[0288] Neurocognitive Disorders and Alzheimer's Disease
[0289] 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 Wernicke-Korsakoff syndrome) are
accompanied by dementia.
[0290] 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).
[0291] 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.
[0292] 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
Wernicke-Korsakoff syndrome.
[0293] 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.
[0294] 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 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 invention improve
the ability of a subject with neurocognitive disorder to undertake
everyday activities.
[0295] 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 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.
[0296] 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 psychiatric symptoms of neurocognitive
disorders.
[0297] 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).
[0298] 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).
[0299] 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).
[0300] 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).
[0301] 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 (Aricept.RTM.), galantamine (Razadyne.RTM.) and
rivastigmine (Exelon.RTM.), and memantine.
[0302] Parkinson's Disease
[0303] 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 impairment. 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.
[0304] 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.
[0305] 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 impairment 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.
[0306] 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 embodiments, the compositions of the invention improve
attention, working memory, verbal fluency and/or anxiety.
[0307] In other preferred embodiments, the compositions of the
invention prevent, reduce or alleviate cognitive dysfunctions
comorbid with Parkinson's disease.
[0308] 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 impairment 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.
[0309] 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.
[0310] 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).
[0311] 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 Parkinson's disease.
[0312] 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.
[0313] Other Central Nervous System Disorders
[0314] 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.
[0315] Huntington's Disease
[0316] Huntington's disease is an inherited brain condition, caused
by an inherited faulty gene, which damages certain nerve cells in
the brain. This brain damage gets progressively worse over time and
can affect movement, cognition (perception, awareness, thinking,
judgement) and behaviour. Early features of the disease can include
personality changes, mood swings, fidgety movements, irritability
and altered behaviour.
[0317] In certain embodiments, the compositions of the invention
are for use in treating or preventing Huntington's disease. In
certain embodiments, the compositions of the invention manage the
symptoms of Huntington's disease, such as irritability or excessive
movement. In certain embodiments, the compositions of the invention
treat the depression associated with Huntington's disease and/or
improve symptoms such as social withdrawal, lack or interest and
sleep disturbance. In certain embodiments, the compositions of the
invention improve memory and ability to concentrate on tasks. In
certain embodiments, the compositions of the invention treat
disabling abnormal movements. In certain embodiments, the
compositions of the invention treat behavioural problems,
antisocial behaviour, irritability and psychosis associated with
Huntington's disease. In certain embodiments, the compositions of
the invention induce neuroprotection and prevent nerve damage. In
certain embodiments, the compositions of the invention increase the
levels of dopamine and/or the levels of dopamine-containing
cells.
[0318] Neurochemical Factors, Neuropeptides and Neurotransmitters
and the Microbiota-Gut-Brain Axis
[0319] 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 ([14], [32], [10], [33]). The
experiments performed by the inventors indicate that behavioural
changes can be triggered by administration of Blautia strains. 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 Blautia strains. 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 Blautia strains may be also achieved for
these disorders and conditions. Administration of Blautia stercoris
may be particularly effective for triggering behavioural changes
associated with central nervous system disorders or conditions. In
certain embodiments, administration of Blautia wexlerae may be
particularly effective for triggering behavioural changes
associated with central nervous system disorders or conditions.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] The signalling of the microbiota-gut-brain axis is modulated
by levels of .gamma.-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.
[0324] 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 and Huntington's disease.
[0325] 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 permeability and/or barrier function. In
certain other embodiments, the histamine acts as a neurotransmitter
linked to central processes.
[0326] 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
hormones.
[0327] 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 C-reactive protein; IL-1 family cytokines; IL-1.beta.;
IL-2; IL-4; IL-6; IL-8; IL-10; IL-12p40; IL-17; IL-17A; IL-21;
IL-23; TNF-.alpha. and IFN-.gamma.. 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-.alpha.. In preferred embodiments, the
compositions of the invention modulate the levels of IFN-.gamma..
In some embodiments, the compositions of the invention modulate the
IFN-.gamma.:IL-10 ratio. In certain preferred embodiments, the
compositions of the invention decrease the IFN-.gamma.:IL-10 ratio.
In preferred embodiments, the compositions of the invention
decrease the levels of the pro-inflammatory cytokines TNF-.alpha.
and IFN-.gamma.. 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.
[0328] 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.
[0329] 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.
[0330] 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
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
abnormalities). 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.
[0331] 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 permeability 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.
[0332] 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 harmful 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,
Ruminococcus genera and/or the Alcaligenaceae family. In certain
embodiments, the compositions of the invention increase the level
of Lactobacillus plantarum and/or Saccharomyces boulardii.
[0333] 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, 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.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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
abnormally and persistently elevated, expansive, or irritable
mood.
[0338] 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
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: 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.
[0339] In preferred embodiments, the compositions of the invention
improve spatial and/or non-spatial memory deficits in a subject. In
preferred embodiments, the 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.
[0340] In preferred embodiments, the compositions of the invention
prevent, reduce or alleviate at least one comorbidity associated
with a CNS disorder disclosed herein.
[0341] 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 Health Questionnaire (GHQ); the
Depression Anxiety and Stress Scale (DASS); 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 (BAI); the Hopkins
Symptom Checklist (HSCL-90); the Hospital Anxiety and Depression
Scale (HADS); the 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).
[0342] 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 examples, the compositions of the invention
may improve reciprocal social interactions; olfactory
communication; ultrasonic vocalisation; motor stereotypes (such as
circling and vertical jumping), repetitive behaviour such as
self-grooming and diffing; and perseverance in spatial tasks.
[0343] 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 Fmr1 gene mutant mice
strains).
[0344] Butyrate is a short chain fatty acid that acts as a histone
deacetylase inhibitor, is capable of signalling through G-protein
coupled receptors and is implicated in the regulation of metabolic
pathways.
[0345] The Examples demonstrate that Blautia hydrogenotrophica
increases the intestinal levels of butyrate when administered in
oral compositions. The Examples also demonstrate that Blautia
hydrogenotrophica is useful for treating central nervous system
disorders and conditions. This effect of Blautia hydrogenotrophica
may be mediated by butyrate.
[0346] Butyrate has been linked to histone deacetylation in the
hippocampus and frontal cortex of the brain [34] and has been
implicated in Huntington's disease, Parkinson's disease,
Alzheimer's disease and autism [35].
[0347] In certain embodiments, the Blautia hydrogenotrophica strain
for use in the invention is a butyrate producer. In certain
embodiments, the Blautia hydrogenotrophica strain for use in the
invention synthesise butyrate by the acetyl-CoA, glutarate,
4-aminobutyrate and/or lysine pathways. In certain embodiments, the
Blautia hydrogenotrophica strain for use in the invention
metabolises complex polysaccharides (e.g. starch and xylan) to
produce acetyl-CoA, which can subsequently be used to synthesise
butyrate. In certain embodiments, the Blautia hydrogenotrophica
strain for use in the invention produces butyrate by bacterial
fermentation in the colon.
[0348] In certain preferred embodiments, the compositions of the
invention comprising Blautia hydrogenotrophica modulate the levels
of butyrate. In certain embodiments, compositions of the invention
comprising Blautia hydrogenotrophica increase the levels of
butyrate.
[0349] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica is a histone deacetylase
(HDAC) inhibitor. In certain embodiments, the composition of the
invention comprising Blautia hydrogenotrophica inhibits histone
deacetylation in the hippocampus and frontal cortex of the brain.
In certain embodiments, the composition of the invention comprising
Blautia hydrogenotrophica increases histone acetylation and
promotes the expression of pro-survival, pro-regenerative and
pro-plasticity genes. In certain embodiments, the composition of
the invention comprising Blautia hydrogenotrophica rescues histone
acetylation, prevents neuronal cell death and extends lifespan (for
example in Huntington's disease). In certain embodiments, the
composition of the invention comprising Blautia hydrogenotrophica
protects neurones from cell death (for example in Parkinson's
disease). In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica restores histone acetylation
and increases the expression of learning associated genes (e.g. for
treating or preventing Alzheimer's disease). In certain
embodiments, these epigenetic modifications may be potential
psychiatric treatments.
[0350] In certain embodiments, the HDAC inhibitor activity of the
composition of the invention comprising Blautia hydrogenotrophica
influences the transcription of numerous genes involved in neuronal
survival, plasticity and regeneration. In certain embodiments, the
composition of the invention comprising Blautia hydrogenotrophica
increases the acetylation around the promoters of neurotrophic
factors. In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica increases the acetylation
around the promoter of BDNF, GDNF and NGF. In certain embodiments,
the composition of the invention comprising Blautia
hydrogenotrophica increases the expression of BDNF, GDNF and NGF.
In certain embodiments, the composition of the invention comprising
Blautia hydrogenotrophica increases the expression of immediate
early genes involved in plasticity, including c-Fos and Homer1a. In
certain embodiments, the expression of these factors is altered in
the brain.
[0351] In certain embodiments, the deacetylase inhibitory activity
of the composition of the invention comprising Blautia
hydrogenotrophica maintains acetylation of non-histone proteins. In
certain embodiments, the acetylation affects the enzymatic and
metabolic activity of many proteins. For example, HDAC inhibitors
have been shown to maintain acetylation and activation of the
transcription factor Sp1 during oxidative stress, enhancing the
protective adaptive response to promote cell survival. In certain
embodiments, the composition of the invention comprising Blautia
hydrogenotrophica prevents oxidative stress in vivo.
[0352] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica restores blood-brain barrier
(BBB) integrity and/or tight junction protein expression (e.g.
claudin 5 and/or occluding) in the frontal cortex, hippocampus and
striatum. In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica restores and/or maintains BBB
integrity. In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica promotes and/or maintains
tight junction expression. Therefore, in certain embodiments, the
composition of the invention comprising Blautia hydrogenotrophica
can establish and/or maintain a barrier to inflammatory mediators,
neurochemical factors, neuropeptides and neurotransmitters
associated with central nervous system disorders, in particular
neurodevelopmental disorders and/or neuropsychiatric
conditions.
[0353] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica inhibits neuro-inflammation.
In certain embodiments, the composition of the invention comprising
Blautia hydrogenotrophica increases the levels of IL-1RA (an
inhibitor of the pro-inflammatory IL-1.beta.). In certain
embodiments, the composition of the invention comprising Blautia
hydrogenotrophica decreases the levels pro-inflammatory IL-1.beta.
and/or TNF.alpha.. In certain embodiments, the composition of the
invention comprising Blautia hydrogenotrophica increases IL-4
expression, which increases the levels of IL-1RA.
[0354] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica is an anti-inflammatory agent.
In certain embodiments, the composition of the invention comprising
Blautia hydrogenotrophica inhibits nuclear factor .kappa.B
(NF-.kappa.B) activation. Accordingly, the composition of the
invention comprising Blautia hydrogenotrophica may modulate the
expression of early immune inflammatory response genes, including
IL-1B, TNF.alpha., IL-2, IL-6, IL-8, IL-12, inducible nitric acid
synthase, cyclooxygenase-2, intercellular adhesion molecule-1, T
cell receptor-.alpha. and IHC class II molecules.
[0355] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica affects mitochondrial
activity. Accordingly, in certain embodiments, the composition of
the invention comprising Blautia hydrogenotrophica treats and/or
prevents Alzheimer's disease, Parkinson's disease, Huntington's
disease, mitochrondial encephalopathy and/or
adrenoleukodystrophy.
[0356] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica affects the signalling through
GPCRs. Accordingly, in certain embodiments, the composition of the
invention comprising Blautia hydrogenotrophica treats and/or
prevents Parkinson's disease.
[0357] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica affects histone acetylation.
Accordingly, in certain embodiments, the composition of the
invention comprising Blautia hydrogenotrophica treats and/or
prevents Alzheimer's disease, Parkinson's disease, and/or
Huntington's disease.
[0358] In certain embodiments, the composition of the invention
comprising Blautia hydrogenotrophica affects microbiome
homeostasis. Accordingly, in certain embodiments, the composition
of the invention comprising Blautia hydrogenotrophica treats and/or
prevents central nervous system disorders and conditions.
[0359] In certain embodiments, the compositions of the invention
comprising Blautia hydrogenotrophica may trigger improvement in
behavioural changes associated with central nervous system
disorders or conditions.
[0360] In certain embodiments, the effects of Blautia
hydrogenotrophica may be independent of butyrate. For example, the
Examples demonstrate that administration of Blautia
hydrogenotrophica, but not butyrate alone, significantly increases
horizontal and vertical activity of mice and time spent in the
centre of the open field model suggesting a role in reducing
anxiety. Specifically, Blautia hydrogenotrophica displays efficacy
in reducing anxiety-like and stereotyped behaviour, while the
efficacy of butyrate is limited to reducing stereotyped
behaviour.
[0361] Modes of Administration
[0362] Preferably, the compositions of the invention 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. Generally, the
compositions of the invention are administered orally, but they may
be administered rectally, intranasally, or via buccal or sublingual
routes.
[0363] In certain embodiments, the compositions of the invention
may be administered as a foam, as a spray or a gel.
[0364] In certain embodiments, the compositions of the invention
may be administered as a suppository, such as a rectal suppository,
for example in the form of a theobroma oil (cocoa butter),
synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin,
polyethylene glycol, or soap glycerin composition.
[0365] 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.
[0366] 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.
[0367] 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 strain of the invention 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.
[0368] 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.
[0369] 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.
[0370] The compositions of the invention may be administered to a
patient that has been identified as having an abnormal gut
microbiota. For example, the patient may have reduced or absent
colonisation by Blautia, in particular Blautia stercoris or Blautia
wexlerae. For example, the patient may have reduced or absent
colonisation by Blautia, in particular Blautia stercoris, Blautia
wexlerae or Blautia hydrogenotrophica.
[0371] The compositions of the invention may be administered as a
food product, such as a nutritional supplement.
[0372] Generally, the compositions of the invention are for the
treatment of humans, although they may be used to treat 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 performance of animals. If
administered to animals, oral gavage may be used.
[0373] Compositions
[0374] Generally, the composition of the invention comprises
bacteria. In preferred embodiments of the invention, the
composition is formulated in freeze-dried form. For example, the
composition of the invention may comprise granules or gelatin
capsules, for example hard gelatin capsules, comprising a bacterial
strain of the invention.
[0375] 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, references [36], [ ], and [38].
[0376] Alternatively, the composition of the invention may comprise
a live, active bacterial culture.
[0377] 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-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 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.
[0378] In some embodiments, the composition comprises a mixture of
live bacterial strains and bacterial strains that have been
killed.
[0379] 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 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 [39] and [40].
[0380] The composition may be administered orally and may be in the
form of a tablet, capsule or powder. Encapsulated products are
preferred because Blautia are anaerobes. Other ingredients (such as
vitamin C, for example), may be included as oxygen scavengers and
prebiotic substrates 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.
[0381] The composition may be formulated as a probiotic.
[0382] 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.
[0383] A suitable daily dose of the bacteria, for example for an
adult human, may be from about 1.times.10.sup.3 to about
1.times.10.sup.11 colony forming units (CFU); for example, from
about 1.times.10.sup.7 to about 1.times.10.sup.10 CFU; in another
example from about 1.times.10.sup.6 to about 1.times.10.sup.10
CFU.
[0384] In certain embodiments, the composition contains the
bacterial strain in an amount of from about 1.times.10.sup.6 to
about 1.times.10.sup.11 CFU/g, respect to the weight of the
composition; for example, from about 1.times.10.sup.8 to about
1.times.10.sup.10 CFU/g. The dose may be, for example, 1 g, 3 g, 5
g, and 10 g.
[0385] Typically, a probiotic, such as the composition of the
invention, is optionally combined with at least one suitable
prebiotic compound. 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. Known prebiotics include commercial products such
as inulin and transgalacto-oligosaccharides.
[0386] 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). 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 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.
[0387] 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.
[0388] The compositions of the invention may comprise
pharmaceutically acceptable excipients or carriers. Examples of
such suitable excipients may be found in the reference [41].
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art and are described, for example, in
reference [42]. 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.
[0389] 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 term
"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.
[0390] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the genus Blautia and do
not contain bacteria from any other genera, or which comprise only
de minimis or biologically irrelevant amounts of bacteria from
another genera. Thus, in some embodiments, the invention provides a
composition comprising one or more bacterial strains of the genus
Blautia, which does not contain bacteria from any other genera or
which comprises only de minimis or biologically irrelevant amounts
of bacteria from another genera, for use in therapy.
[0391] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Blautia
stercoris or Blautia wexlerae and do not contain bacteria from any
other species, or which comprise only de minimis or biologically
irrelevant amounts of bacteria from another species.
[0392] Thus, in some embodiments, the invention provides a
composition comprising one or more bacterial strains of the species
Blautia stercoris or Blautia wexlerae, 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.
[0393] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Blautia
hydrogenotrophica 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 Blautia hydrogenotrophica,
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.
[0394] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Blautia
stercoris or Blautia wexlerae and do not contain bacteria from any
other Blautia species, or which comprise only de minimis or
biologically irrelevant amounts of bacteria from another Blautia
species. Thus, in some embodiments, the invention provides a
composition comprising one or more bacterial strains of the species
Blautia stercoris or Blautia wexlerae, which does not contain
bacteria from any other Blautia species or which comprises only de
minimis or biologically irrelevant amounts of bacteria from another
Blautia species, for use in therapy.
[0395] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Blautia
hydrogenotrophica and do not contain bacteria from any other
Blautia species, or which comprise only de minimis or biologically
irrelevant amounts of bacteria from another Blautia species. Thus,
in some embodiments, the invention provides a composition
comprising one or more bacterial strains of the species Blautia
hydrogenotrophica, which does not contain bacteria from any other
Blautia species or which comprises only de minimis or biologically
irrelevant amounts of bacteria from another Blautia species, for
use in therapy.
[0396] In certain embodiments, the compositions of the invention
contain a single bacterial strain or species and do not contain any
other bacterial strains or species. 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.
[0397] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the genus Blautia, 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.
[0398] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the species Blautia
stercoris or Blautia wexlerae, 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.
[0399] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the species Blautia
hydrogenotrophica, 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.
[0400] In some embodiments, the compositions of the invention
comprise more than one bacterial strain. For example, in some
embodiments, the compositions of the invention comprise more 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. The invention comprises
any combination of the foregoing.
[0401] In some embodiments, the composition comprises a microbial
consortium. For example, in some embodiments, the composition
comprises the Blautia bacterial strain as part of a microbial
consortium. For example, in some embodiments, the Blautia 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 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 Blautia in combination with 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.
[0402] In some embodiments, the composition of the invention
additionally comprises a bacterial strain that has the same safety
and therapeutic efficacy characteristics as strain MRX006, but
which is not MRX006 deposited as NCIMB 42381, or which is not a
Blautia stercoris. In some embodiments, the composition of the
invention additionally comprises a bacterial strain that has the
same safety and therapeutic efficacy characteristics as strain
MRX008, but which is not MRX008 deposited as NCIMB 42486, or which
is not a Blautia wexlerae.
[0403] In some embodiments, the composition of the invention
additionally comprises a bacterial strain that has the same safety
and therapeutic efficacy characteristics as the Blautia
hydrogenotrophica strain deposited as DSM 14294, but which is not
the Blautia hydrogenotrophica strain deposited as DSM 14294, or
which is not a Blautia hydrogenotrophica.
[0404] In some embodiments, the composition of the invention does
not comprise a bacterial strain of 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 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.
[0405] 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 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.
[0406] In some embodiments, the bacterial strain for use in the
invention is obtained from human adult 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.
[0407] As mentioned above, in some embodiments, the one or more
Blautia 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.
[0408] The compositions for use in accordance with the invention
may or may not require marketing approval.
[0409] 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.
[0410] 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.
[0411] The compositions of the invention can comprise
pharmaceutically acceptable excipients, diluents or carriers.
[0412] In certain embodiments, the invention provides a
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 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; and/or chronic pain; motor neuron disease; Huntington's
disease; Guillain-Barre syndrome and/or meningitis.
[0413] 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 disorder;
post-traumatic stress disorder; dementia; Alzheimer's; Parkinson's
disease; 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.
[0414] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein the amount of the bacterial
strain is from about 1.times.10.sup.3 to about 1.times.10.sup.11
colony forming units per gram with respect to a weight of the
composition.
[0415] 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.
[0416] 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.
[0417] 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.
[0418] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising a diluent selected from the
group consisting of ethanol, glycerol and water.
[0419] 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.
[0420] In certain embodiments, the invention provides the above
pharmaceutical composition, further comprising at least one of a
preservative, an antioxidant and a stabilizer.
[0421] 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.
[0422] 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.
[0423] 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.
[0424] The composition of the present invention may, in some
embodiments, be provided as a pharmaceutical formulation. For
example, the composition may be provided as a tablet or capsule. In
some embodiments, the capsule is a gelatine capsule
("gel-cap").
[0425] In some embodiments, the compositions of the invention are
administered orally. Oral administration may involve swallowing, so
that the compound enters the gastrointestinal tract, and/or buccal,
lingual, or sublingual administration by which the compound enters
the blood stream directly from the mouth.
[0426] Pharmaceutical formulations 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.
[0427] In some embodiments the pharmaceutical formulation is an
enteric formulation, i.e. a gastro-resistant formulation (for
example, resistant to gastric pH) that is suitable for delivery of
the composition of the invention to the intestine by oral
administration. Enteric formulations may be particularly useful
when the bacteria or another component of the composition is
acid-sensitive, e.g. prone to degradation under gastric
conditions.
[0428] In some embodiments, the enteric formulation comprises an
enteric coating. In some embodiments, the formulation is an
enteric-coated dosage form. For example, the formulation 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.
[0429] In some embodiments, the enteric formulation is
intrinsically enteric, for example, gastro-resistant without the
need for an enteric coating. Thus, in some embodiments, the
formulation is an enteric formulation that does not comprise an
enteric coating. In some embodiments, the formulation is a capsule
made from a thermogelling material. In some embodiments, the
thermogelling 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 forming
polymer. In some embodiments, the capsule comprises a shell and the
shell comprises hydroxypropylmethylcellulose and does not comprise
any film forming polymer (e.g. see [43]). In some embodiments, the
formulation is an intrinsically enteric capsule (for example,
Vcaps.RTM. from Capsugel).
[0430] In some embodiments, the formulation 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.
[0431] Culturing Methods
[0432] The bacterial strains for use in the present invention can
be cultured using standard microbiology techniques as detailed in,
for example, references [44], [ ] and [46].
[0433] The solid or liquid medium used for culture may be YCFA agar
or YCFA medium. YCFA medium may include (per 100 ml, approximate
values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO.sub.3 (0.4
g), cysteine (0.1 g), K.sub.2HPO.sub.4 (0.045 g), KH.sub.2PO.sub.4
(0.045 g), NaCl (0.09 g), (NH.sub.4).sub.2SO.sub.4 (0.09 g),
MgSO.sub.4.7H.sub.2O (0.009 g), CaCl.sub.2 (0.009 g), resazurin
(0.1 mg), hemin (1 mg), biotin (1 .mu.g), cobalamin (1 .mu.g),
p-aminobenzoic acid (3 .mu.g), folic acid (5 .mu.g), and
pyridoxamine (15 .mu.g).
[0434] Bacterial Strains for Use in Vaccine Compositions
[0435] 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 permeability 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.
[0436] General
[0437] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., references [47] and [48]-[54], etc.
[0438] The term "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.
[0439] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0440] In certain embodiments the term "modulate" means increase or
activate. In alternative embodiments, the term "modulate" means
decrease or suppress.
[0441] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0442] 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
determined using software programs known in the art, for example
those described in section 7.7.18 of ref. [55]. A preferred
alignment is determined by the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is disclosed in ref
[56].
[0443] 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 performed in an alternative
order, if appropriate.
[0444] 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).
MODES FOR CARRYING OUT THE INVENTION
[0445] The present study aimed to assess the effect of live
biotherapeutics on the treatment of psychiatric and neurological
disorders in two different mouse models that display behavioural
characteristics associated with neurodevelopmental and psychiatric
disorders. In particular, the study focuses 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 MRX006 versus
vehicle treatment across anxiety, depression, and cognitive and
social domains of behaviour in the three mouse models were
investigated. In addition, physiological and anatomical analyses
were performed as well as detection of circulating cytokine and
oxytocin levels.
[0446] 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 live biotherapeutics tested in the
Examples have shown effective treatment of at least one core
symptom of autistic spectrum disorder, so these strains and related
Blautia 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.
Example 1--Assessing Anxiety, Depression, Cognitive and Social
Domains of Behaviour in C57BL/6 Mice
Example 1a--Materials and Methods for the C57BL/6 Mice Experiments
Mice
[0447] Male C57BL/6 mice were purchased from Harlan UK. The animals
were housed in a temperature- and humidity-controlled room on a 12
hr dark cycle (lights on from 7:00-19:00 hr). All experiments were
conducted in accordance with the European Directive 2010/63/EEC,
the requirements of S.I. No 543 of 2012, and approved by the Animal
Experimentation Ethics Committee of University College Cork.
[0448] Strain
[0449] MRX006: Blautia stercoris bacterium deposited under
accession number NCIMB 42381.
[0450] Biotherapeutic was provided in glycerol stock. Live
biotherapeutics were grown in the facility in anaerobic
conditions.
[0451] Live Biotherapeutic Administration
[0452] Dosing with MRX006 or vehicle (control) commenced when the
mice were 7 weeks old. These mice were treated once daily with
MRX006 or phosphate buffer solution (PBS) for 4 weeks before the
beginning of the behavioural battery. Mice were further treated
once daily during the behavioural battery. MRX006 (1.times.10.sup.9
CFU oral administration) was dissolved in PBS prior to
administration.
[0453] Administration Schedule
[0454] The treatment groups for the study are shown below. The
vehicle for oral administration is PBS. Daily oral administration
occurs via oral gavage.
TABLE-US-00001 Group Treatment Number 1 Naive (no gavage) 12 2
Control (PBS, oral gavage) 12 3 MRX006 (oral gavage in PBS) 12
[0455] The Naive group was not handled until the beginning of the
behavioural battery.
[0456] Fecal Collection
[0457] Fresh fecal samples were collected from individual mice
every week until the end of the study. At least 20 mg of fresh
faeces were placed in a microcentrifuge tube, place immediately on
ice and then stored at -80.degree. C.
[0458] Experimental Design and Methods
[0459] As outlined above, dosing with MRX006 commenced when the
mice were 7 weeks old. The initial dosing took place for 4 weeks
before the behavioural experiments. The behavioural battery
occurred in the following order: marble burying and 3 chamber tests
at week 5; the elevated plus maze and tail suspension tests at week
6; the open field and novel object recognition tests at week 8; the
stress-induced hyperthermia test at week 9; the fear conditioning
test at week 10; and the forced swim test at week 11. The
fluorescein gut permeability assay was performed at week 9.
Finally, in weeks 12, the mice were culled and dissected for brain,
proximal and distal colon, adrenal and spleen regions, along with
plasma samples.
[0460] Graphical Design and Statistical Analysis
[0461] All graphs were generated on graphpad prism software
(version 5). Data were analysed using IBM SPSS Statistic 22.0
(EEUU). Data distribution was analysed using the Kolmogorov-Smirnov
normality test. Data comparing vehicle group versus MRX006 groups
were analysed using two-way ANOVA and Fisher's least significant
difference (LSD) post hoc test. Data comparing vehicle group versus
naive mice were analysed by unpaired Student t test. Non-normally
distributed data were analysed by the Kruskal-Wallis and
non-parametric Mann-Whitney U test. P<0.05 was the criterion for
statistical significance.
Example 1b--Assessment of Social Interaction Behaviour--the Three
Chamber Tests
[0462] Rationale
[0463] 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
is used to characterize and demonstrate changes in this behavioural
readout. The test allows mice to freely explore between an
inanimate object or sex-matched conspecific mice.
[0464] In addition, the 3-chamber test (3-CHT) is a test used to
assess cognition in the form of general sociability and interest in
social novelty in rodent models. Rodents normally prefer to spend
more time with another rodent (sociability) than with an object.
Moreover, rodents prefer to investigate a novel mouse versus a
familiar mice (social novelty). Based on these inclinations, the
3-CHT can help identify rodents with deficits in sociability and/or
social novelty.
[0465] Methods
[0466] Animals are placed in a rectangular apparatus divided into
three chambers (left and right and a smaller centre chamber) by
partitions with small circular openings allowing easy access to all
compartments. The test is composed of three sequential 10 min
trials: (1) habituation (the test animal is allowed to explore the
three empty chambers); (2) sociability (an unfamiliar animal is
placed in an inner mesh wire cage in either the left or right
chambers); (3) social novelty preference (a novel animal is placed
into the previously empty inner cage in the chamber, opposite the
now familiar animal). Naive animals should have no preference for
either chamber in the habituation phase, a preference for the mouse
in the sociability phase, and a preference for the novel mouse in
the social novelty phase. An increase in the discrimination ratio
would suggest an increase in social behaviour. All animals are age-
and sex-matched, with each chamber cleaned and lined with fresh
bedding after each 30 minute trial. For each of the three stages,
behaviour is recorded by a video camera mounted above the
apparatus.
[0467] Results
[0468] Student t test within groups revealed that all groups spent
more time investigating a mouse versus an object (**p<0.01)
suggesting no deficits in sociability (FIG. 1). Daily gavage did
not affect sociability. Interestingly, MRX006 enhanced the time
spent investigating a novel versus a familiar mice, suggesting
increased social novelty (p<0.05; FIG. 1).
[0469] Conclusions
[0470] Chronic treatment with MRX006 enhanced preference for social
novelty in C57BL/6 mice in the three chamber test.
Example 1c--Assessing Depression-Like Behaviour--the Forced Swim
Test (FST)
[0471] Rationale
[0472] The forced swim test (FST) is the most widely used
experimental paradigm to assess antidepressant activity. Naive
animals will display escape behaviour in the form of swimming,
climbing and diving before adapting an immobile floating posture.
The duration of immobility is indicative of behavioural despair.
Antidepressant drugs decrease the time spent immobile in this
test.
[0473] Methods
[0474] Mice are forced to swim for 6 min in a glass cylinder
(24.times.21 cm) filled with 23-25.degree. C. tap water to a depth
of 17 cm. The FST was videotaped from a ceiling camera. The
behavioural parameter scored is immobility during the last 4 min of
the 6-min test.
[0475] Results
[0476] Daily gavage increased immobility time suggesting
depressive-like behaviour compared to naive mice (t Student test, t
(190=4.565; p<0.01; FIG. 2). Chronic treatment with MRX006
significantly reduced immobility suggesting antidepressant-like
effects compared to the vehicle group (F (2.29)=14.992;
**p<0.01).
[0477] Conclusions
[0478] Chronic treatment with MXR006 induced antidepressant-like
behaviour in the forced swim test.
Example 1d--Assessing Depression-Like Behaviour--the Tail
Suspension Test
[0479] Rationale
[0480] The tail suspension test is a well-characterized test used
to assess antidepressant-like behaviour. The time spend immobile is
an index of depression-like behaviour. Treatment with
antidepressant drugs decreases the time spent immobile.
[0481] Methods
[0482] Mice are suspended to an elevated bar (60 cm) by a piece of
adhesive tape attached 1 cm before the tip of their tail for a
period of 6 min. The mice are suspended in such a way that they
cannot escape or hold on to nearby surfaces. During this test, six
minutes in duration, the resulting escape oriented behaviours are
quantified. The behavioural parameter scored is time spent
immobile. The test was video-recorded by a tripod camera and the
time of immobility was scored manually by an investigator blind to
the experimental conditions.
[0483] Results
[0484] Daily handling for gavage (Student t test, t (20)=0.9405;
p=0.3582) and chronic treatment with MRX006 (one-way ANOVA, F
(2.30)=2.014; p=0.152) did not induce any significant effect in the
tail suspension test (FIG. 3).
[0485] Conclusion
[0486] Chronic treatment with MRX006 did not induce any observable
anti-depressant-like behaviour in C57BL/6 mice in the tail
suspension test.
Example 1e--Assessing Cognition--the Fear Conditioning Test
[0487] Rationale
[0488] Contextual fear conditioning is used as a measure of
hippocampus-dependent memory. Fear conditioning is a form of an
associative learning which measures the freezing response displayed
by the animal to an unconditioned stimuli (US), such as a shock
with a conditioned stimulus (CS), a particular tone or light or
smell. The measurement of freezing levels was used to assess the
animal response to the US and CS stimuli. This test measures how
efficiently the mice forget what they have acquired on the
acquisition day. The test assesses the anxiety of mice towards
conditioned stimuli associated with unconditioned stimuli and the
speed at which the mice show reduced anxiety and/or stress
(freezing levels) in the presence of conditioned stimuli in the
repeated absence of paired unconditioned stimuli.
[0489] Methods
[0490] The apparatus for this test consisted of chambers with a
light above. Each chamber is located inside a larger chamber, which
protects from outside light and noise. On the first day (training
or acquisition stage), mice were placed into the chamber and their
freezing behaviour was recorded for 3 min (baseline), followed by
up to 6 light/tone [conditioned stimulus (CS--30 s)] and footshock
[unconditioned stimulus (US--2 s)] pairings with an interval of up
to 2 min. Pairings consisted of the cue [e.g., a combined light
(.about.260 lx) and tone exposure (80 dB)] for 20 s and an electric
footshock during the last 2 s of the cue. Foot shock increases
freezing behaviour. The intensity of the current was 0.6 mA. The
minimal current that induces a freezing response was used. Two
minutes after the last pairing, mice were returned to their normal
housing conditions. At 24 and 48 h after conditioning (days 2
(retrieval stage) and 3 (extinction stage), respectively), the same
experimental procedure was repeated in absence of footshocks to
test for memory retention and extinction of the conditioned fear
memory (extinction phase). Contextual memory retention is
characterized by freezing behaviour when the animal is placed in
the context (i.e. the footshock chamber) in the absence of a foot
shock.
[0491] Results
[0492] Freezing levels were measured in the acquisition phase
(exposure to both CS and US coupled together), retrieval and
extinction phase (CS not coupled with US) (see FIG. 4). In this
study, daily gavage induced a significant increase in freezing
levels (phase 1 #p<0.05; phase 2 ###p<0.001; phase 3-4
##p<0.01) in the acquisition phase as compared to naive animals.
MRX006 chronic treatment did not alter freezing levels when
compared to the vehicle group during the acquisition phase. The
acquisition phase was followed by the retrieval phase which took
place 24 hours after the training session (CS not followed by US).
The data showed a significant increase in freezing levels in phase
1 (exposure to the first tone) in the vehicle group compared to the
naive group (p<0.05). Interestingly, a significant reduction in
the freezing levels was observed at phase 1 in MRX006-treated mice
(*p<0.05) when compared to the vehicle group. Overall, in the
retrieval phase, MRX006-treated displayed a trend for decreased
freezing levels when compared to the vehicle group, suggesting that
chronic treatment with MRX006 may enhance learning. The retrieval
phase was followed by the extinction phase (24 hours later, CS and
no US). This test measures how efficiently the mice forget what
they have acquired on the acquisition day. In phase 3 of extinction
phase the vehicle group displayed increased freezing levels when
compared to the naive group (p<0.05). Chronic treatment with
MRX006 did not induce any significant change in the extinction
phase.
[0493] Conclusion
[0494] Chronic treatment with MRX006 reduced freezing levels in the
retrieval phase suggesting MRX006 may enhance learning. Overall,
chronic treatment with MRX006 did not observably alter
significantly fear conditioned behaviour.
Example 1f--Assessing Cognition--the Novel Object Recognition (NOR)
Test
[0495] Rationale
[0496] The protocol used was adapted from Bevins and Besheer
(2006), and used to test cognition, particularly recognition
memory. This test is based on the spontaneous tendency of rodents
to spend more time exploring a novel object than a familiar one.
The choice to explore the novel object reflects the use of learning
and recognition memory. In addition, improved memory is a
reflection of reduced depression-like behaviour.
[0497] Methods
[0498] The protocol used was adapted from Bevins and Besheer
(2006). It was conducted over 3 days. On Day 1, the animals were
allowed to acclimate to the testing environment for 10 minutes,
which was a large container equipped with an overhead camera. No
bedding was used and the container was wiped with 70% ethanol
between each animal. On Day 2, the animals were allowed to
acclimate to the test apparatus for 10 minutes. Following this
period, the animal was removed from the container and two identical
objects were introduced to the environment. The animal was returned
to the container and allowed to explore for a further 10 minutes.
The objects were cleansed before each trial with a 70% ethanol
solution. Following the training period, the rodent was removed
from the environment for a delay period of 24 hours. On Day 3, the
rodent was returned to the container, which this time contained
only 1 familiar object from the day previous and 1 novel object.
Activity of the animal with the 2 objects was recorded for 5
minutes. The amount of time that the rodent spent exploring each
object was recorded by manual observation and a discrimination
index (DI) value corresponding to time spent interacting with the
novel object over total interaction time was generated. A decrease
in DI compared to control rats indicates a deficit in this type of
memory.
[0499] Results
[0500] Daily handling for gavage did not affect recognition memory
in C57Bl/6 mice (FIG. 5). Indeed, both groups show a pattern for
preference exploring more a novel versus a familiar object
(although did not reach statistically significance). Mice
chronically treated with MRX006 display significantly increased
preference exploring a novel object versus a familiar one
(p<0.05) (FIG. 5).
[0501] Conclusion
[0502] Chronic treatment with MRX006 resulted in C57Bl/6 mice
spending significantly more time investigating a novel versus a
familiar object suggesting enhance recognition memory.
Example 1g--Assessing Anxiety-Like Behaviour--the Marble Burying
Test
[0503] Rationale
[0504] 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.
[0505] Methods
[0506] Mice were individually placed into a novel polypropylene
cage (35.times.28.times.18.5 cm, L.times.W.times.H), containing
standard rodent (hard wood) bedding (5 cm) and 20 marbles on top of
it (five rows of marbles regularly spaced 2 cm away from the walls
and 2 cm apart). Experiments were conducted under a light intensity
of 1000 lux. 30 minutes later, mice were removed from these cages
and the number of marbles buried for more than 2/3rds of their
surface was scored.
[0507] Results
[0508] Student t test (vehicle versus naive; t (20)=0.1308;
p=0.8973) and one way ANOVA analysis (F (2.38)=0.992; p=0.384)
revealed that neither chronic treatment with MRX006 nor daily
gavage altered the number of marbles buried suggesting no
alterations in anxiety-like baselines (FIG. 6).
[0509] Conclusions
[0510] Chronic treatment with MRX006 did not alter observably
anxiety-like behaviour in C57Bl/6 mice in the marble burying
test.
Example 1h--Assessing Anxiety-Like Behaviour--the Elevated Plus
Maze Test
[0511] Rationale
[0512] 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 arms of the maze. An increase in open arm activity (duration)
reflects anti-anxiety behaviour.
[0513] Methods
[0514] The set up consisted of a grey plastic cross-shaped maze 1
meter elevated from the floor, comprising two open (aversive) and
two closed (safe) arms (50.times.5.times.15 cm walls). Experiments
occurred under red light (7 lux). Mice were placed into the centre
of the maze facing an open arm (to avoid direct entrance into a
closed arm) and were allowed to explore the arena for a duration of
six minutes. Experiments were videotaped using a ceiling camera to
allow for measuring several behavioural parameters. The apparatus
was cleaned with 70% (vol/vol) ethanol after each subject to
prevent olfactory cues from the previous mouse. Time spent in the
open/closed arms, time spent in the center, and the number of
transitions were analysed manually. The percentage of time spent
and the number of entries in each arm was measured for anxiety-like
behaviour and locomotor activity, respectfully. Entrance into an
arm was defined as all four paws inside the arm. An increase in
open arm activity (duration) reflects anti-anxiety behaviour.
[0515] Results
[0516] Student t test analysis revealed that daily gavage did not
affect the time spent in open arms (FIG. 7). One-way ANOVA analysis
revealed that chronic treatment with MRX006 did not alter the
behaviour in the elevated plus maze when compared to the control
group (FIG. 7). Specifically, chronic treatment with Mrx006 did not
alter the time spent in open arms and in closed arms.
[0517] Conclusions
[0518] Chronic treatment with MRX006 did not observably alter
behaviour of C57Bl/6 mice in the elevated plus maze.
Example 1i--Assessing Anxiety Levels in the Stress Induced
Hyperthermia (SIH) Test
[0519] Rationale
[0520] The SIH paradigm is a well-characterised index of anxiety.
In this test, the stress is triggered simply by the measurement of
rectal temperature.
[0521] Methods
[0522] Briefly, animals were singly housed 1 d before the test.
Rectal temperature was measured twice with a 15 min interval using
a lubricated temperature-sensitive probe. Due to the stress
experienced during the first temperature measurement, the
temperature of the second measurement (T2) is higher than that of
the first (T1). This difference in temperature (.DELTA.T=T2-T1) is
defined as the SIH response. The SIH response is reduced by
different classes of anxiolytics.
[0523] Results
[0524] Daily handling for gavage increased .DELTA.T suggesting
anxiety-like behaviour (Student t test, t (19)=2.121, p=0.047).
Chronic treatment with MRX006 did not induce changes in .DELTA.T
when compared to the vehicle group (one-way ANOVA, F (2.29)=1.215;
p=0.312) (FIG. 8).
[0525] Conclusions
[0526] Chronic treatment with MRX006 did not observably alter the
stress induced change in temperature in the stress induced
hyperthermia test in C57Bl/6 mice.
Example 1j--Physiological Analysis--Plasma Oxytocin Levels
[0527] Methods and Results
[0528] Oxytocin levels were measured in naive, vehicle and MRX006
treated mice (FIG. 9). Oxytocin peptide, which acts in the central
nervous systems of males and females is critical for a variety of
complex social behaviours including affiliation, sexual behaviour,
social recognition, aggression and trust. Radioimmunoassay (RIA) is
a sensitive method for measuring very small quantities of peptides
and metabolites in the blood. Samples were prepared for RIA and
dispatched for oxytocin measure through RIA technique (RIAgnosis,
Sinzing, Germany). Chronic treatment with MRX006 in C57Bl/6 mice
showed a reduction of oxytocin levels (p<0.05).
Example 1k--Physiological Analysis--Stress-Induced Corticosterone
Plasma Levels
[0529] Methods and Results
[0530] Corticosterone is a major rodent hormone released in
response to stress. In this study corticosterone level changes were
measured at baseline and following the forced swim test (acute
stress exposure) in naive, vehicle and MRX6-treated mice (FIG. 10).
Corticosterone measurements were carried out at different time
points, namely at T0 (before forced swim test), and 30 min (T30),
90 min (T90) and 120 min (T120) post-exposure to forced swim test.
The data showed a significant increase in stress-induced
corticosterone levels in the vehicle group compared to the naive
group at T30 (p<0.05). Interestingly, a significant increase in
stress-induced corticosterone levels was observed at T30 in
MRX6-treated mice when compared to the control group. No
significant changes were observed in the other time points. These
results may suggest an increased sensitivity to stress in both the
vehicle and the MRX006 treated group.
Example 1l--Physiological Analysis--In Vivo Gastrointestinal
Permeability Assay
[0531] Rationale
[0532] This procedure is used to assess in vivo intestinal
motility. Gut permeability was quantified between the different
chronic treatments through the quantification of Fluorescein
isothiocynate (FITC) in the blood post oral administration of the
fluorescein derivative. It is an established method to quantify the
gut permeability, based on the principal of leaking of the orally
administered fluorescein derivative through the gut into the
peripheral system.
[0533] Methods
[0534] Test mice were singly-housed and fasted overnight. The
following morning (.about.9 am), mice were administered FITC
dextran ((600 mg/kg) by oral gavage. Two hours later, 100 .mu.l of
blood sample was collected in heparin-coated capillary tubes and
transferred to a darkened eppendorf and placed on ice. Samples were
centrifuged 3500.times.g for 15 minutes, plasma was aspirated and
samples were stored at -80.degree. D for long storage.
[0535] Undiluted plasma was used to quantify FITC concentration. 25
.mu.l of FITC was pipetted in duplicated in 384 well plate (Greiner
bio one). FITC was measured with a Victor spectrometer between the
ranges of 490 nm-520 nm. For a standard curve, a serial dilution of
FITC was prepared in PBS (pH7.4). An increase in absorbance is
indicative of a decrease in barrier integrity.
[0536] Results
[0537] The data showed a trend towards an increase in intestine
permeability with daily gavages (p=0.051) but it failed to reach
significance (FIG. 11). Overall intestine permeability between the
MRX006 groups remained unaltered.
[0538] Conclusions
[0539] Chronic treatment with MRX006 displayed no observable effect
on gut permeability.
Example 1m--Physiological Analysis--Organ Weight and Colon
Length
[0540] Daily handling for gavage and chronic treatment with MRX006
did not induce changes in caecum weight, spleen weight, and adrenal
weight (FIG. 12).
[0541] Conclusions from the C57B/6 Mouse Model
[0542] Chronic treatment with MRX006 induced antidepressant-like
effects in the forced swim test, a widely used test to screen
antidepressant-like activity. In addition, chronic treatment with
MRX006 enhanced social behaviour in C57Bl/6 mice, which spend more
time interacting with novel versus familiar mice in the 3-chamber
test, indicating that MRX006 reversed social impairments, a core
symptom of autism spectrum disorders. Furthermore, chronic
treatment with MRX006 tended to reduce freezing levels in the fear
conditioning test indicating that administration of this strain
improves cognitive functions of memory and reduces anxiety in
C57Bl/6 mice.
[0543] Further studies are required to characterise the effects of
MRX006 when acutely or sub-chronically administered on oxytocin
levels and corticosterone levels. The evidence indicates that
MRX006 modulates signalling of the hypothalamic pituitary axis
(HPA).
[0544] Therefore, administration of MRX006 causes antidepressant
effects, enhances social novelty and pro-cognitive effects.
Example 2--the BTBR Mouse Model
[0545] 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 (Meyza
and Blanchard, 2017). 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.
Example 2a--Materials and Methods for BTBR Mouse Model
[0546] Mice
[0547] Male BTBR mice were bred in house. The animals were housed
in a temperature- and humidity-controlled room on a 12 hr dark
cycle (lights on from 7:00-19:00 hr). All experiments were
conducted in accordance with the European Directive 2010/63/EEC,
the requirements of S.I. No 543 of 2012, and approved by the Animal
Experimentation Ethics Committee of University College Cork.
[0548] Strain
[0549] MRX006: Blautia stercoris bacterium deposited under
accession number NCIMB 42381.
[0550] Biotherapeutic was provided in glycerol stock. Live
biotherapeutics were grown in the facility in anaerobic
conditions.
[0551] Live Biotherapeutic Administration
[0552] Dosing with MRX006 or vehicle commenced when the mice were 8
weeks old. These mice were treated once daily with MRX006 or
phosphate buffer solution (PBS) for 3 weeks before the beginning of
the behavioural battery. Mice were further treated once daily
during the behavioural battery. MRX006 (1.times.107 to 1.times.109
CFU oral administration) was dissolved in PBS prior to
administration.
[0553] Administration Schedule
[0554] The treatment groups for the study are shown below. The
vehicle for oral administration is PBS. Daily oral administration
occurs via oral gavage.
TABLE-US-00002 Group Treatment Number 1 Control (PBS, oral gavage)
10 2 MRX006 (oral gavage in PBS) 10
[0555] Fecal Collection
[0556] Fresh fecal samples were collected from individual mice
every week until the end of the study. At least 20 mg of fresh
faeces were placed in a microcentrifuge tube, place immediately on
ice and then stored at -80.degree. C.
[0557] Experimental Design and Methods
[0558] As outlined above, dosing with MRX006 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 test at week 4; the
elevated plus maze at week 5; the open field and novel object
recognition tests, and the social transmission of food preference
tests at week 6; the female urine sniffing and social interaction
tests at week 7, and the forced swimming test at week 9. The
carmine red gastrointestinal motility assay and gastrointestinal
permeability assay tail bleeds occurred during weeks 8 and 9
respectively. Finally, in weeks 10 to 11, the mice were killed for
splenocyte stimulation and ex vivo measurement of FITC in the ileum
and colon.
[0559] The effects of live biotherapeutic treatment in the BTBR
model on stereotyped, social and depression-like behaviours, along
with gastrointestinal parameters (permeability and motility) are
outlined in the following examples.
[0560] Group 1, listed in the table above, represents the control
BTBR mice, which would be expected to show phenotypes associated
with autistic spectrum disorders. Any effect of treatment on the
behavioural symptoms of autistic spectrum disorders would be
identified by differences between Group 1 and Group 2.
[0561] Graphical Design and Statistical Analysis
[0562] All graphs were generated on graphpad prism software
(version 5). Data were analysed using IBM SPSS Statistic 22.0
(EEUU). Data distribution was analysed using the Kolmogorov-Smirnov
normality test. Data comparing vehicle group versus the MRX006
group were analysed using one-way ANOVA and Fisher's least
significant difference (LSD) post hoc test. If ANOVA did not reveal
a significant effect of treatment, a priori pairwise comparisons
test against the control group was conducted. Non-normally
distributed data were analysed by the Kruskal-Wallis and
non-parametric Mann-Whitney U test. P<0.05 was the criterion for
statistical significance.
Example 2b--Assessment of Social Behaviours--the Three Chamber
Social Interaction Test
[0563] Rationale
[0564] 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.
[0565] Methods
[0566] Animals are placed in a rectangular apparatus divided into
three chambers (left and right and a smaller centre chamber) by
partitions with small circular openings allowing easy access to all
compartments. The test is composed of three sequential 10 min
trials: (1) habituation (the test animal is allowed to explore the
three empty chambers); (2) sociability (an unfamiliar animal is
placed in an inner mesh wire cage in either the left or right
chambers); (3) social novelty preference (a novel animal is placed
into the previously empty inner cage in the chamber, opposite the
now familiar animal). Naive animals should have no preference for
either chamber in the habituation phase, a preference for the mouse
in the sociability phase, and a preference for the novel mouse in
the social novelty phase. An increase in the discrimination ratio
would suggest an increase in social behaviour. All animals are age-
and sex-matched, with each chamber cleaned and lined with fresh
bedding after each 30 minute trial. For each of the three stages,
behaviour is recorded by a video camera mounted above the
apparatus.
[0567] Results
[0568] For assessing sociability, student's t-test within groups
revealed an increased preference for a novel conspecific (CS) mouse
relative to an object for the MRX006 group [t(22)=5.281;
P<0.0001](FIG. 13A). For assessing social novelty, student's
t-test within groups revealed an increased preference for a novel
conspecific in the vehicle group only [t(22)=3.452; P<0.001].
ANOVA of interaction time with the novel conspecific did not reveal
an effect of treatment [F(3,47)=2.492; P=0.43; FIG. 13B]. However,
a priori pairwise comparisons test revealed that treatment with
MRX006 (t(22)=0.7497; P=0.4614) decreased interaction time with a
novel conspecific when compared to the vehicle group. ANOVA of
percentage time spent investigating the novel conspecific revealed
an effect of treatment [F(3,47)=2.942; P=0.0433; FIG. 13C].
Post-hoc comparisons revealed treatment with MRX006 decreased the
percentage time investigating a novel conspecific (p<0.05).
[0569] Two way ANOVA analysis [Object/Conspecific (CS):
[F(1,47)=21.164; P<0.0001; Treatment: F(1,47)=0.56; P=0.815;
Object/CS.times.Treatment: F(1,47)=5.414; P=0.025] followed by post
hoc analysis revealed that MRX006 treated mice spent more time
investigating the conspecific versus the object (p<0.01; FIG.
13D). Two-way ANOVA analysis [Familiar vs novel: F(1,47)=3.454;
P=0.070; Treatment: F(1,47)=0.360; P=0.552; F/Nx Treatment:
F(1,47)=8.627; P=0.005] followed by post hoc comparisons revealed
that mice treated with MRX006 spent significantly less time
investigating a novel versus a familiar mouse (p<0.05; FIG.
13E). By contrast, vehicle mice spent more time investigating the
novel versus the familiar conspecific (p<0.05; FIG. 13E).
Percentage analysis revealed that MRX006 treated mice spent less
time interacting with the novel conspecific when compared with the
vehicle group (t=2.480 df=22; P=0.0213; FIG. 13F).
[0570] Conclusions
[0571] Chronic treatment with MRX006 reduces social novelty and
decreases social cognition of BTBR mice in the three chamber
test.
Example 2c--Assessment of Social Behaviours--Forced Intruder
Test
[0572] Rationale
[0573] This procedure evaluates social interaction behaviour
between rodents. By placing an intruder mouse into the resident
mouse's home-cage, one can assess social interaction and aggressive
behaviour.
[0574] Methods
[0575] Each session consisted of placing an intruder mouse into a
resident mouse's home-cage for a period of 10 minutes. Experiments
were videotaped using a ceiling camera to allow for measuring
several behavioural parameters. The amount of time that the animals
spent interacting was recorded.
[0576] Results
[0577] ANOVA of interaction time did not reveal an effect of
treatment [F(3,45)=2.327; P=0.088; FIG. 14]. Similarly, a priori
pairwise comparisons test revealed that treatment with MRX006
(t=1.425 df=22; P=0.1682) did not affect social interaction
behaviour when compared to the vehicle group.
[0578] Conclusions
[0579] Chronic treatment with MRX006 does not influence social
behaviour of BTBR mic in the social interaction test.
Example 2d--Assessment of Stereotyped Behaviours--the Marble
Burying Test
[0580] Rationale
[0581] This test assesses for repetitive, compulsive and anxious
behaviour. A higher number of marbles buried is indicative of
greater anxious or stereotyped behaviours. Indeed, Mice treated
with pharmacological agents such as anxiolytics show decreased
marble burying behaviour, compared to the control mice.
[0582] Methods
[0583] Mice were individually placed into a novel polypropylene
cage (35.times.28.times.18.5 cm, L.times.W.times.H), containing
standard rodent (hard wood) bedding (5 cm) and 20 marbles on top of
it (five rows of marbles regularly spaced 2 cm away from the walls
and 2 cm apart). Experiments were conducted under a light intensity
of 1000 lux. 30 minutes later, mice were removed from these cages
and the number of marbles buried for more than 2/3rds of their
surface was scored.
[0584] Results
[0585] There was no effect of treatment as determined by ANOVA on
the number of marbles buried [F(3,45)=1.64; P=0.193]. However, a
priori pairwise comparisons test revealed that MRX006 (t=2.276
df=21, p<0.05) decreased the number of marbles buried (FIG.
15).
[0586] Conclusions
[0587] Treatment with MRX006 reduces repetitive behaviour in BTBR
mice in the marble burying test.
Example 2e--Assessment of Stereotyped Behaviours--the Grooming
Test
[0588] Rationale
[0589] 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.
[0590] Methods
[0591] Mice were individually placed into a novel glass beaker (500
mL), which was covered with a Plexiglas top. Experiments were
conducted under a light intensity of 60 lux. Experiments were
videotaped using a hand-held camera attached to a tripod stand.
Grooming behaviour was recorded for 20 minutes.
[0592] Results
[0593] There was a significant effect of live biotherapeutics as
determined by ANOVA [F(3,47)=4.174; P=0.011] on grooming activity.
Post-hoc comparisons revealed that treatment with MRX006
significantly reduced the amount of time spent grooming relative to
the vehicle group (p<0.05) (FIG. 16). Similarly, a priori
pairwise comparisons test revealed that MRX006 (t=2.895 df=22,
p<0.01) decreased the time spent grooming compared to the
vehicle group.
[0594] Conclusions
[0595] Chronic treatment with MRX006 reduces repetitive behaviours
in BTBR mice in the grooming test.
Example 2f--Assessment of Anxiety-Like Behaviours--the Elevated
Plus Maze
[0596] Rationale
[0597] 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 arms of the maze. An increase in open arm activity (duration)
reflects anti-anxiety behaviour.
[0598] Methods
[0599] The set up consisted of a grey plastic cross-shaped maze 1
meter elevated from the floor, comprising two open (aversive) and
two closed (safe) arms (50.times.5.times.15 cm walls). Experiments
occurred under red light (7 lux). Mice were placed into the centre
of the maze facing an open arm (to avoid direct entrance into a
closed arm) and were allowed to explore the arena for a duration of
five minutes. Experiments were videotaped using a ceiling camera to
allow for measuring several behavioural parameters. The percentage
of time spent and the number of entries in each arm was measured
for anxiety-like behaviour and locomotor activity, respectfully.
Entrance into an arm was defined as all four paws inside the
arm.
[0600] Results
[0601] ANOVA analysis revealed no effects of live biotherapeutic
treatment on percentage of time spent in closed arms
[F(3.47)=0.885; P=0.457; FIG. 17A). Kruskal Wallis non-parametric
analysis of percentage time spent in open arms revealed no effect
of treatment [chi-squared=1.220; df=3; P=0.748; FIG. 17B]. ANOVA of
the number of entries into the closed arms revealed no effect of
treatment [F(3,44)=1.82; P=0.159; FIG. 17C]. Kruskal Wallis
non-parametric analysis of number of the entries into the open arms
revealed no effect of treatment [chi-squared=2.045; df=3; P=0.563;
FIG. 17D].
[0602] Conclusions
[0603] Chronic treatment with MRX006 has no effect on anxiety-like
behaviour in BTBR mice in the elevated plus maze.
Example 2g--Assessment of Anxiety-Like Behaviours--the Open Field
Arena
[0604] Rationale
[0605] The open field arena is used to assess the response of
exposure to a novel stressful environment and locomotor activity.
Naive 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 the centre represents a
decrease in anxiety-like behaviour.
[0606] Methods
[0607] Mice were individually placed into an open field arena
(43.times.35.times.25, L.times.w.times.h) and allowed to explore
for 10 minutes. Experiments occurred under a light intensity of 60
lux. Experiments were videotaped using a ceiling camera to allow
for measuring several behavioural parameters using Ethovision
software. The distance travelled was scored for locomotor
activity.
[0608] Results
[0609] ANOVA of distance moved did not reveal an effect of
treatment on locomotor activity in the open field arena
[F(3,47)=0.317; P=0.813; FIG. 18A and FIG. 18D]. ANOVA of time
spent in the outer zone did not reveal an effect of treatment
[F(3,46)=2.217; P=0.100; FIG. 18B]. However, a priori pairwise
comparisons test revealed that MRX006 treatment (t=2.791 df=21;
p<0.05; FIG. 18E) decreased the time spent in the outer zone of
open field arena. ANOVA of time spent in the inner zone did not
reveal an effect of treatment [F(3,46)=2.217; P=0.100; FIG. 18C].
However, apriori pairwise comparisons test revealed that MRX006
treatment (t=2.791 df=21; p<0.05; FIG. 18F) increased the time
spent in the inner zone of open field arena.
[0610] Conclusions
[0611] Chronic treatment with MRX006 reduces anxiety-like behaviour
in BTBR mice in the open field arena test.
Example 2h--Assessment of Depression-Like Behaviour--the Forced
Swim Test
[0612] Rationale
[0613] The forced swim test (FST) is the most widely used
experimental paradigm to assess antidepressant activity. Naive
animals will display escape behaviour in the form of swimming,
climbing and diving before adapting an immobile floating posture.
The duration of immobility is indicative of behavioural despair.
Antidepressant drugs decrease the time spent immobile in this
test.
[0614] Methods
[0615] Mice are forced to swim for 6 min in a glass cylinder
(24.times.21 cm) filled with 23-25.degree. C. tap water to a depth
of 17 cm. The FST was videotaped from a ceiling camera. The
behavioural parameter scored is immobility during the last 4 min of
the 6-min test.
[0616] Results
[0617] ANOVA of immobility time did not reveal an effect of
treatment [F(3,46)=1.309; P=0.284; FIG. 19].
[0618] Conclusions
[0619] Chronic treatment with MXR006 does not influence immobility
time of BTBR mice in the forced swimming test.
Example 2i--Assessment of Depression-Like Behaviour--the Female
Urine Sniffing Test
[0620] Rationale
[0621] The female urine sniffing test (FUST) is used to assess
anhedonic-like behaviour in rodents. A reduction in sniffing time
suggests social avoidance/anhedonia while an increase represents an
increase in social behaviour/hedonic behaviour.
[0622] Methods
[0623] Experimental mice are singly housed one week prior to the
test. During the test, a cotton tip applicator, dipped in sterile
water, is placed into the home cage and mice are allowed to
sniff/investigate for three minutes. Following this three minute
test, the cotton tip applicator is removed. 45 minutes later, a new
cotton tip applicator is dipped into female urine (collected from
female mice of the same strain that are in the estrous stage of
their cycle), and placed into the cage. Mice are allowed to
sniff/investigate this for a further three minutes. The amount of
time spent sniffing the water and urine is recorded.
[0624] Results
[0625] For the vehicle group, student's t-test revealed a
significant increase in the time spent sniffing urine relative to
the time spent sniffing water [t(16)=2.611; P=0.0189; FIG. 20A].
For exposure to water, ANOVA of time spent sniffing did not reveal
an effect of treatment in the water group [F(3,35)=0.875; P=0.464].
For exposure to urine, ANOVA of time spent sniffing did not reveal
an effect of treatment [F(3,34)=2.153; P=0.114]. However, a priori
comparison revealed that chronic treatment with MRX006 (t=3.602
df=16; P=0.0024) increased the time spent sniffing urine when
compared to the vehicle group.
[0626] Two-way ANOVA analysis [Urine: [F(1,36)=44.118;
P<0.0001]; Treatment: [F(1,36)=12.335; P=0.001]; Urine x
treatment: [F(1,36)=9.236; P=0.005] followed by post hoc tests
revealed that mice treated with MRX006 spent more time sniffing
urine compared with the vehicle group (*p<0.01; FIG. 20B).
Importantly, the vehicle mice spent more time sniffing urine than
water as expected (#p<0.05).
[0627] Conclusions
[0628] Chronic treatment with MRX006 significantly increases the
time spent sniffing female urine in BTBR mice in the female
sniffing urine test.
Example 2j--Assessment of Depression-Like Behaviour--the Novel
Object Recognition Test
[0629] Rationale
[0630] The protocol used was adapted from Bevins and Besheer
(2006), and used to test recognition memory.
[0631] Improved memory is a reflection of reduced depression-like
behaviour.
[0632] Methods
[0633] The protocol used was adapted from Bevins and Besheer
(2006). It was conducted over 3 days. On Day 1, the animals were
allowed to acclimate to the testing environment for 10 minutes,
which was a large container equipped with an overhead camera. No
bedding was used and the container was wiped with 70% ethanol
between each animal. On Day 2, the animals were allowed to
acclimate to the test apparatus for 10 minutes. Following this
period, the animal was removed from the container and two identical
objects were introduced to the environment. The animal was returned
to the container and allowed to explore for a further 10 minutes.
The objects were cleansed before each trial with a 70% ethanol
solution. Following the training period, the rodent was removed
from the environment for a delay period of 24 hours. On Day 3, the
rodent was returned to the container, which this time contained
only 1 familiar object from the day previous and 1 novel object.
Activity of the animal with the 2 objects was recorded for 5
minutes. The amount of time that the rodent spent exploring each
object was recorded by manual observation and a discrimination
index (DI) value corresponding to time spent interacting with the
novel object over total interaction time was generated. A decrease
in DI compared to control rats indicates a deficit in this type of
memory.
[0634] Results
[0635] Student t-test within groups did not reveal a
side-preference for either object A or B on day one of the novel
object recognition test (FIG. 21A). Student t-test within groups
did not reveal a preference for the novel object relative to the
familiar object. For the novel object, ANOVA of interaction time
did not reveal an effect of treatment [F(3,46)=0.122; P=0.946; FIG.
21B]. In addition, no effect of treatment on discrimination index
was revealed by ANOVA analysis [F(3,47)=0.535; P=0.661; FIG.
21C].
[0636] Conclusions
[0637] Chronic treatment with MRX006 has no effect on cognitive
behaviour in BTBR mice in the novel object recognition test.
Example 2k--In Vivo Gastrointestinal Permeability Assay
[0638] Rationale
[0639] This procedure is used to assess in vivo intestinal
motility.
[0640] Methods
[0641] Test mice were singly-housed and fasted overnight. The
following morning (.about.9 am), mice were administered FITC
dextran ((600 mg/kg) by oral gavage. Two hours later, 100 .mu.l of
blood sample was collected in heparin-coated capillary tubes and
transferred to a darkened eppendorf and placed on ice. Samples were
centrifuged 3500.times.g for 15 minutes, plasma was aspirated and
samples were stored at -80.degree. D for long storage.
[0642] Undiluted plasma was used to quantify FITC concentration. 25
.mu.l of FITC was pipetted in duplicated in 384 well plate (Greiner
bio one). FITC was measured with a Victor spectrometer between the
ranges of 490 nm-520 nm. For a standard curve, a serial dilution of
FITC was prepared in PBS (pH7.4). An increase in absorbance is
indicative of a decrease in barrier integrity.
[0643] Results
[0644] Intestinal barrier function was assessed through oral
administration of the fluorescent compound, fluorescein
isothiocyante (FITC), followed by subsequent tail bleeds to assess
levels of FITC in plasma. ANOVA of FITC concentrations did not
reveal a significant effect of treatment [F(3,47)=1.366; P=0.266;
FIG. 23].
[0645] Conclusion
[0646] Chronic treatment with MRX006 did not influence intestinal
permeability in BTBR mice.
Example 2l--Ex Vivo Gastrointestinal Permeability Assay
[0647] Rationale and Methods
[0648] The permeability of the ileum and colon was assessed ex vivo
using Ussing chambers. Colon and ileum were excised from mice and
collected into 5 mL tubes containing Kreb's buffer. Both colon and
ileum were cut along the mesenteric line and mounted onto the
Ussing chamber apparatus. For colon, 4 mLs of Krebs solution
containing D-glucose were added into both sides of the Ussing
chamber apparatus. For ileum, 4 mLs of Krebs solution containing
D-mannitol was added into the muscosal side, while an equal volume
of Krebs with D-glucose was added to the serosal side. The chambers
were oxygenated with carbogen gas (95% O2 and 5% CO2) and kept at
37.degree. C. to maintain tissue integrity. 2.5 mg/mL FITC-dextran
was added to the mucosal chamber. Samples were taken from the
serosal chamber at timepoints 0 min (baseline), 60 min, 90 min and
120 mins. 25 .mu.l of FITC was pipetted in duplicated in 384 well
plate (Greiner bio one). FITC was measured with a Victor
spectrometer between the ranges of 490 nm-520 nm. For a standard
curve, a serial dilution of FITC was prepared in PBS (pH7.4).
[0649] Results
[0650] In the ex vivo intestinal permeability assay, repeated
measures ANOVA revealed an effect of time for both the colon
[F(3,87)=64.197; P<0.0001] and the ileum [F(3,87)=34.572;
P<0.0001]. Repeated measures ANOVA did not reveal an effect of
treatment with respect to time for either the colon [F(9,87)=1.184;
P=0.316; FIG. 23A] or ileum [F(9,87)=0.810; P=0.609; FIG. 22B].
[0651] Conclusions
[0652] Chronic treatment with MRX006 does not influence the
permeability of the colon or ileum.
Example 2m--In Vivo Gastrointestinal Motility Assay
[0653] Rationale
[0654] This procedure is used to assess in vivo intestinal
motility.
[0655] Methods
[0656] Mice are singly housed prior to the commencement of the
test. Mice were orally gavaged with a non-absorbable, coloured dye
(Carmine Red). The time to excretion of the first coloured faecal
bolus was recorded and used as an index of peristaltic motility of
the whole intestine.
[0657] Results
[0658] Mice were administered a non-absorbable, coloured dye
(Carmine Red) by oral gavage. The time to excretion of the first
coloured faecal bolus was recorded and used as an index of
peristaltic motility of the whole intestine. ANOVA of motility time
revealed no effect of treatment [F(3,47)=2.097; P=0.114]. However,
a priori pairwise comparisons test revealed that mice treated with
MRX006 (t=2.270 df=22, p<0.05; FIG. 24) display altered
intestinal motility when compared to the vehicle group.
[0659] Conclusions
[0660] Chronic treatment with MRX006 showed reduced intestinal
motility in BTBR mice.
Example 2n--Stress-Induced Circulating Corticosterone
Determination
[0661] Rationale
[0662] Exposure to the FST results in a robust activation of the
HPA axis, with an increase in the levels of the stress hormone,
corticosterone. Plasma corticosterone concentrations taken prior
to, and after exposure to the FST, were used as an index of
stress-induced activation of the hypothalamic pituitary adrenal
(HPA) axis.
[0663] Methods
[0664] On the day of the FST, mice were removed from their
home-cage and moved to a surgical room where a basal blood sample
was taken. A scalpel blade was used to remove the very tip (1 mm)
of the tail. Blood was the collected using a heparinised capillary
tube and then transferred to a microcentrifuge tube. Blood samples
were also taken 30, 60, 90 and 120 minutes following exposure to
the FST to assess for peak and recovery corticosterone levels.
Blood was kept on ice and then centrifuged at 2,500.times.g for 15
minutes at 4.degree. C. Plasma corticosterone was assessed by
ELISA, following vendor instructions (ENZO Corticosterone ELISA,
ADI-900-097, Enzo Life Sciences).
[0665] Results
[0666] Repeated measures ANOVA revealed a significant effect of
time [F(4,164)=127.127; P<0.0001; FIG. 25]. Post-hoc comparisons
revealed a significant increase in circulating corticosterone at
the 30-minute time point for all groups. Repeated measures ANOVA
did not reveal a significant effect of treatment with respect to
time [F(12,164)=0.561; P=0.871].
[0667] Conclusions
[0668] Chronic treatment with MRX006 does not influence
stress-induced corticosterone levels in BTBR mice exposed to the
forced swimming test.
Example 2o--Organ Weight and Colon Length
[0669] ANOVA of organ weight as a percentage of body weight did not
reveal an effect of treatment for the adrenals [F(3,44)=1.480;
P=0.234; FIG. 26A}, spleen [F(3,43)=0.779; P=0.513; FIG. 26B] or
caecum [F(3,44)=0.441; P=0.725; FIG. 26C]. ANOVA of colon length
did not reveal an effect of treatment [F(3,46)=0.826; P=0.487; FIG.
26D].
[0670] Conclusions
[0671] Treatment with MRX006 does not influence selective
anatomical markers.
Example 2p--Weight Monitoring
[0672] Animal body weights were assessed once per week over the
duration of the experiment to determine whether any of the
bacterial strains were influencing this particular parameter.
Repeated measures ANOVA revealed a significant effect of time
[F(11,484)=111.217; P<0.0001; FIG. 27]. Repeated measures ANOVA
did not reveal an effect of treatment with respect to time
[F(33,484)=0.581; P=0.971].
[0673] Conclusions
[0674] Chronic treatment with MRX006 does not influence body weight
in BTBR mice.
[0675] Conclusions from the BTBR Mouse Model
[0676] The main findings of this study were that treatment with
MRX006 attenuated stereotyped and anxiety-related behaviours.
Specifically, MRX006 reduced the number of marbles buried in the
marble burying test as well as reducing the amount of time that
animals spent grooming. Moreover, treatment with this live
biotherapeutic increased the amount of time spent in the centre of
the open field, corresponding with a decrease in the amount of time
spent in the periphery, which is indicative of an anxiolytic-like
effect. However, no effects on anxiety-like behaviour were observed
in the elevated plus maze test. The ability of MRX006 to improve
stereotyped and anxiety related behaviours in BTBR mice is
promising and indicates that it may be an effective
therapeutic.
[0677] MRX006 also increased the time spent sniffing urine from
female mice. The female urine sniffing test was originally designed
as a test to assess hedonic-like behaviour in rodents, with
increases in the time spent sniffing urine interpreted as an
increase in reward seeking behaviour (Malkesman et al., 2010).
Given that BTBR mice are not reported to display a depressive-like
phenotype, it is unlikely that the observed increase in time spent
sniffing urine in the current experiments following treatment with
MRX006 reflects an improvement in hedonic behaviour. Rather, it may
be that MRX006 is increasing the ability of these mice to recognise
and process social information (i.e. female pheromones). However,
no differences in social behaviour among the groups were observed
in the 3 chamber test and social interaction test. Treatment with
MRX006 reduced the amount of time that mice spent investigating a
novel conspecific mouse relative to a familiar conspecific.
[0678] With the exception of intestinal motility, the live
biotherapeutic assessed in the current study did not affect the
several physiological parameters measured. For instance, no effect
of the live biotherapeutic was observed in stress-induced
corticosterone secretion, anatomical weight, intestinal
permeability or total body weight. In the intestinal motility
assay, treatment with MRX006 prolonged the time taken for the first
red pellet to appear following oral gavage with carmine red dye.
Such results suggest that MRX006 prolongs intestinal motility.
Example 3--the Maternal Immune Activation (MIA) Mouse Model
[0679] 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 (Crawley 2012).
Example 3a--Materials and Methods for MIA Mouse Model
[0680] Mice
[0681] Maternal immune activation (environmental ASD mouse model)
protocol was conducted as previously described (Hsiao, McBride et
al. 2013). Briefly, pregnant C57BL/6N mice (ENVIGO, UK) were
injected i.p. on E12.5 with saline or 20 mg/kg poly(I:C) according
to methods described in (Hsiao, McBride et al. 2013). These mice
are listed in the experiments below as MIA mice. Male mice started
behaviour at 8 weeks old. The animals were housed in a temperature-
and humidity-controlled room on a 12 hr dark cycle (lights on from
7:00-19:00 hr). All experiments were conducted in accordance with
the European Directive 2010/63/EEC, the requirements of S.I. No 543
of 2012, and approved by the Animal Experimentation Ethics
Committee of University College Cork.
[0682] Strain
[0683] MRX006: Blautia stercoris bacterium deposited under
accession number NCIMB 42381.
[0684] Live biotherapeutics were grown in the facility in anaerobic
conditions.
[0685] Live Biotherapeutic Administration
[0686] Dosing with MRX006 or vehicle commenced when the mice were 8
weeks old. These mice were treated once daily with MRX006 or
phosphate buffer solution (PBS) for 3 weeks before the beginning of
the behavioural battery. Mice were further treated once daily for 5
weeks during the behavioural battery. MRX006 (1.times.107 to
1.times.109 CFU oral administration) was dissolved in PBS prior to
administration.
[0687] Administration Schedule
[0688] The treatment groups for the study are shown below. The
vehicle for oral administration is PBS. Daily oral administration
occurs via oral gavage.
TABLE-US-00003 Group Treatment Number 1 Control (PBS, oral gavage)
9 2 Vehicle MIA (PBS, oral gavage) 15 3 MRX006 MIA (oral gavage in
PBS) 13
[0689] Fecal Collection
[0690] Fresh fecal samples were collected from individual mice
every week until the end of the study. At least 20 mg of fresh
faeces were placed in a microcentrifuge tube, place immediately on
ice and then stored at -80.degree. C.
[0691] Experimental Design and Methods
[0692] As outlined above, dosing with MRX006 or vehicle commenced
when the mice were 8 weeks old. The behavioural battery occurred in
the following order: the open field arena at week 4, the marble
burying test at week 5; social transmission of food preference test
at week 6, and the female urine sniffing test at week 7. The
carmine red gastrointestinal motility assay and gastrointestinal
permeability assay tail bleeds occurred during weeks 7 and 8
respectively. Finally, in week 9, the mice were killed for
splenocyte stimulation and ex vivo measurement of FITC in the ileum
and colon.
[0693] The effects of live biotherapeutic treatment in the MIA
model on stereotyped, social and depression-like behaviours, along
with gastrointestinal parameters (permeability and motility) are
outlined in the following examples.
[0694] Group 2, listed in the table above, represents the maternal
immune activation mice, the mothers of which were treated with poly
(I:C) during pregnancy. These mice would be expected to show
phenotypes associated with autistic spectrum disorders compared to
the control mice (Group 1)--this control ensures that the poly
(I:C) administration did cause the expected behavioural symptoms in
the maternal mouse offspring. Any effect of treatment on the
behavioural symptoms of autistic spectrum disorders would be
identified by differences between Group 2 and Group 3.
[0695] Graphical Design and Statistical Analysis
[0696] All graphs were generated on graphpad prism software
(version 5). Data were analysed using IBM SPSS Statistic 22.0
(EEUU). Data distribution was analysed using the Kolmogorov-Smirnov
normality test. Data comparing vehicle group versus the MRX006
group were analysed using one-way ANOVA and Fisher's least
significant difference (LSD) post hoc test. If ANOVA did not reveal
a significant effect of treatment, a priori pairwise comparisons
test against the control group was conducted. Non-normally
distributed data were analysed by the Kruskal-Wallis and
non-parametric Mann-Whitney U test. P<0.05 was the criterion for
statistical significance.
Example 3b--Assessment of Stereotyped Behaviours--the Marble
Burying Test
[0697] Rationale
[0698] This test assesses for repetitive, compulsive and anxious
behaviour. A higher number of marbles buried is indicative of
greater anxious or stereotyped behaviours. Indeed, Mice treated
with pharmacological agents such as anxiolytics show decreased
marble burying behaviour, compared to the control mice.
[0699] Methods
[0700] Mice were individually placed into a novel polypropylene
cage (35.times.28.times.18.5 cm, L.times.W.times.H), containing
standard rodent (hard wood) bedding (5 cm) and 20 marbles on top of
it (five rows of marbles regularly spaced 2 cm away from the walls
and 2 cm apart). Experiments were conducted under a light intensity
of 1000 lux. 30 minutes later, mice were removed from these cages
and the number of marbles buried for more than 2/3rds of their
surface was scored.
[0701] Results
[0702] Student's t-test analysis between the control group and the
vehicle MIA group revealed that the vehicle MIA mice buried more
marbles compared to the control group (t(21)=2.751, P=0.011; FIG.
28). ANOVA of the number of marbles buried revealed a significant
effect of treatment [F(3,48)=18.39; P<0.001]. Post-hoc
comparisons revealed that chronic treatment with MRX006 decreased
the number of marbles buried (p<0.01; FIG. 28).
[0703] Conclusions
[0704] The vehicle MIA group showed significantly more marbled
buried than the control group, indicating that the MIA model
successfully triggered autistic spectrum disorder-like symptoms in
the mice. Chronic treatment with MRX006 reduces repetitive,
compulsive and anxious behaviour in MIA mice.
Example 3c--Assessment of Social Behaviours--Social Transmission of
Food Preference
[0705] Rationale
[0706] Social transmission food preference is a relevant test of
olfactory memory that is used in mice to assess social behaviour.
In this test, observer mice interact with a demonstrator mouse that
has recently eaten novel food. When observer mice are presented
with a choice between the food eaten by the demonstrator and some
other novel food, observer mice should prefer the food eaten by the
demonstrator. Reduced food preference would indicate reduced
sociability.
[0707] Methods
[0708] This test was performed as previously described (Desbonnet,
Clarke et al. 2015). Briefly, 18 hours prior to testing, mice were
deprived of food, whereas water was available ad libitum. Food
choices consisted of either 1% ground cinnamon or 2% powdered cocoa
made with grounded mouse chow. A demonstrator mouse was randomly
selected from each cage and the tail was marked using a blue marker
to enable identification during subsequent social interactions.
Demonstrator food containers were weighed before and after the 1
hour sampling sessions. A minimum of 0.2 g of consumed food was
required for inclusion in the test. Demonstrator mice were placed
back into their respective home cages for a 30 minute interaction
period with cage-mates. Subsequently, cage-mates were individually
tested for preference of cued food or novel food. Containers were
weighed immediately before and after each choice session. Observed
mice were then placed back into their respective home cages and the
choice session was repeated 24 hours later. The test mice should
smell the cinnamon or cocoa off the demonstrator mouse as a social
cue, and preferentially choose the same food when given a choice
between the two.
[0709] Results
[0710] Student's t-test of percentage food preference revealed no
difference between control and vehicle MIA groups at either the 0
hour (t(22)=0.3325, P=0.7427) or 24 hour (t(21)=0.2878, P=0.7763)
assessment. ANOVA of demonstrator cued food preference revealed no
significant difference when observers were exposed to food choice
at the 0 hour [F(3,48)=1.49, P=0.228; FIG. 29A] or 24 hour
assessment [F(3,47)=2.66, P=0.059; FIG. 29B]. Treatment with MRX006
did not alter preference for cued food in the social transmission
of food preference test.
[0711] Conclusions
[0712] The vehicle MIA group did not display reduced social
transmission food preference (the MIA vehicle displayed no
alteration in food preference compared to the control), suggesting
the MIA model has not triggered the reduced sociability phenotype.
Chronic treatment with MRX006 had not effect on food preference.
However, as the MIA model appears not to have caused a reduced
sociability phenotype in this test, it is not possible to determine
the effects of chronic treatment with MRX006 on sociability in the
social transmission of food preference test.
Example 3d--Assessment of Anxiety-Like Behaviour--the Open Field
Arena
[0713] Rationale
[0714] The open field arena is used to assess the response of
exposure to a novel stressful environment and locomotor activity.
Naive 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 the centre represents a
decrease in anxiety-like behaviour.
[0715] Methods
[0716] Mice were individually placed into an open field arena
(43.times.35.times.25, L.times.w.times.h) and allowed to explore
for 10 minutes. Experiments occurred under a light intensity of 60
lux. Experiments were videotaped using a ceiling camera to allow
for measuring several behavioural parameters using Ethovision
software. The distance travelled was scored for locomotor
activity.
[0717] Results
[0718] Student's t-test revealed no significant difference in the
total distance moved between control and vehicle MIA groups
(t(22)=0.9357, P=0.3596). ANOVA of total distance moved revealed a
significant effect of treatment [F(3,47)=4.36, P=0.003, FIG. 30A].
Post hoc comparisons revealed that treatment with MRX006 reduced
total distance travelled relative to vehicle treated animals
(p<0.05). Student's t-test revealed a significant increase in
the time spent in the outer zone of the open field by the vehicle
MIA group relative to the control group (t(21)=3.337, P=0.003).
ANOVA of time spent in the outer zone of the open field revealed no
effect of treatment [F(3,47)=0.093, FIG. 30B]. Student's t-test
revealed a significant decrease in the time spent in the inner zone
by the vehicle MIA groups relative to the control mice
(t(21)=3.337, P=0.003). ANOVA of time spent in the inner zone
revealed no effect of treatment [F(3,47)=0.93, P=0.96, FIG.
30C].
[0719] Conclusions
[0720] Treatment with MRX006 decreases the distance travelled by
MIA mice in the open field arena. Therefore, MRX006 may be
attenuating stress-induced locomotor activity caused by exposure to
the open field arena.
Example 3e--Assessment of Depression-Like Behaviour--the Female
Urine Sniffing Test
[0721] Rationale
[0722] The female urine sniffing test (FUST) is used to assess
anhedonic-like behaviour in rodents. A reduction in sniffing time
suggests social avoidance/anhedonia while an increase represents an
increase in social behaviour/hedonic behaviour.
[0723] Methods
[0724] Experimental mice are singly housed one week prior to the
test. During the test, a cotton tip applicator, dipped in sterile
water, is placed into the home cage and mice are allowed to
sniff/investigate for three minutes. Following this three minute
test, the cotton tip applicator is removed. 45 minutes later, a new
cotton tip applicator is dipped into female urine (collected from
female mice of the same strain that are in the estrous stage of
their cycle), and placed into the cage. Mice are allowed to
sniff/investigate this for a further three minutes. The amount of
time spent sniffing the water and urine is recorded.
[0725] Results
[0726] Mann-Whitney U-test revealed that both control [Mann-Whitney
U value=7, P=0.0123] and vehicle MIA [Mann Whitney U value=57;
P=0.0201] groups spent more time sniffing urine than water (FIG.
31). For time spent sniffing urine, Kruskal-Wallis non-parametric
analysis did not reveal an effect of treatment [df=4,
P=0.3293].
[0727] Conclusions
[0728] Chronic treatment with MRX006 does not influence
depressive-like behaviour in MIA mice in the female sniffing urine
test.
Example 3f--In Vivo Intestinal Motility Assay
[0729] Rationale
[0730] 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.
[0731] Methods
[0732] Mice are singly housed prior to the commencement of the
test. Mice were orally gavaged with a non-absorbable, coloured dye
(Carmine Red). The time to excretion of the first coloured faecal
bolus was recorded and used as an index of peristaltic motility of
the whole intestine.
[0733] Results
[0734] Student's t-test analysis revealed that vehicle MIA group do
not exhibit altered intestinal motility (red pellet detected in
less time) when compared to the control group (t(22)=0.006,
P=0.9950). ANOVA of motility time revealed no effect of treatment
[F(3,50)=0.99; P=0.404, FIG. 32].
[0735] Conclusions
[0736] In this experiment, the vehicle MIA group did not exhibit
altered intestinal motility compared to the control. Chronic
treatment with MRX006 did not affect intestinal motility compared
to the control or vehicle MIA groups.
Example 3g--Organ Weight and Colon Length
[0737] For colon length, student's t-test did not reveal any
significant difference between vehicle MIA and control groups
(t(21)=1.26, P=0.26). ANOVA of colon of length did not reveal an
effect of treatment [F(3,49)=0.69, P=0.57; FIG. 33A]. For caecum
weight as a percentage of body weight, student's t-test did not
reveal a significant difference between vehicle MIA and control
groups (t(22)=0.56, P=0.58). ANOVA revealed no significant effect
of treatment upon caecum weight [F(3,48)=0.84, P=0.48, FIG. 33B].
For spleen weight as a percentage of body weight, student's t-test
did not reveal a significant difference between vehicle MIA and
control groups (t(22)=0.64, P=0.53). ANOVA revealed no significant
effect of treatment upon spleen weight [F(3,48)=2.25, P=0.09, FIG.
33C).
[0738] Conclusions
[0739] Treatment with MRX006 does not influence colon length or
organ weight in the MIA mouse model of autism.
[0740] Discussion of Results from the MIA Mouse Model
[0741] Chronic treatment with MRX006 was able to reverse the
phenotype observed in the marble burying test in MIA mice. Chronic
treatment with MRX006 was able to reduce the number of marbles
buried suggesting a reduction in stereotyped-like behaviour.
Furthermore, chronic treatment with MRX006 decreased the distance
travelled without having any effect on time spent in the inner and
outer zones in the open field arena. Consequently, treatment with
MRX006 may attenuate stress-induced locomotor activity caused by
exposure to the open field arena. No significant effect of
treatment was observed in the social transmission food and the
female urine sniffing tests suggesting no directly observable
effects in social and depressive-like behaviour in the MIA mouse
model. The live biotherapeutic tested did not affect intestinal
motility or permeability. Therefore, the MIA model has proven
useful for assessing stereotyped-like, repetitive and anxious
behaviour, but it did not recreate a number of other symptoms
associated with autistic spectrum disorders. Nevertheless, the
results display that chronic treatment with MRX006 may have a
positive impact on the symptoms of autistic spectrum disorders.
[0742] Overall Conclusions Regarding MRX006 in the Treatment of
Autistic Spectrum Disorders
[0743] MRX006 was shown to be effective in the treatment of
stereotyped and anxiety-like behaviours in both the BTBR and MIA
mouse model. Therapies that reverse behavioural and biological
phenotypes in mouse models of autism are expected to be effective
against human disease.
[0744] 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 MRX006 live biotherapeutic has shown
effective treatment of at least one core symptom of autistic
spectrum disorder, so it and related Blautia and B. stercoris
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.
Example 4--Measurement of Circulating Cytokines in BTBR Mice
[0745] Methods and Results
[0746] Blood plasma was collected from trunk blood on the day of
the culls from each animal at the end of the experiments.
Circulating cytokines were assessed in plasma samples from vehicle
and MRX006 groups using a commercially available
electrochemiluminescence multiplex system (MSD, Gaithersburg, MSD,
USA). The following cytokines were assayed for: IL-1.beta., IL-4,
IL-6, IL-10, IL-17A, IL-21, IL-23, TNF-.alpha. and IFN-.gamma..
Multiplex analysis revealed that levels of IL-1.beta., IL-4,
IL-17A, IL-21 and IL-23 were below the limits of detection in both
vehicle and MRX006 treated animals. For circulating TNF-.alpha.,
student's t-test did not reveal a significant effect of treatment
with MRX006 (t(21)=0.4264, P=0.6742, FIG. 34A). For circulating
IFN-, student's test did not reveal a significant effect of
treatment with Mrx006 (t(17)=0.4103, P=0.6867, FIG. 34B). For
circulating IL-6, Student's t-test did not reveal a significant
effect of treatment with MRX006 (t(11)=0.020, P=0.98, FIG. 34C).
For circulating IL-10, chronic treatment with MRX006 causes a
non-significant increase in IL-10 levels (t(13)=1.396, P=0.1861,
FIG. 34D).
[0747] Conclusions
[0748] While there was no significant effect of MRX006 in terms of
regulating circulating cytokine concentrations, there was a clear
non-significant trend for an increase in circulating IL-10
following treatment with the live biotherapeutic. Such results
suggest that MRX006 possesses immune-regulatory properties and can
increase the production of anti-inflammatory cytokines. While the
multiplex assay was performed upon plasma samples that contained
basal, unstimulated cytokine concentrations, it will be interesting
to assess whether MRX006 is capable of modulating IL-10 and other
cytokines under stimulated conditions.
Example 5--Assessing the Effects of Subchronic Treatment with
MRX006 Upon Central and Peripheral Oxytocin Levels in C57BL/6
Mice
[0749] The bacterial strains were prepared and administered as
outlined in the Examples above. The C57BL/6 mice were treated with
live biotherapeutic for six days in 7 experimental treatment groups
each with 10-12 mice. Subsequently, the hypothalamus was dissected
from the mice and the levels of oxytocin in the hypothalamus were
detected by radioimmunoassay (RIA), In addition, levels of oxytocin
in the plasma were detected by RIA. Furthermore, the levels of
oxytocin receptors, interleukins and other inflammatory markers,
and vasopressin hormones were detected by RIA and other analytical
methods.
Example 6--Stability Testing
[0750] A composition described herein containing at least one
bacterial strain described herein is stored in a sealed container
at 25.degree. C. or 4.degree. C. and the container is placed in an
atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95%
relative humidity. After 1 month, 2 months, 3 months, 6 months, 1
year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%,
70%, 80% or 90% of the bacterial strain shall remain as measured in
colony forming units determined by standard protocols.
Example 7--Administration of Another Live Biotherapeutic in the MIA
Mouse Model
Example 7a--Materials and Methods for MIA Mouse Model
[0751] The mice, live biotherapeutic administration and fecal
collection used in this Example are identical to those used in
Example 3 above.
[0752] Strain
[0753] MRX008: Blautia wexlerae, bacteria deposited under accession
number NCIMB 42486.
[0754] Administration Schedule
[0755] The treatment groups for the study are shown below. The
vehicle for oral administration is PBS. Daily oral administration
occurs via oral gavage.
TABLE-US-00004 Group Treatment Number 1 Control (PBS, oral gavage)
11 2 Vehicle MIA (PBS, oral gavage) 10 3 MRX008 MIA (oral gavage in
PBS) 11
[0756] Experimental Design and Methods
[0757] Dosing with MRX008 or vehicle commenced when the mice were 8
weeks old. The behavioural battery occurred in the following order:
marble burying test at week 5; social transmission of food
preference at week 6 and the forced swimming test at week 8. The
carmine red gastrointestinal motility assay and gastrointestinal
permeability assay tail bleeds occurred during weeks 7 and 8
respectively. Finally, in week 9, the mice were killed for
splenocyte stimulation and ex vivo measurement of FITC in the ileum
and colon.
[0758] The effects of live biotherapeutic treatment in the MIA
model on stereotyped, social and depression-like behaviours, along
with gastrointestinal parameters (permeability and motility) are
outlined in the following examples.
[0759] Group 2, listed in the table above, represents the maternal
immune activation mice, the mothers of which were treated with poly
(I:C) during pregnancy. These mice would be expected to show
phenotypes associated with autistic spectrum disorders compared to
the control mice (Group 1)--this control ensures that the poly
(I:C) administration did cause the expected behavioural symptoms in
the maternal mouse offspring. Any effect of treatment on the
behavioural symptoms of autistic spectrum disorders would be
identified by differences between Group 2 and Group 3.
[0760] Graphical Design and Statistical Analysis
[0761] All graphs were generated on graphpad prism software
(version 5). Data were analysed using IBM SPSS Statistic 22.0
(EEUU). Data distribution was analysed using the Kolmogorov-Smirnov
normality test. Data comparing vehicle group versus the MRX008
group were analysed using one-way ANOVA and Fisher's least
significant difference (LSD) post hoc test. If ANOVA did not reveal
a significant effect of treatment, a priori pairwise comparisons
test against the control group was conducted. Non-normally
distributed data were analysed by the Kruskal-Wallis and
non-parametric Mann-Whitney U test. P<0.05 was the criterion for
statistical significance.
Example 7b--Assessment of Stereotyped Behaviours--the Marble
Burying Test
[0762] Rationale and Methods
[0763] See Example 3b above.
[0764] Results
[0765] Student's t-test analysis between the control group and the
vehicle MIA group revealed that the vehicle MIA mice buried more
marbles compared to the control group (t(19)=3.00, P=0.007; FIG.
35). ANOVA of the number of marbles buried revealed an effect of
treatment [F(3,42)=6.37, P=0.001]. Chronic treatment with MRX008
showed a reduction in the number of marbles buried relative to the
vehicle MIA group.
[0766] Conclusions
[0767] The vehicle MIA group showed significantly more marbles
buried than the control group, indicating that the MIA model
successfully triggered autistic spectrum disorder-like symptoms in
the mice. There is a trend towards a reduction in repetitive,
compulsive and anxious behaviour in MIA mice upon chronic treatment
with MRX008.
Example 7c--Assessment of Social Behaviours--Social Transmission of
Food Preference
[0768] Rationale and Methods
[0769] See Example 3c above.
[0770] Results
[0771] ANOVA of demonstrator cued food preference revealed no
significant difference when observers were exposed to food choice
immediately after demonstrator interaction (T0) (F(3,34)=0.38,
P=0.77; FIG. 36A) or 24 hrs later (F(3,34)=0.85, P=0.48; FIG. 36B),
irrespective of vehicle or MRX008 administration.
[0772] Conclusions
[0773] The vehicle MIA group did not display reduced social
transmission food preference (the MIA vehicle displayed no
alteration in food preference compared to the control), suggesting
the MIA model has not triggered the reduced sociability phenotype.
Accordingly, it is not possible to determine the effects of chronic
treatment with MRX008 on sociability using the MIA mouse model.
Example 7d--Assessment of Depression-Like Behaviours--the Forced
Swimming Test
[0774] Rationale
[0775] The forced swim test (FST) is the most widely used
experimental paradigm to assess antidepressant activity ([57]). In
this test, mice are forced to swim for 6 min and the behavioural
parameter scored is immobility during the last 4 min of the 6-min
test. Naive animals will display escape behaviour in the form of
swimming, climbing and diving before adapting an immobile floating
posture. The duration of immobility is indicative of behavioural
despair. Antidepressant drugs decrease the time spent immobile in
this test.
[0776] Methods
[0777] Mice are forced to swim for 6 min in a glass cylinder
(24.times.21 cm) filled with 23-25.degree. C. tap water to a depth
of 17 cm. The FST was videotaped from a ceiling camera. The
behavioural parameter scored is immobility during the last 4 min of
the 6-min test.
[0778] Results
[0779] Student's t test analysis revealed no significant
differences on immobility time between the control group and
vehicle MIA group (t=0.8968 df=20; 0.3805). ANOVA of immobility
time did not reveal an effect of treatment with MRX008, although
there appears to be a slight trend towards a reduction in time
spent immobile after administration of MRX008 [F(3,42)=1.803;
P=0.1625; FIG. 37].
[0780] Conclusions
[0781] The vehicle MIA group did not display increased immobility
time in the forced swim test (the MIA vehicle displayed no
alteration in immobility time compared to the control), suggesting
the MIA model has not increased depressive-like symptoms.
Accordingly, it is not possible to determine the effects of chronic
treatment with MRX008 on depressive-like behaviour using the MIA
mouse model.
Example 7e--In Vivo Intestinal Permeability Assay
[0782] Rationale
[0783] 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 affects intestinal
permeability.
[0784] Methods
[0785] Test mice were single caged and food was removed overnight.
Next day (at around 9 am) mice were administered by oral gavage
with FITC dextran (Fluroscein-isothiocynate; MW: 4 kDa, Sigma;
concentration: 600 mg/kg per animal of 80 mg/ml FITC in PBS
(pH7.4)). Two hours following FITC administration, 100 .mu.l of
blood sample, from tail bleeds, was collected in heparin-coated
capillary tubes and transferred to amber eppendorf and placed on
ice. Samples were centrifuged 3500.times.g for 15 minutes, plasma
was aspirated and samples were stored at -80.degree. D for long
storage.
[0786] Undiluted plasma was used to quantify FITC concentration. 25
.mu.l of FITC was pipetted in duplicated in 384 well plate (Greiner
bio one). FITC was measured with a Victor spectrometer between the
ranges of 490 nm-520 nm. For a standard curve, a serial dilution of
FITC was prepared in PBS (pH7.4).
[0787] In addition, after the cull of the mice in week 9, ex vivo
measurements of FITC in the ileum and colon are performed.
[0788] Results
[0789] Student's t test analysis revealed no differences between
the control group and the MIA vehicle group (t(20)=0.56, P=0.58;
FIG. 38). ANOVA of FITC concentrations did not reveal a significant
effect of treatment [F(3,39)=2.23, P=0.08].
[0790] Conclusions
[0791] In this experiment, the vehicle MIA group did not display
altered intestinal permeability (the MIA vehicle displayed no
alteration in permeability compared to the control). Furthermore,
chronic treatment with MRX008 did not affect intestinal
permeability in MIA mice.
Example 7f--In Vivo Intestinal Motility Assay
[0792] Rationale and Methods
[0793] See Example 3f above.
[0794] Results
[0795] Student's t test analysis revealed that the vehicle MIA
group exhibited increased intestinal motility (red pellet detected
in less time) when compared to the control group (t(19)=3.00,
P=0.007). ANOVA of motility time revealed no effect of treatment
[F(3,38)=0.74, P=0.54; FIG. 39].
[0796] Conclusions
[0797] In this experiment, the vehicle MIA group displayed
increased intestinal motility compared to the control. Chronic
treatment with MRX008 did not affect intestinal motility compared
to the control.
Example 8--Administration of Another Live Biotherapeutic in the
BTBR Mouse Model
Example 8a--Materials and Methods for BTBR Mouse Model
[0798] The mice, live biotherapeutic administration and fecal
collection used in this Example are identical to those used in
Example 2 above.
[0799] Strain
[0800] MRX008: Blautia wexlerae, bacteria deposited under accession
number NCIMB 42486.
[0801] Administration Schedule
[0802] The treatment groups for the study are shown below. The
vehicle for oral administration is PBS. Daily oral administration
occurs via oral gavage.
TABLE-US-00005 Group Treatment Number 1 Control (PBS, oral gavage)
10 2 MRX008 (oral gavage in PBS) 10
[0803] Experimental Design and Methods
[0804] As outlined above, dosing with MRX008 commenced when the
mice were 8 weeks old. The initial dosing took place for 3 weeks
before the behavioural experiments encompassing tests of
sociability, anxiety, stereopathy and cognition. The behavioural
battery occurred in the following order: marble burying test at
week 4; the elevated plus maze at week 5; the open field and novel
object recognition tests, and the social transmission of food
preference tests at week 6; the female urine sniffing and social
interaction tests at week 7, and the forced swimming test at week
9. The carmine red gastrointestinal motility assay and
gastrointestinal permeability assay tail bleeds occurred during
weeks 8 and 9 respectively. Finally, in weeks 10 to 11, the mice
were killed for splenocyte stimulation and ex vivo measurement of
FITC in the ileum and colon.
[0805] The effects of live biotherapeutic treatment in the BTBR
model on stereotyped, social and depression-like behaviours, along
with gastrointestinal parameters (permeability and motility) are
outlined in the following examples.
[0806] Group 1, listed in the table above, represents the control
BTBR mice, which would be expected to show phenotypes associated
with autistic spectrum disorders. Any effect of treatment on the
behavioural symptoms of autistic spectrum disorders would be
identified by differences between Group 1 and Group 2.
[0807] Graphical Design and Statistical Analysis
[0808] All graphs were generated on graphpad prism software
(version 5). Data were analysed using IBM SPSS Statistic 22.0
(EEUU). Data distribution was analysed using the Kolmogorov-Smirnov
normality test. Data comparing vehicle group versus the MRX008
group were analysed using one-way ANOVA and Fisher's least
significant difference (LSD) post hoc test. If ANOVA did not reveal
a significant effect of treatment, a priori pairwise comparisons
test against the control group was conducted. Non-normally
distributed data were analysed by the Kruskal-Wallis and
non-parametric Mann-Whitney U test. P<0.05 was the criterion for
statistical significance.
Example 8b--Assessment of Social Behaviours--Social Transmission of
Food Preference
[0809] Rationale
[0810] Social transmission food preference is a relevant test of
olfactory memory that is used in mice to assess social behaviour.
In this test, observer mice interact with a demonstrator mouse that
has recently eaten novel food. When observer mice are presented
with a choice between the food eaten by the demonstrator and some
other novel food, observer mice should prefer the food eaten by the
demonstrator. Reduced food preference would indicate reduced
sociability.
[0811] Methods
[0812] This test was performed as previously described (Desbonnet,
Clarke et al. 2015). Briefly, 18 hours prior to testing, mice were
deprived of food, whereas water was available ad libitum. Food
choices consisted of either 1% ground cinnamon or 2% powdered cocoa
made with grounded mouse chow. A demonstrator mouse was randomly
selected from each cage and the tail was marked using a blue marker
to enable identification during subsequent social interactions.
Demonstrator food containers were weighed before and after the 1
hour sampling sessions. A minimum of 0.2 g of consumed food was
required for inclusion in the test. Demonstrator mice were placed
back into their respective home cages for a 30 minute interaction
period with cage-mates. Subsequently, cage-mates were individually
tested for preference of cued food or novel food. Containers were
weighed immediately before and after each choice session. Observed
mice were then placed back into their respective home cages and the
choice session was repeated 24 hours later. The test mice should
smell the cinnamon or cocoa off the demonstrator mouse as a social
cue, and preferentially choose the same food when given a choice
between the two.
[0813] Results
[0814] ANOVA of demonstrator cued food preference revealed no
significant difference when observers were exposed to food choice
immediately after demonstrator interaction (T0) (F(3,36)=1.123;
P=0.354; FIG. 40A) or 24 hrs later (F(3,38)=0.138; P=0.936; FIG.
40B).
[0815] Conclusions
[0816] Treatment with MRX008 did not affect the sociability of BTBR
mice in the social transmission food preference test.
Example 8c--Assessment of Social Behaviours--Forced Intruder
Test
[0817] Rationale and Methods
[0818] See Example 2c above.
[0819] Results
[0820] ANOVA of interaction time did not reveal an effect of
treatment [F(3,36)=1.905; P=0.1462; FIG. 41].
[0821] Conclusions
[0822] Treatment with MRX008 did not influence social behaviour of
BTBR mice in the social interaction test.
Example 8d--Assessment of Stereotyped Behaviours--the Marble
Burying Test
[0823] Rationale and Methods
[0824] See Example 2d above.
[0825] Results
[0826] ANOVA of the number of marbles buried did not reveal a
significant effect of treatment [F(3,39)=0.835; P=0.483; FIG. 42],
although, chronic treatment with MRX008 does display a trend
towards a reduction in number of marbles buried by BTBR mice.
[0827] Conclusions
[0828] Chronic treatment with MRX008 did not significantly affect
repetitive, compulsive and anxious behaviour in BTBR mice, although
it does display a trend towards reduced levels of this
behaviour.
Example 8e--Assessment of Anxiety-Like Behaviour--the Elevated Plus
Maze
[0829] Rationale and Methods
[0830] See Example 2f above.
[0831] Results
[0832] ANOVA of percentage time spent in closed arms revealed no
effect of treatment [F(3,39)=0.556; P=0.647; FIG. 43A]. Mice
treated with MRX008 appear to spent more time in the open arms
compared to the vehicle group (FIG. 43B). In line with this,
chronic treatment with MRX008 appears to increased the number of
entries into open arms compared to the vehicle group (FIG. 43D).
ANOVA of the number of entries into the closed arms revealed no
effect of treatment [F(3,39)=0.556; P=0.647; FIG. 43C].
[0833] Conclusions
[0834] Chronic treatment with MRX008 shows a non-significant trends
towards anti-anxiety behaviour in BTBR mice in the elevated plus
maze.
Example 8f--Assessment of Anxiety-Like Behaviour--the Open Field
Arena
[0835] Rationale and Methods
[0836] See Example 2g above.
[0837] Results
[0838] ANOVA of distance moved did not reveal a significant effect
of treatment upon locomotor activity in the open field arena
[F(3,37)=1.325; P=0.282, FIG. 44A], although MRX008 appeared to
reduce the distance moved, suggesting a reduction in stress-induced
locomotor activity. ANOVA of time spent in the outer zone did not
reveal an effect of treatment [F(3,37)=1.598; P=0.208; FIG. 44B]. A
priori pairwise comparison revealed that treatment with Mrx008
decreased the time spent in the inner zone [t=2.388 df=17;
P=0.0288; FIG. 44C].
[0839] Conclusions
[0840] Chronic treatment with MRX008 shows a trend towards a
reduction in stress-induced locomotor activity, but did show a
reduction of time in the inner zone implicating anxiety-like
behaviour.
Example 8g--Assessment of Depression-Like Behaviour--the Forced
Swimming Test
[0841] Rationale and Methods
[0842] See Example 2h above.
[0843] Results
[0844] ANOVA of immobility time did not reveal an effect of
treatment on immobility time of BTBR mice in the FST
[F(3,38)=1.879; P=0.151; FIG. 45], although chronic treatment with
MRX008 does cause a trend towards a reduction in the time spent
immobile suggesting anti-depressant behaviour.
[0845] Conclusions
[0846] Treatment with MRX008 non-significantly reduces immobility
time of BTBR mice in the forced swimming test implicating an
anti-depressant effect of treatment.
Example 8h--Assessment of Depression-Like Behaviour--the Female
Urine Sniffing Test
[0847] Rationale and Methods
[0848] See Example 2i above.
[0849] Results
[0850] For the vehicle group, Mann-Whitney U test revealed a
significant increase in the time spent sniffing urine relative to
the time spent sniffing water [t=2.976 df=18; P=0.0081]. For
exposure to water, Kruskal Wallis non-parametric analysis of time
spent sniffing did not reveal an effect of treatment in the water
group [Chi squared: 6.352; df=3; P=0.096]. For exposure to urine,
Kruskal Wallis non-parametric analysis of time spent sniffing did
not reveal an effect of treatment [Chi squared: 3.639; df=3;
P=0.303, FIG. 46].
[0851] Conclusions
[0852] Treatment with MRX008 had no effect upon the time spent
sniffing urine in BTBR mice.
Example 8i--In Vivo Gastrointestinal Motility Assay
[0853] Rationale and Methods
[0854] See Example 2m above.
[0855] Results
[0856] ANOVA of motility time revealed no effect of treatment
[F(3,39)=2.072; P=0.121; FIG. 47].
[0857] Conclusions
[0858] Treatment with MRX008 did not influence intestinal
motility.
Example 8j--Organ Weight and Colon Length
[0859] ANOVA of organ weight as a percentage of body weight did not
reveal an effect of treatment for the adrenals [F(3,37)=0.208;
P=0.890; FIG. 48A], spleen F(3,35)=0.629; P=0.601; FIG. 48B] or
caecum [F(3,37)=0.883; P=0.460; FIG. 48C]. ANOVA of colon length
did not reveal an effect of treatment [F(3,37)=5.635; P=0.003; FIG.
48D].
[0860] Overall Conclusions Regarding MRX008 in the Treatment of
Autistic Spectrum Disorders
[0861] The experiments disclosed herein display evidence that
administration of another Blautia species (namely Blautia wexlerae
MRX008) may be applicable for the treatment of neurodevelopmental
and neuropsychiatric disorders in mice models. In particular,
treatment with MRX008 displayed trends towards potential
anti-anxiolytic effects as well as anti-depressive effects in the
elevated plus maze and forced swim tests, respectively, in the BTBR
mouse model, although the open field arena assay suggested MRX008
did not affect anxiety-like behaviour. In addition, the MRX008 may
reduce stereotyped, repetitive and anxious behaviour as shown by
the marble burying test in both the MIA and BTBR mouse models.
Treatment with the MRX008 biotherapeutic did not alter the several
physiological parameters measured in these studies.
[0862] 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 MRX008 live biotherapeutic has shown
effective treatment of at least one core symptom of autistic
spectrum disorder, so it and related Blautia and B. wexlerae
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.
Example 9--Assessing the Effects of Subchronic Treatment with
MRX008 Upon Central and Peripheral Oxytocin Levels in C57BL/6
Mice
[0863] The bacterial strains were prepared and administered as
outlined in the Examples above. The C57BL/6 mice were treated with
live biotherapeutic for six days in 7 experimental treatment groups
each with 10-12 mice. Subsequently, the hypothalamus was dissected
from the mice and the levels of oxytocin in the hypothalamus were
detected by radioimmunoassay (RIA), In addition, levels of oxytocin
in the plasma were detected by RIA. Furthermore, the levels of
oxytocin receptors, interleukins and other inflammatory markers,
and vasopressin hormones were detected by RIA and other analytical
methods.
Example 10--Assessing the Effects of Chronic Treatment with MRX006
on Gene Expression Levels of Oxytocin, Vasopressin and their
Respective Receptors in the Hypothalamus and the Amygdala of BTBR
Mice
[0864] Chronic treatment with MRX006 increases the level of gene
expression of oxytocin and vasopressin in the hypothalamus of BTBR
mice (see FIGS. 49C and D). The effect on levels of oxytocin and
vasopressin receptors in this tissue are shown in FIGS. 49A and
B.
[0865] The effects of chronic treatment with MRX006 on the level of
gene expression of oxytocin, vasopressin or their respective
receptors in the amygdala of BTBR mice is shown in FIG. 50.
[0866] Therefore, chronic treatment with MRX006 increases the
expression of vasopressin and oxytocin in the hypothalamus of BTBR
mice. This striking result provides a correlation between chemical
changes in the brain and behavioural changes upon administration of
a live biotherapeutic. This is the first time any study has
reported that a live biotherapeutic is capable of altering the
central oxytocin/vasopressin system, with a concurrent change in
social, anxiety-like and stereotyped behaviour with an improvement
in gastrointestinal function.
Example 11--Administration of Blautia hydrogenotrophica in the
C57BL/6 and BTBR Mouse Models
[0867] In behavioural experiments using BTBR mice as a model for
autism spectrum disorder and other neurological disorders, C57BL/6
mice administered both PBS and LYO were used as controls to confirm
that the BTBR mice model effectively demonstrated increased
anxiety, reduced social aversion, and increased stereotypes
behaviours. This allowed an assessment of the effect of bacterial
treatment on these ASD related behavioural defects.
Example 11a--Assessment of Anxiety-Like Behaviour--the Open Field
Arena
[0868] Rationale and Methods
[0869] See Example 2g above. The horizontal activity is the
distance travelled by the mouse in the open field arena. The
vertical activity is the number of occasions on which the mouse
reared onto the hind legs. A higher frequency of these behaviours
indicates increased locomotion and exploration and/or a lower level
of anxiety. An increase frequency of these behaviours in the
central area of the arena indicates high exploratory behaviour and
low anxiety levels.
[0870] PBS is the negative control for the butyrate administration
as the butyrate was administered in PBS. LYO is the negative
control for the administration of the Blautia hydrogenotrophica.
After the first analyses (FIGS. 51 A, C and E), the values for the
negative controls of PBS and LYO in both the C57BL/6 and BTBR
models are combined and averaged to provide a simplified comparison
in the second analysis (FIGS. 51 B, D and F).
[0871] Results
[0872] Horizontal Activity
[0873] As would be expected from an anxiety and/or autism-related
model, BTBR mice display significantly reduced horizontal activity
compared to C57BL/6 mice. The LYO negative control showed no effect
on the horizontal activity in C57BL/6 mice compared to the PBS
control. Compared to the PBS control within the first 30 minutes,
BTBR mice treated with the LYO control or butyrate alone showed no
significant difference in distance travelled, although in the
second 30 minutes, the LYO control reduced the distance travelled
by BTBR mice. However, mice treated with the bacterial strain
showed a significant increase in distance travelled compared to the
BTBR control mice (FIG. 51A).
[0874] To provide a further comparison between the controls and the
experimental values, as outlined above, the values for the PBS and
LYO controls were combined in the second analysis. Similarly to the
first analysis, the administration of butyrate did not affect the
horizontal activity. However, the administration of the bacterial
strain significantly increased the horizontal activity compared to
the BTBR model control (FIG. 51B).
[0875] Vertical Activity
[0876] BTBR mice display significantly reduced vertical activity
(rearing) compared to C57BL/6 mice. The LYO negative control showed
no effect on the vertical activity in C57BL/6 mice compared to the
PBS control. Compared to the BTBR PBS control mice, BTBR mice
treated with butyrate alone showed no difference in rearing, while
the LYO control reduced the vertical activity of BTBR mice.
However, mice treated with the bacterial strain showed a
significant increase in vertical activity compared to the BTBR LYO
control mice (FIG. 51C).
[0877] To provide a further comparison between the controls and the
experimental values, as outlined above, the values for the PBS and
LYO controls were combined in the second analysis. Similarly to the
first analysis, the administration of butyrate did not affect the
vertical activity of BTBR mice. However, the administration of the
bacterial strain significantly increased the vertical activity
compared to the BTBR control (FIG. 51D).
[0878] % distance travelled in the centre of the open field in the
first 5 minutes
[0879] As would be expected, in the first five minutes of the
analysis BTBR mice showed an increased percentage of their distance
travelled in the centre of the open field arena compared to C57BL/6
mice.
[0880] This is reflective of the reduced overall distance travelled
by the BTBR mice, which display increased anxious behaviour, and
the fact that within the first 5 minutes of the assay, the more
anxious BTBR mice are more likely to familiarise themselves with
their initial environment rather than enter an exploratory phase
(FIG. 51E).
[0881] To provide a further comparison between the controls and the
experimental values, as outlined above, the values for the PBS and
LYO controls were combined in the second analysis (FIG. 51F).
[0882] % time spent in the centre of the open field arena
[0883] When considering the entire time of the analysis, BTBR mice
show a reduced percentage time spent in the centre of the arena
compared to C57BL/6 mice. This is reflective of the increased
anxiety and reduced horizontal activity of the BTBR mice. Neither
the LYO control nor butyrate alone affected the time spent in the
centre of the arena. However, the administration of the bacterial
strain significantly increased the amount of time spent in the
centre of the arena compared to the LYO control in BTBR mice (FIG.
51G).
[0884] To provide a further comparison between the controls and the
experimental values, as outlined above, the values for the PBS and
LYO controls were combined in the second analysis. Similarly to the
first the administration of butyrate did not affect the time spent
in the centre of the field compared to the BTBR control. The
administration of the bacterial strain increased the amount of time
spent in the centre of the open field.
[0885] Conclusions
[0886] The chronic treatment with a composition of Blautia
hydrogenotrophica increases exploratory activity and reduces
anxiety-like behaviour in the BTBR mouse model in the open field
arena test. Critically, administration of the bacterial strain to
the BTBR mouse model increased horizontal and vertical activity and
increased the total amount of time spent in the centre of the arena
compared to the BTBR control. Accordingly, this bacterial strain
has anxiolytic effects and improves exploratory behaviour in a
mouse model representative of central nervous system disorders
(e.g. autism spectrum disorders).
Example 11b--Assessment of Stereotyped Behaviours--the Marble
Burying Test
[0887] Rationale and Methods
[0888] See Example 2d above. PBS is the negative control for the
administration of butyrate. LYO is the negative control for the
administration of the Blautia hydrogenotrophica strain. After the
first analyses (FIG. 52 A), the values for the negative controls of
PBS and LYO in both the C57BL/6 and BTBR models are combined and
averaged to provide a simplified comparison in the second analysis
(FIG. 52B).
[0889] Results
[0890] As would be expected, the BTBR model mice displayed an
increase in repetitive behaviour showing significantly more marbles
buried compared to the C57BL/6 model control (FIG. 52B). The
administration of butyrate and the bacterial strain reduced the
number of marbles buried (FIGS. 52A and B).
[0891] Conclusion
[0892] Administration of butyrate and/or the bacterial strain
reduces the number of marbles buried, indicating a reduction in
anxious or stereotyped behaviours.
Example 11c--Assessment of Stereotyped Behaviours--the Digging
Test
[0893] Rationale and Methods
[0894] Similar to the rationale of the marble burying test,
increased digging behaviour corresponds to an increase in
repetitive and stereotyped behaviour.
[0895] Results
[0896] As would be expected, there was a significant increase in
time spent digging in the BTBR model compared to the C57BL/6
control strain (FIG. 53A). However, the number of digging bouts was
not significantly different between the C57BL/6 and BTBR strains
(FIG. 53B). Therefore, it is not possible to assess the role of the
bacterial strain or butyrate in preventing repetitive behaviour in
this analysis.
Example 11d--Assessment of Stereotyped Behaviours--the
Self-Grooming Test
[0897] Rationale and Methods
[0898] See Example 2e above. PBS is the negative control for the
administration of butyrate. LYO is the negative control for the
administration of the Blautia hydrogenotrophica. After the first
analyses (FIGS. 54A, C and E), the values for the negative controls
of PBS and LYO in both the C57BL/6 and BTBR models were combined
and averaged to provide a simplified comparison in the second
analysis (FIGS. 54B, D and F).
[0899] Results
[0900] In line with the BTBR model for stereotyped behaviours, the
BTBR mice showed increased time spent grooming as well as increased
numbers of grooming bouts compared to the C57BL/6 mouse model, in
both the PBS and LYO controls. Administration of butyrate alone
showed a reduction in the time spent grooming, the number of
grooming bouts, and the time spent grooming per bout, compared to
the PBS control. Administration of the bacterial strain reduced the
time spent grooming per grooming bout (FIGS. 54A, C and E).
[0901] To provide a further comparison between the controls and the
experimental values, as outlined above, the values for the PBS and
LYO controls were combined in the second analysis. This second
analysis provided similar results to those of the first analysis
(FIGS. 54B, D and F).
[0902] Conclusion
[0903] Administration of butyrate or Blautia hydrogenotrophica
reduces the amount of time spent grooming per bout of grooming.
Example 11e--Overall Conclusion of the Blautia hydrogenotrophica
Experiments
[0904] In the open field arena test, Blautia hydrogenotrophica
significantly improved the exploratory behaviour of the BTBR mice.
Furthermore, this bacterial strain reduced anxiety-like behaviour
of these mice. Accordingly, it is clear that administration of this
bacteria modulates the behaviour of the BTBR mice which display
autism-like characteristics. Therefore, one would expect these
bacteria to be useful in the treatment and/or prevention of central
nervous system disorders or conditions, including
neurodevelopmental and/or a neuropsychiatric disorders or
conditions.
[0905] Administration of this bacterial strain also appears to
reduce the amount of time performing stereotyped behaviour per
grooming bout in the self-grooming test.
Example 11f--Overall Conclusion of the Butyrate Experiments
[0906] The data from the stereotyped behaviour assays point towards
a therapeutic role for butyrate in central nervous system
disorders.
[0907] The administration of butyrate reduced the number of marbles
buried compared to the BTBR control and returned the average number
to a level similar to that observed in the C57BL/6 wild-type
control mice. In addition, the administration of butyrate alone
reduced the overall time spend grooming and number of grooming
bouts compared to the BTBR control.
[0908] These results provide telling indications regarding a role
for butyrate in reducing repetitive and stereotyped behaviours in
animal models.
Example 12--Effects of Bacterial Lyophilisate on SCFA Production
Healthy Rats
[0909] The effects of chronic administration of a lyophilisate of
Blautia hydrogenotrophica strain DSM 14294 on SCFA production in
healthy HIM rats were studied and the results are reported in FIG.
55. Further details regarding the experiments are provided above in
the descriptions of the figure. FIG. 55 shows that administration
of BH induces a significant increase in acetate as well as in
butyrate production.
Example 13--Efficacy of B. hydrogenotrophica Studied in Human
Microbiota Associated Rat (HMA Rat) Model
[0910] Summary
[0911] Groups of 16 germ-free rats (comprising 8 rats in the
control group and 8 rats in the treatment group) were inoculated
with the faecal microbiota from a human IBS subject (IBS-HMA rats).
Three successive experiments were carried out using faecal samples
from 3 different IBS patients. Two other groups of rats (n=10) were
inoculated with faecal samples of healthy subject (n=2 subjects; 2
groups of healthy-HMA rats) as visceral sensitivity control. Thus,
there were 24 IBS-microbiota associated rats (control), 24 IBS
microbiota associated rats treated with Blautix and 20
healthy-microbiota associated rats. Half of the IBS-HMA rats were
then administered for 28 days with composition comprising the
bacterial strain of B. hydrogenotrophica according to the invention
while the other half animals received a control solution.
[0912] Strain
[0913] Blautia hydrogenotrophica (BH) strain DSM 14294.
[0914] Composition and Administration
[0915] BH lyophilisate was suspended in sterile mineral solution to
a concentration of 10.sup.10 bacteria per ml. Two ml of this
suspension was administered daily per IBS-HMA rat, by oral gavage,
for a 28 days period.
[0916] The control solution was the sterile mineral solution that
was administered daily (2 ml per rat) by oral gavage to the control
group of IBS-HMA rats.
[0917] Rats
[0918] Germ-Free male Fisher rats (aged 10 weeks) were inoculated
with human faecal microbiota from an IBS subject (IBS-HMA rats).
Sixteen rats were inoculated with the same human faecal inoculum.
Three successive experiments were performed with faecal samples
from three different IBS subjects. Two other groups of ten rats
were inoculated with faecal sample from 2 healthy subjects
(normo-sensitivity control groups).
[0919] Study Design
[0920] Day -14--Inoculation of Germ-free rats with human faecal
microbiota.
[0921] Days 0 to 28--Daily dose of BH lyophilisate (assay group),
or control solution (control group) by oral gavage
[0922] Between days 14 and 22--operation to implant electrode into
the abdomen (for distension assay)
[0923] Days 22-28--Adaptation of the rats to avoid stress
associated with distension test.
[0924] Day 28--distension assay and euthanasia of animals to
collect the caecal samples for sulphides and short chain fatty acid
(SCFA) analysis.
[0925] Days 0, 14 and 28--Collection of faecal samples for
microbial analysis: qPCR for evaluating BH population and other
commensal groups of microorganisms and enumeration of functional
groups of microorganisms using selective media and strictly
anaerobic method.
[0926] Results
[0927] FIG. 56 presents the results of qPCR analysis of the B.
hydrogenotrophica population in faecal samples from IBS-HMA rats
receiving control solution or BH lyophilisate. A significant
increase in the BH population was observed at the end of the
administration period (D 28) in rats receiving the BH lyophilisate,
which confirms successful delivery of BH in the colon.
[0928] FIG. 57 reports on the impact of administration of BH on the
main fermentative metabolites, short chain fatty acids, in caecal
samples of IBS-HMA rats. Administration of BH-resulted in a
significant increase in acetate concentration as well as in a
significant increase in butyrate concentration (FIG. 57B).
Example 14--Assessment of Social Interaction Behaviour in the Three
Chamber Test
[0929] Rationale and Methods
[0930] See Example 1b above. In this experiment, the data recorded
is the exploration time, which is defined by the sniffing time of
the cylinders (containing an object, a congener) during the first
5-min period and during the 10-min session.
[0931] Read-outs: [0932] Test 1, sociability (congener vs. object):
[0933] Index of sociability: % sniffing time of the congener (if
>50%: sociability, i.e. preference for the congener vs. the
object) [0934] Other read-outs, indices of exploratory behaviour:
exploration time of the congener, the object, total [0935] Test 2,
social novelty preference (new congener vs. familiar congener):
[0936] Index of social novelty preference (or aversion): % sniffing
time of the new congener [0937] Other read-outs, indices of
exploratory behaviour: exploration time of the new and familiar
congeners, total
[0938] Results
[0939] Test 1: Sociability (FIG. 58A):
[0940] In the C57BL/6 mice the sociability is not different in PBS
and in LYO treated mice. As would be expected, BTBR mice showed
reduced sociability in the PBS control. Unexpectedly BTBR mice
displayed improved sociability when treated with LYO. However, the
differences between BTBR-PBS vs. C57-PBS, BTBR-LYO vs. C57-LYO and
BTBR-PBS vs. BTBR-LYO are not significant. Interestingly,
administration of butyrate improved sociability in BTBR mice
(significantly different between BTBR-PBS vs. BTBR-BUT.
Administration of Blautia hydrogenotrophica increased sociability
compared to the bacterial PBS control.
[0941] Test 2: Social Novelty (FIG. 58B):
[0942] There is a social novelty preference in C57BL/6 mice treated
with PBS, but this preference is reduced in C57BL/6 mice
administered LYO (these differences are not significant). In the 10
min session, BTBR mice showed reduced social novelty preference
compared to C57BL/6 mice when treated with either PBS or LYO. The
differences between differences BTBR-PBS vs. C57-PBS, BTBR-LYO vs.
C57 LYO and BTBR-PBS vs. BTBR-LYO are not significant. The results
shown in FIG. 58B are therefore difficult to interpret.
[0943] Overall Conclusions Regarding Blautia hydrogenotrophica in
the Treatment of Autistic Spectrum Disorders
[0944] The experiments disclosed herein display evidence that
administration of another Blautia species (namely Blautia
hydrogenotrophica) may be applicable for the treatment of
neurodevelopmental and neuropsychiatric disorders in mice models.
In particular, treatment with Blautia hydrogenotrophica reduced
anxiety-like, stereotyped and repetitive behaviour, and increased
sociability in mice.
[0945] 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 Blautia hydrogenotrophica live
biotherapeutic has shown effective treatment of at least one core
symptom of autistic spectrum disorder, so it and related Blautia
and B. hydrogenotrophica 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.
Example 15--Assessing the Effects of Chronic Treatment with MRX006
on Gene Expression Levels of Oxytocin and its Respective Receptors
in the Hypothalamic Cell Lines
[0946] Chronic treatment with MRX006 significantly increases the
level of mRNA expression of oxytocin and its receptor in
hypothalamic cell lines (FIGS. 59A and B).
[0947] This striking result provides a correlation between chemical
changes in the brain and behavioural changes upon administration of
MRX006. This is the first time any study has reported that a live
biotherapeutic is capable of altering the central oxytocin system,
with a concurrent change in social, anxiety-like and stereotyped
behaviour with an improvement in gastrointestinal function.
Example 16--the BALBc Mouse Model
Example 16a--Materials and Methods for BALBc Mouse Model
[0948] Mice
[0949] BALBc (Envigo, UK) adult male mice were group housed under a
12 h light-dark cycle (lights on from 7:00-19:00 hr); standard
rodent food and water were available ad libitum. All experiments
were conducted in accordance with the European Directive
2010/63/EEC, the requirements of SI. No 543 of 2012, and approved
by the Animal Experimentation Ethics Committee of University
College Cork. Animals were 8 weeks old at the start of the
experiment.
[0950] Strain
[0951] MRX006: Blautia stercoris bacterium deposited under
accession number NCIMB 42381.
[0952] The bacteria were provided in glycerol stock and grown in
the facility in anaerobic conditions.
[0953] MRX006 Administration
[0954] Animals were allowed to habituate to their holding room for
one week after arrival into the animal unit. Dosing with MRX006 or
vehicle commenced when the mice were 8 weeks old. MRX006
(1.times.10.sup.7 to 1.times.10.sup.9 CFU) was dissolved in PBS
prior to administration. The mice received oral gavage (200 .mu.L
dose) of MRX006 at a dose of 1.times.10.sup.9 CFU for 6 consecutive
days between 15:00 and 17:00. On day 7, the animals were
decapitated and tissues were harvested for experimentation.
[0955] Tissue Collection
[0956] 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.degree. C. for
further analysis. The brain was quickly excised, dissected and each
brain region was snap-frozen on dry ice and stored at -80.degree.
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 1 cm of tissue from the caecum were used)
were mounted into the Ussing chambers for intestinal permeability
assay. A further 1 cm of ileum and colon tissue was taken for tight
junction gene expression analysis. The caecum was removed, weighed
and stored at -80.degree. C. for SCFAs analysis.
[0957] Statistical Analysis
[0958] 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 16b--Assessing the Effects of Chronic Treatment with MRX006
on Ex Vivo Gastrointestinal Permeability and Tight Junction
Expression
[0959] Methods
[0960] 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 cm.sup.2) as described previously (Hyland and
Cox, 2005 [58]) with oxygenated (95% O2, 5% CO2) Krebs buffer
maintained at 37.degree. C. 4 kDa FITC-dextran was added to the
mucosal chamber at a final concentration of 2.5 mg/mL; 200 .mu.L
samples were collected from the serosal chamber every 30 min for
the following 3 h.
[0961] Results
[0962] Using the passage of FITC from the luminal to the serosal
side of the Ussing chamber as an index of gut permeability (as
described in Example 21), it was determined that MRX006 had no
effect on ileum or colon tissue permeability. FIGS. 60A and 61A
demonstrate that chronic treatment with MRX006 does not influence
the permeability of the colon or ileum.
[0963] MRX006 had no effect on mRNA expression of the tight
junction protein (involved in maintaining the integrity of the gut
barrier) occludin, the enzyme IDO-1 (Indoleamine-pyrrole
2,3-dioxygenase-1 the first and rate-limiting enzyme in the
tryptophan/kynurenine pathway), nor TPH1 (Tryptophan hydroxylase 1,
an isoform of the enzyme tryptophan hydroxylase, responsible for
the synthesis of serotonin) in ileum or colon tissue (FIGS. 60 and
61 B, C and E). MRX006 did however increase TJP-1 (Tight Junction
Protein 1, a tight junction protein) mRNA expression in the ileum,
but not the colon (FIGS. 60D and 61D).
[0964] Discussion
[0965] MRX006 had no effect on ileum or colon permeability, but did
increase TJP1 expression. TJP1 is one of a number of tight junction
proteins associated with maintaining gut integrity, and while we
did see this increase in mRNA expression, this may not necessarily
reflect the protein expression of this tight junction nor its
incorporation into the endothelium. The finding that the 6 day
treatment with MRX006 does not alter permeability suggests that it
does not negatively impact on gut permeability and integrity.
MRX006 also did not alter IDO-1 nor TPH1 suggesting that it does
not alter serotonin production nor the tryptophan/kynurenine
pathway in the gut.
[0966] These data demonstrate that chronic treatment with MRX006
does not alter the gut permeability and does not affect the
integrity of the gut barrier. This shows that the ability of MRX006
attenuate stereotyped and anxiety-related behaviours does not lead
to a deficiency in the gut barrier integrity.
Example 16c--Assessing the Effects of Chronic Treatment with MRX006
on Caecal Short Chain Fatty Acid Production
[0967] Methods
[0968] 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.degree. 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 SCFA was
analyzed using a Varian 3500 GC flame-ionization system, fitted
with a with a ZB-FFAP column (30 m.times.0.32 mm.times.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 pmol/g.
[0969] Results
[0970] Short chain fatty acids (SCFAs) are produced when
non-digestible fibres from the diet are fermented by bacteria in
the gut. 6 days of MRX006 administration had no effect on acetate
(t12=0.959, p=0.357), propionate (t12=1.033, p=0.322), isobutyrate
(t12=1.859, p=0.090), butyrate (t12=0.857, p=0.408), isovalearate
(t12=1.757, p=0.107) or valearate (t12=0.434, p=0.672), when
compared to vehicle PBS administration (FIG. 62).
[0971] Discussion
[0972] The administration of MRX006 had no effect on caecal SCFA
production. This suggests that the 6 day regime of MRX006 did not
alter the fermentation, or the bacteria responsible for the
fermentation of non-digestible fibres from the diet.
Example 16d--Assessing the Effects of Chronic Treatment with MRX006
on Cytokine Expression from Splenocytes
[0973] Rationale Methods
[0974] The ex-vivo splenocyte assay involves challenging the
splenocytes (cells isolated from the spleen--a main organ involved
in immune defence), with a bacterio- or viral-mimetic
challenge.
[0975] Spleens were collected immediately in 5 mL RPMI media
following sacrifice and cultured immediately. Spleen cells were
first homogenised in the RPMI media. The homogenate step was
followed by RBC lysis step where the cells were incubated for 5
mins in 1 ml of RBC lysis buffer (11814389001 ROCHE, Sigma). 10 ml
of the media was added to stop the lysis and followed by 200 g
centrifugation for 5 mins. This was followed by final step where
the cells were passed through 40 um strainer. The homogenate was
then filtered over a 40 um strainer, centrifuged at 200 g for 5 min
and resuspended in media. Cells were counted and seeded
(4,000,000/mL media). After 2.5 h of adaptation, cells were
stimulated with lipopolysaccharide (LPS-2 .mu.g/ml) or concanavalin
A (ConA-2.5 .mu.g/ml) for 24 h. Following stimulation, the
supernatants were harvested to assess the cytokine release using
Proinflammatory Panel 1 (mouse) V-PLEX Kit (Meso Scale Discovery,
Maryland, USA) for TNF.alpha., IL-10, IL-1.beta., Interferon
.gamma., CXCL2 and IL6. The analyses were performed using MESO
QuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S
600.
[0976] Results
[0977] MRX006 had no effect on splenocyte release of
proinflammatory (IFN.gamma., TNF.alpha., IL-1.beta.) nor
anti-inflammatory (IL-10, IL-6) or CXCL1 (marker of immune response
activation) in response to LPS (mimicking a bacterial infection) or
concavalin A (mimicking a viral infection) stimulation (FIG.
63).
[0978] Discussion
[0979] MRX006 also had no effect on cytokine expression from
splenocytes following a challenge with LPS or Concavalin A. This
demonstrates that the 6-day MRX006 administration did not
negatively influence the innate peripheral immune response. This
shows that MRX006 treatment does not activate the systemic immune
activation.
Example 16e--Assessing the Effects of Chronic Treatment with MRX006
on Plasma Levels of Amino Acids
[0980] Rationale and Methods
[0981] At the end of the experiment trunk blood was collected for
amino acid analysis in the plasma. This would give an index of the
biosynthesis and catabolism of essential amino acids by changes in
microbiota.
[0982] 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.degree. C. for
further analysis. Plasma was diluted with 0.2 mol/L sodium citrate
buffer, pH 2.2 to yield 250 nmol of each amino acid residue.
Samples were diluted with the internal standard norleucine, to give
a final concentration of 125 nm/mL. Amino acids were quantified
using a Jeol JLC-500/V amino acid analyser (Jeol Ltd, Garden City,
Herts, UK) fitted with a Jeol Na+ high performance cation exchange
column.
[0983] Results
[0984] MRX006 decreased proline and phenylalanine levels in the
plasma.
[0985] Discussion
[0986] Plasma levels of amino acids were largely unaltered
following 6 day administration of MRX006. There are nine essential
amino acids that cannot be synthesised de novo and must be supplied
directly in the diet or by breakdown of the diet. These include
valine, phenylalanine, threonine, tryptophan, methionine, leucine,
isoleucine, lysine, and histidine. Six other amino acids are
considered conditionally essential in the human diet, meaning their
synthesis can be limited under special pathophysiological
conditions, such as prematurity in the infant or individuals in
severe catabolic distress. These six amino acids include arginine,
cysteine, glycine, glutamine, proline, and tyrosine. Five amino
acids are dispensable in humans, meaning they can be synthesized in
sufficient quantities in the body. These five are alanine, aspartic
acid, asparagine, glutamic acid and serine.
[0987] In this study the essential amino acid phenylalanine, and
proline another important amino acid were decreased following
MRX006 administration, suggesting that this probiotic may play a
role in metabolism of key amino acids from the diet.
Example 16f--Assessing the Effects of Chronic Treatment with MRX006
on Neurotransmitter Levels in the Brainstem
[0988] Methods
[0989] Neurotransmitter concentration was analysed by HPLC on
samples from the brainstem. Briefly, brainstem tissue was sonicated
in 500 .mu.l of chilled mobile phase spiked with 4 ng/40 .mu.l 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.times.g at
4.degree. C. and 40 .mu.l 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 Gastorr Degasser (ISS, UK). A reverse-phase column
(Kinetex 2.6 u C18 100.times.4.6 mm, Phenomenex) maintained at
30.degree. C. was employed in the separation (Flow rate 0.9
ml/min). The glassy carbon working electrode combined with an
Ag/AgCl reference electrode (Shimdazu) operated a +0.8 V and the
chromatograms generated were analyzed using Class-VP 5 software
(Shimadzu). The neurotransmitters were identified by their
characteristic retention times as determined 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.
[0990] Results
[0991] 6 days administration of MRX006 had no effect on levels of
noradrenaline, dopamine, serotonin, 5-HIAA (5-hydroxy-indole-acetic
acid; a metabolite of 5-HT (5-hydroxy-tryptamine (serotonin)), or
serotonin turnover (the ratio of 5-HIAA:5-HT) as determined by
unpaired 2-tailed t-test (FIG. 65). Noradrenaline (t12=0.307,
p=0.764), dopamine (t12=0.957, p=0.357), serotonin (t12=0.745,
p=0.074), 5-HIAA (t12=0.379, p=0.711) levels or serotonin turnover
in brainstem (t12=0.683, p=0.507).
[0992] Discussion
[0993] Neurotransmitter levels in the brainstem were unaltered
following 6-day MRX006 administration. These data suggest that
MRX006 does not negatively impact on behaviours that are governed
by monoamine levels at the level of the brainstem.
Example 16g--Assessing the Effects of Chronic Treatment with MRX006
on Central and Gastrointestinal Gene Expression
[0994] Rationale
[0995] Expression of genes for neurotransmitter receptors
[serotonin receptor 1a(5-HT1a), dopamine D1 receptor, GABAB
receptor subunit B1, GABAA receptor, NMDA2A and NMDA2B receptor],
inflammatory markers [IL-1, IL6, CD11b, TNF.alpha. and TLR4], and
endocrine markers [corticosterone releasing factor (CRF),
corticosterone releasing factor receptors 1 and 2 (CRFR1, CRFR2),
brain-derived neurotrophin factor (BDNF), vasopressin receptor,
oxytocin receptor, glucocorticoid receptor and mineralocorticoid
receptor] were analysed in brain tissue from the amygdala,
prefrontal cortex and hippocampus.
[0996] Methods
[0997] Total RNA was extracted using the mirVana.TM. 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
NanoDrop.TM. spectrophotometer (Thermo Fisher Scientific Inc.,
Wilmington, Del., 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/.mu.L) (1)(2)(1)(10) was added as part
of RT master mix, incubated for 25.degree. C. for 10 min,
37.degree. C. for 2 h, 85.degree. C. for 5 min and stored at
4.degree. C. Quantitative PCR was carried out using probes (6
carboxy fluorescein--FAM) designed by Applied Biosystems to mouse
specific targeted genes, while using .beta.-actin as an endogenous
control. Amplification reactions contained 1 .mu.l cDNA, 5 .mu.l of
the 2.times.PCR Master mix (Roche), 900 nM of each primer and were
brought to a total of 10 .mu.l by the addition of RNase-free water.
All reactions were performed in triplicate using 96-well plates on
the LightCycler.RTM.480 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 R-actin and transformed using
the 2-.DELTA..DELTA.CT method and presented as a fold change vs.
control group.
[0998] Results
[0999] FIG. 66 shows that MRX006 had no effect on hippocampal gene
expression of the neurotransmitter receptors serotonin 1a (5-HT1a)
(t.sub.11=0.742, p=0.474), dopamine D1 receptor (t.sub.10=1.426,
p=0.184), GABA.sub.B receptor B1 subunit (t.sub.12=1.871, p=0.086),
GABA.sub.A receptor (t.sub.12=0.017, p=0.987), NMDA receptor
subunit 2A (t.sub.11=1.275, p=0.229), NMDA receptor subunit 2B
(t.sub.11=1.39, p=0.192).
[1000] FIG. 67 shows that MRX006 had no effect on amygdalar gene
expression of the neurotransmitter receptors dopamine D1 receptor
(t.sub.11=0.429, p=0.677), GABA.sub.B receptor B1 subunit
(t.sub.11=0.998, p=0.344), GABA.sub.A receptor (t.sub.11=1.145,
p=0.277), NMDA receptor subunit 2A (t.sub.12=0.852, p=0.411), NMDA
receptor subunit 2B (t.sub.12=0.395, p=0.707).
[1001] FIG. 68 shows that MRX006 had no effect on prefrontal cortex
gene expression of the neurotransmitter receptors dopamine D1
receptor (t.sub.11=0.583, p=0.571), GABA.sub.B receptor B1 subunit
(t.sub.12=1.304, p=0.217), GABA.sub.A receptor (t.sub.10=2.043,
p=0.068), NMDA receptor subunit 2A (t.sub.11=0.177, p=0.104), NMDA
receptor subunit 2B (t.sub.11=1.235, p=0.243).
[1002] There was no effect of MRX006 on mRNA expression of
neurotransmitter receptors in any of the brain regions investigated
(FIGS. 66-68).
[1003] In the hippocampus and amygdala (FIGS. 69 and 70) there was
no effect on mRNA expression of the various inflammatory markers.
MRX006 had no effect on Hippocampal gene expression of the
inflammatory markers IL-13 (t.sub.10=1.346, p=0.208), IL-6
(t.sub.12=1.041, p=0.308), CD11b (t.sub.12=1.195, p=0.255),
TNF.alpha. (t.sub.11=0.816, p=0.342), TLR4 (t.sub.12=0.521,
p=0.612). MRX006 had no effect on amygdalar gene expression of the
inflammatory markers IL-1.beta. (t.sub.11=1.53, p=0.988), IL-6
(t.sub.11=1.145, p=0.217), CD11b (t.sub.11=1.143, p=0.275), TLR4
(t.sub.11=0.971, p=0.532).
[1004] In the prefrontal cortex, MRX006 decreased mRNA expression
for TLR4 without any changes in other inflammatory markers (FIG.
71). MRX006 significantly decreased mRNA expression of TLR4
(t.sub.12=2.639, p=0.0216) in the prefrontal cortex, but had no
further effect on the prefrontal cortex gene expression for IL-6
(t.sub.11=1.145, p=0.217) or CD11b (t.sub.11=2.175, p=0.523).
[1005] MRX006 significantly decreased vasopressin receptor mRNA
expression in hippocampus (t.sub.12=2.389, p=0.0342), but had no
further effect on mRNA expression for endocrine markers CRF
(t.sub.12=0.767, p=0.458), CRFR1 (t.sub.12=0.174, p=0.865), CRFR2
(t.sub.11=0.238, p=0.816), BDNF (t.sub.12=1.548, p=0.148), oxytocin
receptor (t.sub.12=0.762, p=0.461), glucocorticoid receptor
(t.sub.12=0.607, p=0.556), mineralocorticoid receptor
(t.sub.12=0.67, p=0.516) (FIG. 72).
[1006] MRX006 had no effect on mRNA expression of amygdalar
endocrine markers CRFR1 (t.sub.12=0.226, p=0.825), CRFR2
(t.sub.11=0.78, p=0.451), BDNF (t.sub.12=0.201, p=0.844),
vasopressin receptor (t.sub.12=0.756, p=0.465), oxytocin receptor
(t.sub.11=0.167, p=0.87), glucocorticoid receptor (t.sub.11=1.027,
p=0.327), mineralocorticoid receptor (t.sub.11=1.448, p=0.175)
(FIG. 73).
[1007] MRX006 had no effect on mRNA expression of the prefrontal
cortex endocrine markers CRFR1 (t.sub.12=1.666, p=0.122), CRFR2
(t.sub.11=1.179, p=0.261), BDNF (t.sub.11=1.065, p=0.310), oxytocin
receptor (t.sub.11=1.037, p=0.322), glucocorticoid receptor
(t.sub.12=1.185, p=0.259), mineralocorticoid receptor
(t.sub.11=1.910, p=0.083) (FIG. 74).
[1008] In the amygdala and the prefrontal cortex (FIGS. 73 and 74),
there were no changes in mRNA expression of any endocrine markers,
while in the hippocampus (FIG. 72) there was a decrease in mRNA
expression of vasopressin receptor without any effect on the other
endocrine markers analysed.
[1009] Discussion
[1010] The central gene expression for inflammatory, endocrine and
neurotransmitter receptors were mostly unaltered following 6-day
MRX006 administration.
[1011] Overall Conclusions Regarding MRX006 Administration on
Physiological Parameters
[1012] Overall these data confirm that MRX006 administration does
not negatively impact on systemic and central physiological events.
These data suggest that MRX006 may have a high tolerability profile
with minimal non-desirable side-effects.
Example 17--the Maternal Immune Activation (MIA) Mouse Model
[1013] The MIA mice used are the same as described in Example
3a.
Example 17a--Assessing the Effects of Chronic Treatment with MRX006
on In Vivo Gastrointestinal Permeability in MIA Model
[1014] The permeability of the ileum and colon was assessed in vivo
using Ussing chambers as described in Example 2k. FIG. 75
demonstrates that chronic treatment with MRX006 does not influence
the permeability of the colon or ileum in MIA model.
[1015] This confirms that chronic treatment with MRX006 does not
alter the gut permeability, which shows that the beneficial social
behaviour, reduce anxiety-like behaviour and stereotype behaviour
effects of MRX006 do not lead to a deficit in the integrity of the
gut.
Example 17b--Assessment of Social Behaviours--the Three Chamber
Social Interaction Test
[1016] The 3-Chamber Social Interaction Test (3-CSIT) was conducted
as described on example 1a, however this data was manually scored
by a researcher that was blinded to treatment. The data in example
1a was automatically generated by computer tracking software which
cannot distinguish between interaction with the mouse and just
being in the same chamber as the mouse.
[1017] In the social novelty test (FIG. 76), there was no
MIA-induced deficit in social discrimination and MRX006 had no
further effect on social novelty.
[1018] FIG. 77 shows that in the sociability test, MRX006 was able
to reverse MIA-induced deficits in social behaviour. This is
similar to data seen in the BTBR model where MRX006 could reverse
deficits in sociability.
Example 17c--Assessment of Social Behaviours--the Grooming Test
[1019] The grooming test was conducted as described in example 2e.
Chronic treatment with MRX006 did not lead to a change in
repetitive behaviours in MIA mice in the grooming test (FIG.
78).
Example 17d--Assessment of Social Behaviours--the Elevated Plus
Maze
[1020] The elevated plus maze test was conducted as described in
example 2f. Treatment with MRX006 has no effect on anxiety-like
behaviour in MIA mice in the elevated plus maze (FIG. 79).
Example 17e--Assessment of Social Behaviours--the Forced Swim
Test
[1021] The forced swim test was conducted as described in example
2h. Chronic treatment with MRX006 did reduce the immobility time of
MIA mice in the forced swimming test (FIG. 80).
Example 17f--Stress-Induced Circulating Corticosterone
Determination
[1022] The levels of corticosterone were measured as described in
example 2n. Chronic treatment with MRX006 does not influence
stress-induced corticosterone levels in MIA mice exposed to the
forced swimming test (FIG. 81).
[1023] Conclusions
[1024] Treatment with MRX006 reversed MIA-induced deficits in
social behaviour and reduced the immobility time in the forced
swimming test. This demonstrates the ability of MRX006 to improve
the sociability and antidepressant activity.
[1025] In addition to the results described above in example 3,
MRX006 has been demonstrated to have a positive impact on the
symptoms of autistic spectrum disorders.
TABLE-US-00006 Sequences (Blautia stercoris strain GAM6-1 16S
ribosomal RNA gene, partial sequence- HM626177) SEQ ID NO: 1 1
tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg
61 gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac
agttggaaac 121 ggctgctaat accgcataag cgcacggtat cgcatgatac
agtgtgaaaa actccggtgg 181 tatgagatgg acccgcgtct gattagctag
ttggaggggt aacggcccac caaggcgacg 241 atcagtagcc ggcctgagag
ggtgaacggc cacattggga ctgagacacg gcccagactc 301 ctacgggagg
cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc 361
gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt
421 acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac
gtagggggca 481 agcgttatcc ggatttactg ggtgtaaagg gagcgtagac
ggaagagcaa gtctgatgtg 541 aaaggctggg gcttaacccc aggactgcat
tggaaactgt ttttcttgag tgccggagag 601 gtaagcggaa ttcctagtgt
agcggtgaaa tgcgtagata ttaggaggaa caccagtggc 661 gaaggcggct
tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg 721
attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc
781 tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt
cgcaagaatg 841 aaactcaaag gaattgacgg ggacccgcac aagcggtgga
gcatgtggtt taattcgaag 901 caacgcgaag aaccttacca agtcttgaca
tcgatctgac cggttcgtaa tggaaccttt 961 ccttcgggac agagaagaca
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1021 gggttaagtc
ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc 1081
actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat
1141 gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg
aagcgagccc 1201 gcgaggggga gcaaatccca aaaataacgt cccagttcgg
actgcagtct gcaactcgac 1261 tgcacgaagc tggaatcgct agtaatcgcg
aatcagaatg tcgcggtgaa tacgttcccg 1321 ggtcttgtac acaccgcccg
tcacaccatg ggagtcagta acgcccgaag tc (consensus 16S rRNA sequence
for Blautia stercoris MRX006 (strain 830)) SEQ ID NO: 2
TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAAC-
CTT
CGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATA-
ACA
GTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGA-
GAT
GGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGG-
TGA
ACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGG-
AAA
CCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGA-
CGG
TACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGA-
TTT
ACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCAT-
TGG
AAACTGTTTTTCTTGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAG-
GAA
CACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTA-
GAT
ACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGCAGCAAACG-
CAA
TAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTG-
GAG
CATGTGGTTTATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAAC-
CTT
TCCTTCGGGACAGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG-
CAA
CGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGG-
AGG
AAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAA-
GGG
AAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCA-
CGA
AGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGT-
CAC
ACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAA-
CTG GGGTGAAGTCTAGGGGGT (Blautia wexlerae strain WAL 14507 16S
ribosomal RNA gene, partial sequence- EF036467) SEQ ID NO: 3 1
caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg
61 gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt
cagaaatggc 121 tgctaatacc gcataagcgc acagagctgc atggctcagt
gtgaaaaact ccggtggtat 181 aagatggacc cgcgttggat tagcttgttg
gtggggtaac ggcccaccaa ggcgacgatc 241 catagccggc ctgagagggt
gaacggccac attgggactg agacacggcc cagactccta 301 cgggaggcag
cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg 361
tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc
421 tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta
gggggcaagc 481 gttatccgga tttactgggt gtaaagggag cgtagacggt
gtggcaagtc tgatgtgaaa 541 ggcatgggct caacctgtgg actgcattgg
aaactgtcat acttgagtgc cggaggggta 601 agcggaattc ctagtgtagc
ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa 661 ggcggcttac
tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt 721
agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca
781 ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg
caagaatgaa 841 actcaaagga attgacgggg acccgcacaa gcggtggagc
atgtggttta attcgaagca 901 acgcgaagaa ccttaccaag tcttgacatc
cgcctgaccg atccttaacc ggatctttcc 961 ttcgggacag gcgagacagg
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1021 gttaagtccc
gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact 1081
ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc
1141 ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag
cgagattgtg 1201 agatggagca aatcccaaaa ataacgtccc agttcggact
gtagtctgca acccgactac 1261 acgaagctgg aatcgctagt aatcgcggat
cagaatgccg cggtgaatac gttcccgggt 1321 cttgtacaca ccgcccgtca
caccatggga gtcagtaacg cccgaagtca gtgacctaac 1381 tgcaaagaag
gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt (consensus 16S
rRNA sequence for Blautia wexlerae strain MRX008) SEQ ID NO: 4
TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCT-
TAT
ACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAA-
CTC
CGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAG-
CCG
GCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA-
TTG
CACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGC-
AGG
GAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGC-
AAG
CGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAA-
CCT
GTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGC-
GTA
GATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGG-
AGC
AAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCG-
GTG
CCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGA-
CCC
GCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGAC-
CGA
TCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGAT-
GTT
GGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTG-
CCA
GGGATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTAC-
AAT
GGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTC-
TGC
AACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTT-
GTA
CACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGA-
A (MRX006 (strain 830) chromosome sequence)- SEQ ID NO: 5 see
electronic sequence listing. (MRX006 (strain 830) plasmid
sequence)- SEQ ID NO: 6 see electronic sequence listing. (Blautia
hydrogenotrophica strain S5a36 16S ribosomal RNA gene, partial
sequence-X95624.1) SEQ ID NO: 7 1 gatgaacgct ggcggcgtgc ttaacacatg
caagtcgaac gaagcgatag agaacggaga 61 tttcggttga agttttctat
tgactgagtg gcggacgggt gagtaacgcg tgggtaacct 121 gccctataca
gggggataac agttagaaat gactgctaat accgcataag cgcacagctt 181
cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag
241 ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag
ggtgaacggc 301 cacattggga ctgagacacg gcccaaactc ctacgggagg
cagcagtggg gaatattgca 361 caatggggga aaccctgatg cagcgacgcc
gcgtgaagga agaagtatct cggtatgtaa 421 acttctatca gcagggaaga
aagtgacggt acctgactaa gaagccccgg ctaattacgt 481 gccagcagcc
gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg 541
gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg
tggactgcat
601 tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt
agcggtgaaa 661 tgcgtagata ttaggaggaa caccagtggc gaaggcggcc
tgctggacgg taactgacgt 721 tgaggctcga aagcgtgggg agcaaacagg
attagatacc ctggtagtcc acgctgtaaa 781 cgatgaatac taggtgtcgg
gtggcaaagc cattcggtgc cgcagcaaac gcaataagta 841 ttcccacctg
gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca 901
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac
961 atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac
aggtggtgca 1021 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt
cccgcaacga gcgcaaccct 1081 tattcttagt agccagcagg tagagctggg
cactctaggg agactgccag ggataacctg 1141 gaggaaggtg gggatgacgt
caaatcatca tgccccttat gatttgggct acacacgtgc 1201 tacaatggcg
taaacaaagg gaagcgaagg ggtgacctgg agcaaatctc aaaaataacg 1261
tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgc
1321 gaatcagaat gtcgcggtga atacgttccc gggtcttgta cacaccgccc
gtcacaccat 1381 gggagtcagt aacgcccgaa gtcagtgacc caaccnaaag
gagggagctg ccgaaggtgg 1441 gactgataac tggggtga
REFERENCES
[1026] [1] Spor et al. (2011) Nat Rev Microbiol. 9(4):279-90.
[1027] [2] Eckburg et al. (2005) Science. 10; 308(5728):1635-8.
[1028] [3] Macpherson et al. (2001) Microbes Infect. 3(12):1021-35
[1029] [4] Macpherson et al. (2002) Cell Mol Life Sci.
59(12):2088-96. [1030] [5] Mazmanian et al. (2005) Cell 15;
122(1):107-18. [1031] [6] Frank et al. (2007) PNAS 104(34):13780-5.
[1032] [7] Scanlan et al. (2006) J Clin Microbiol. 44(11):3980-8.
[1033] [8] Kang et al. (2010) Inflamm Bowel Dis. 16(12):2034-42.
[1034] [9] Machiels et al. (2013) Gut. 63(8):1275-83.[10] Mayer et
al (2014) The Journal of Neuroscience 34(46):15490-15496 [1035]
[11] Cryan and Dinan (2015) Neuropsychopharmacology, 40: 241-2.
[1036] [12] Zhou and Foster (2015) Neuropsychiatric Disease and
Treatment 11: 715-723. [1037] [13] Wang and Kasper (2014) Brain
Behav Immun. 38: 1-12. [1038] [14] WO 2013/050792 [1039] [15] WO
03/046580 [1040] [16] WO 2013/008039 [1041] [17] WO 2014/167338
[1042] [18] Goldin and Gorbach (2008) Clin Infect Dis. 46 Suppl
2:S96-100. [1043] [19] Azad et al. (2013) BMJ. 347:f6471. [1044]
[20] Bravo et al. (2011) Proc Natl Acad Sci USA, 108: 16050-5.
[1045] [21] Kantak et al. (2014) Behav Pharmacol. 25: 71-9. [1046]
[22] Savignac et al. (2014) Neurogastroenterol Motil. 26: 1615-27.
[1047] [23] de Theije et al. (2014) Brain Behav Immun. 37: 197-206.
[1048] [24] Hsiao et al. (2013) Cell, 155: 1451-63. [1049] [25]
Meyza and Blanchard (2017) Neurosci Biobehav Rev. [1050] [26] Liu
et al. (2008) Int J Syst Evol Microbiol 58, 1896-1902. [1051] [27]
Park et al. (2012) Int J Syst Evol Microbiol. 62(Pt 4):776-9.
[1052] [28] Liu et al. (2008) Int J Syst Evol Microbiol. 58(Pt
8):1896-902. [1053] [29] Masco et al. (2003) Systematic and Applied
Microbiology, 26:557-563. [1054] [30] Sr tkova et al. (2011) J
Microbiol. Methods, 87(1):10-6. [1055] [31] Bernalier et al. (1996)
Arch. Microbiol. 166 (3), 176-183. [1056] [32] Wang et al. (2016) J
Neurogastroenterol Motil 22: 589-605. [1057] [33] Li and Zhou
(2016) Neuroscience 324: 131-139. [1058] [34] Hyland and Stanton
(2016) The Gut-Brain Axis: Dietary, Probiotic and Prebiotic
Interventions on the Microbiota (Academic Press). [1059] [35]
Bourassa et al. (2016) Neuroscience Letters 625, 56-63 [1060] [36]
Miyamoto-Shinohara et al. (2008) J. Gen. Appl. Microbiol., 54,
9-24. [1061] [37] Cryopreservation and Freeze-Drying Protocols, ed.
by Day and McLellan, Humana Press. [1062] [38] Leslie et al. (1995)
Appl. Environ. Microbiol. 61, 3592-3597. [1063] [39] Mitropoulou et
al. (2013) J Nutr Metab. (2013) 716861. [1064] [40] Kailasapathy et
al. (2002) Curr Issues Intest Microbiol. 3(2):39-48. [1065] [41]
Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited
by A Wade and P J Weller [1066] [42] Remington's Pharmaceutical
Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) [1067]
[43] US 2016/0067188 [1068] [44] Handbook of Microbiological Media,
Fourth Edition (2010) Ronald Atlas, CRC Press. [1069] [45]
Maintaining Cultures for Biotechnology and Industry (1996) Jennie
C. Hunter-Cevera, Academic Press [1070] [46] Strobel (2009) Methods
Mol Biol. 581:247-61. [1071] [47] Gennaro (2000) Remington: The
Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
[1072] [48] Molecular Biology Techniques: An Intensive Laboratory
Course, (Ream et al., eds., 1998, Academic Press). [1073] [49]
Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic
Press, Inc.) [1074] [50] Handbook of Experimental Immunology, Vols.
I-IV (D. M. Weir and C. C. Blackwell, eds, 1986, Blackwell [1075]
Scientific Publications) [51] Sambrook et al. (2001) Molecular
Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor
Laboratory Press). [1076] [52] Handbook of Surface and Colloidal
Chemistry (Birdi, K. S. ed., CRC Press, 1997) [1077] [53] Ausubel
et al. (eds) (2002) Short protocols in molecular biology, 5th
edition (Current Protocols). [1078] [54] PCR (Introduction to
Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997,
Springer Verlag) [1079] [55] Current Protocols in Molecular Biology
(F. M. Ausubel et al., eds., 1987) Supplement 30 [1080] [56] Smith
& Waterman (1981) Adv. Appl. Math. 2: 482-489. [1081] [57]
Cryan and Mombereau (2004) Mol Psychiatry 9: 326-57. [1082] [58]
Hyland and Cox (2006) Br J Pharmacol. 146(5): 712-722
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220184145A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220184145A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References