U.S. patent application number 17/406339 was filed with the patent office on 2022-03-31 for compositions comprising bacterial strains.
The applicant listed for this patent is 4D Pharma Research Limited. Invention is credited to Suaad AHMED, Anna ETTORRE, Parthena FOTIADOU, Imke Elisabeth MULDER, Helene SAVIGNAC, Samantha YUILLE.
Application Number | 20220096565 17/406339 |
Document ID | / |
Family ID | 1000006016840 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096565 |
Kind Code |
A1 |
MULDER; Imke Elisabeth ; et
al. |
March 31, 2022 |
COMPOSITIONS COMPRISING BACTERIAL STRAINS
Abstract
The invention provides compositions comprising bacterial strains
for treating and preventing a neurodegenerative disorder.
Inventors: |
MULDER; Imke Elisabeth;
(Aberdeen, GB) ; ETTORRE; Anna; (Aberdeen, GB)
; AHMED; Suaad; (Aberdeen, GB) ; FOTIADOU;
Parthena; (Aberdeen, GB) ; YUILLE; Samantha;
(Aberdeen, GB) ; SAVIGNAC; Helene; (Aberdeen,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
4D Pharma Research Limited |
Aberdeen |
|
GB |
|
|
Family ID: |
1000006016840 |
Appl. No.: |
17/406339 |
Filed: |
August 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16713969 |
Dec 13, 2019 |
11123379 |
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17406339 |
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PCT/EP2018/065808 |
Jun 14, 2018 |
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16713969 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/19 20130101; A61K
9/0053 20130101; A61K 35/74 20130101 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 9/00 20060101 A61K009/00; A61K 9/19 20060101
A61K009/19 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 2017 |
GB |
1709466.5 |
Jun 15, 2017 |
GB |
1709533.2 |
Claims
1.-34. (canceled)
35. A pharmaceutical composition comprising a bacterial strain of
the genus Roseburia in a therapeutically effective amount for
reducing a level of a proinflammatory cytokine in a subject when
administered to the subject, and a pharmaceutically acceptable
excipient, diluent, or carrier, wherein: (a) the bacterial strain
is lyophilized, (b) the pharmaceutical composition does not
comprise a therapeutically effective amount of any other strain,
and (c) the pharmaceutical composition is formulated in an enteric
capsule for delivery to an intestine of the subject.
36. The pharmaceutical composition of claim 35, wherein the
proinflammatory cytokine is interleukin 6 (IL-6).
37. The pharmaceutical composition of claim 35, wherein the
therapeutically effective amount comprises from about
1.times.10.sup.3 to about 1.times.10.sup.11 colony forming units
(CFU).
38. The pharmaceutical composition of claim 35, wherein the
pharmaceutical composition comprises from about 1.times.10.sup.6 to
about 1.times.10.sup.11 colony forming units per gram (CFU/g), with
respect to the weight of the pharmaceutical composition.
39. The pharmaceutical composition of claim 35, wherein the
bacterial strain is of the species Roseburia hominis, Roseburia
intestinalis, or Roseburia faecis.
40. The pharmaceutical composition of claim 35, wherein the
bacterial strain comprises a 16S rRNA gene sequence that has at
least 95% sequence identity to the polynucleotide sequence of SEQ
ID NO:3, SEQ ID NO: 5, or SEQ ID NO:6.
41. The pharmaceutical composition of claim 35, 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:3, SEQ ID NO: 5, or SEQ ID NO:6.
42. The pharmaceutical composition of claim 35, wherein the
bacterial strain comprises a 16S rRNA gene sequence of SEQ ID NO:3,
SEQ ID NO: 5, or SEQ ID NO:6.
43. The pharmaceutical composition of claim 35, wherein the
bacterial strain is the strain deposited under accession number
NCIMB 42383, the strain deposited under accession number NCIMB
43043, or a biotype thereof.
44. The pharmaceutical composition of claim 43, wherein the biotype
is a bacterial strain that has the same carbohydrate fermentation
pattern as the strain deposited under accession number NCIMB 42383
or the strain deposited under accession number NCIMB 43043.
45. The pharmaceutical composition of claim 35, wherein the
bacterial strain is capable of at least partially colonizing an
intestine of the subject.
46. The pharmaceutical composition of claim 35, wherein the
pharmaceutical composition is formulated for oral
administration.
47. The pharmaceutical composition of claim 35, wherein the
bacterial strain is live.
48. A pharmaceutical composition comprising a bacterial strain of
the genus Roseburia in a therapeutically effective amount for
reducing a level of the NF.kappa.B promoter activation in a subject
and a pharmaceutically acceptable excipient, diluent, or carrier,
wherein: (a) the bacterial strain is lyophilized, (b) the
pharmaceutical composition does not comprise a therapeutically
effective amount of any other strain, and (c) the pharmaceutical
composition is formulated in an enteric capsule for delivery to an
intestine of the subject.
49. A method of treating a neurodegenerative disorder in a subject
in need thereof comprising administering to the subject a
composition comprising a therapeutically effective amount of a
bacterial strain of the genus Roseburia, wherein the bacterial
strain comprises a 16S rRNA gene sequence that has at least 95%
sequence identity to the polynucleotide sequence of SEQ ID NO:3,
SEQ ID NO: 5, or SEQ ID NO:6, and wherein the administering is
effective to treat the neurodegenerative disorder.
50. The method of claim 49, wherein the administering is effective
to reduce a level of a proinflammatory cytokine in the subject.
51. The method of claim 49, wherein the proinflammatory cytokine is
interleukin 6 (TL-6).
52. The method of claim 49, wherein the therapeutically effective
amount comprises from about 1.times.10.sup.3 to about
1.times.10.sup.11 colony forming units (CFU).
53. The method of claim 49, wherein the composition comprises no
more than de minimis amounts of other bacterial strains.
54. The method of claim 49, wherein the composition further
comprises a pharmaceutically acceptable excipient, diluent, or
carrier.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/713,969, filed Dec. 13, 2019, which is a continuation of
International Application No. PCT/EP2018/065808, filed Jun. 14,
2018, which claims the benefit of Great Britain Application No.
1709466.5, filed Jun. 14, 2017, and Great Britain Application No.
1709533.2, filed Jun. 15, 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 Aug. 7, 2021, is named 56708-728_302_SL.txt and is 4,680,763
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] Dramatic changes in microbiota composition have been
documented in gastrointestinal disorders such as inflammatory bowel
disease (IBD). For example, the levels of Clostridium cluster XIVa
bacteria are reduced in IBD patients whilst numbers of E. coli are
increased, suggesting a shift in the balance of symbionts and
pathobionts within the gut [6-9].
[0006] 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, [10-13]). 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 [14] and [15] for
reviews). The ability of Roseburia hominis to regulate the immune
system has been suggested in [16]. 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 neurodegenerative disorders.
[0007] Recently, there has been increased interest in the art
regarding alterations in the gut microbiome that may play a
pathophysiological role in human brain diseases [17]. Preclinical
and clinical evidence are strongly suggesting a link between brain
development and microbiota [18]. 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 [19], and an imbalance of the Bacteroidetes and
Firmicutes phyla has also been implicated in brain development
disorders [20]. 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
[10]. Roseburia hominis has been proposed for treating a variety of
disorders including asthma, rheumatoid arthritis and multiple
sclerosis [21]
[0008] Like asthma and rheumatoid arthritis, multiple sclerosis is
primarily mediated by the immune system. The immune system attacks
myelinated axons in the central nervous system, destroying the
myelin called plaques or lesions. Demyelination occurs in
particular in the optic nerves, subpial spinal cord, brainstem,
cerebellum, and juxtacortical and periventricular white matter
regions.
[0009] As such, multiple sclerosis has a different pathology to
other neurodegenerative diseases, such as Parkinson's disease,
Alzheimer's disease or dementia. For example, multiple sclerosis is
commonly diagnosed in patients in their 20s and 30s, while many
other neurodegenerative diseases, such as Parkinson's disease,
Alzheimer's and dementia, are diagnosed predominantly in patients
aged over 65 years old.
[0010] Parkinson's disease, like many neurodegenerative diseases,
is primarily mediated by the accumulation of misfolded protein.
Parkinson's disease is a synucleinopathology that involves the
accumulation of .alpha.-synuclein, which aggregate as insoluble
fibrils in Lewy bodies within the cytoplasm of the neuronal body.
The accumulation of .alpha.-synuclein is toxic and impairs the
functions of mitochondria, lysosomes, and endoplasmic reticulum,
and interferes with microtubule transport.
[0011] Nonsteroidal anti-inflammatory drugs (NSAIDs) such as
ibuprofen, have been tested for their efficacy in treating a
variety of neurological diseases, but the clinical impact of NSAIDs
on neurodegenerative diseases like Parkinson's disease remains
unclear. While some studies showed that chronic NSAID use is
protective against Parkinson's disease, other studies could not
confirm the existence of a significant relationship. A recent
meta-analysis indicated that the use of non-aspirin NSAID,
particularly ibuprofen, reduces the risk of PD by 15% while the use
of aspirin did not show any effect [22].
[0012] 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 neurodegenerative disorders. There is a requirement
in the art for new methods of treating neurodegenerative 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
[0013] The inventors have developed new therapies for treating and
preventing neurodegenerative disorders. The inventors have
identified that bacterial strains from the genus Roseburia may be
effective for treating neurodegenerative diseases. As described in
the examples, administration of compositions comprising Roseburia
hominis can protect against reactive oxygen species and prevent
inflammation, thus acting as a neuroprotectant. The inventors have
also identified that treatment with Roseburia hominis can reduce
the activation of proinflammatory molecules, such as NF.kappa.B and
IL-6, by LPS and mutant .alpha.-synuclein A53T. The inventors have
identified that treatment with Roseburia hominis can reduce histone
deacetylation activity and lipid peroxidation in vitro, which can
help to reduce cell death and apoptosis. The inventors have also
identified that Roseburia hominis can produce indole that can
attenuate inflammation and oxidative stress. Furthermore, the
inventors have demonstrated that treatment with Roseburia hominis
can increase kynurenine levels.
[0014] In a first embodiment, the invention provides a composition
comprising a bacterial strain of the genus Roseburia, for use in a
method of treating or preventing a neurodegenerative disorder.
[0015] In particular embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing a disease or
condition selected from the group consisting of: Parkinson's
disease, including progressive supranuclear palsy, progressive
supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal
pressure hydrocephalus, vascular or arteriosclerotic parkinsonism
and drug-induced parkinsonism; Alzheimer's disease, including
Benson's syndrome; multiple sclerosis; Huntington's disease;
amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone
disease; prion disease; spinocerebellar ataxia; spinal muscular
atrophy; dementia, including Lewy body, vascular and frontotemporal
dementia; primary progressive aphasia; mild cognitive impairment;
HIV-related cognitive impairment and corticobasal degeneration.
[0016] In preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing Parkinson's disease,
such as environmental, familial or Parkinson's associated with
general inflammatory status. The inventors have identified that
treatment with Roseburia strains can reduce the activation of
proinflammatory molecules, such as NF.kappa.B and IL-6, by LPS and
mutant .alpha.-synuclein A53T in in vitro models of environmental
and familial Parkinson's. In preferred embodiments, the invention
provides a composition comprising a bacterial strain of the species
Roseburia hominis, for use in the treatment of Parkinson's disease.
Compositions using Roseburia hominis may be particularly effective
for treating Parkinson's.
[0017] In preferred embodiments of the invention, the bacterial
strain in the composition is of Roseburia hominis. Closely related
strains may also be used, such as bacterial strains that have a
16SrRNA 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
Roseburia hominis. 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, 2 or 3. Preferably, the sequence identity
is to SEQ ID NO:3. Preferably, the bacterial strain for use in the
invention has the 16S rRNA sequence represented by SEQ ID NO:3.
[0018] In certain embodiments, the composition of the invention is
for oral administration. Oral administration of the strains of the
invention can be effective for neurodegenerative disorders. Also,
oral administration is convenient for patients and practitioners
and allows delivery to and/or partial or total colonisation of the
intestine.
[0019] In certain embodiments, the composition of the invention
comprises one or more pharmaceutically acceptable excipients or
carriers.
[0020] 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.
[0021] In certain embodiments, the invention provides a food
product comprising the composition as described above.
[0022] In certain embodiments, the invention provides a vaccine
composition comprising the composition as described above.
[0023] Additionally, the invention provides a method of treating or
preventing neurodegenerative disorders, comprising administering a
composition comprising a bacterial strain of the genus
Roseburia.
[0024] In certain embodiments of the invention, the composition is
for use in treating brain injury. The neuroprotective activity of
the compositions of the invention and their ability to reduce
levels of histone deacetylase activity (HDAC) may make them useful
for treating brain injury. In preferred embodiments, the
compositions of the invention are for use in treating stroke, such
as treating brain injury resulting from a stroke.
[0025] In developing the above invention, the inventors have
identified and characterised a bacterial strain that is
particularly useful for therapy. The Roseburia intestinalis strain
of the invention is shown to be effective for treating cancer. The
invention also provides compositions comprising such cells, or
biologically pure cultures of such cells. The invention also
provides a cell of the Roseburia intestinalis strain deposited
under accession number NCIMB 43043, or a derivative thereof, for
use in therapy, in particular for cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0026] FIG. 1: Downregulation of IL-6 secretion
[0027] FIG. 2: Inhibition of .alpha.-synuclein induced NF.kappa.B
promoter activation
[0028] FIG. 3: Inhibition of LPS induced NF.kappa.B promoter
activation
[0029] FIG. 4: Change in antioxidant capacity
[0030] FIG. 5: Change in total anti-oxidant capacity (lipid
oxidation)
[0031] FIG. 6: Change in histone deacetylatase (HDAC) activity
[0032] FIG. 7: Level of Indole production
[0033] FIG. 8: Level of Kyrunenine production
[0034] FIG. 9: Downregulation of IL-6 secretion by MIRX001
[0035] FIGS. 10A-10B: Downregulation of IL-6 secretion by strain A:
Inhibition of IL-6 secretion in U373 (FIG. 10A); Inhibition of IL-6
secretion in U373 (FIG. 10B)
[0036] FIGS. 11A-11B: Inhibition of .alpha.-synuclein induced
NF.kappa.B promoter activation (FIG. 11A) and Inhibition of LPS
induced NF.kappa.B promoter activation by strain A (FIG. 11B)
[0037] FIGS. 12A-12B: Downregulation of IL-6 secretion by strain B:
Inhibition of IL-6 secretion in U373 (FIG. 12A); Inhibition of IL-6
secretion in U373 (FIG. 12B)
[0038] FIGS. 13A-13B: Inhibition of .alpha.-synuclein induced
NF.kappa.B promoter activation (FIG. 13A) and Inhibition of LPS
induced NF.kappa.B promoter activation by strain B (FIG. 13B)
DISCLOSURE OF THE INVENTION
[0039] Bacterial Strains
[0040] The compositions of the invention comprise a bacterial
strain of the genus Roseburia. The examples demonstrate that
bacteria of this genus are useful for treating or preventing
neurodegenerative disorders. The preferred bacterial strains are of
the species Roseburia hominis, Roseburia faecis and Roseburia
intestinalis.
[0041] Examples of Roseburia species for use in the invention
include Roseburia hominis, Roseburia cecicola, Roseburia faecis,
Roseburia intestinalis, and Roseburia inulinivorans. Roseburia
bacteria are slightly curved rod-shaped cells that are strictly
anaerobic and indigenous to the mammalian intestine. They are of
the phylogenetic cluster XIVa within the Firmicutes phylum. The
bacteria are butyrate-producing and are actively motile through
multiple flagella present along the concave side and in a cluster
at one end [23]. Roseburia hominis and Roseburia intestinalis are
recently described examples.
[0042] An example of Roseburia hominis is the strain deposited
under the terms of the Budapest Treaty at National Collections of
Industrial, Food and Marine Bacteria (NCIMB) at NCIMB Ltd, Ferguson
Building, Craibstone Estate, Bucksburn, Aberdeen, UK, AB21 9YA, on
21 Oct. 2004 by the Rowett Research Institute under the accession
number NCIMB 14029.sup.T Roseburia hominis
A2-183.sup.T(DSM=16839.sup.T). Other exemplary Roseburia hominis
strains are described in [24]. GenBank/EMBL/DDBJ accession numbers
for the 16S rRNA gene sequence of strains of Roseburia hominis are
AY804148 and AJ270482 (disclosed herein as SEQ ID NO:1 and SEQ ID
NO:2).
[0043] An example of Roseburia intestinalis is the strain deposited
under the accession number NCIMB 13810 Roseburia intestinalis
L1-82.sup.T(DSM=14610.sup.T). Another example is the Roseburia
intestinalis strain as described in [24]. Reference [24] also
describes exemplary Roseburia faecis and Roseburia inulinivorans
strains.
[0044] The Roseburia hominis bacterium deposited under accession
number NCIMB 42383 was tested in the Examples and is also referred
to herein as strain 433. A 16S rRNA sequence for the 433 strain
that was tested is provided in SEQ ID NO:3. Strain 433 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 "Roseburia hominis 433" and was
assigned accession number NCIMB 42383. GT Biologics Ltd.
subsequently changed its name to 4D Pharma Research Limited.
[0045] WO 2016/203221 describes administration of strain 433 to
mice and shows that it can affect disease processes outside of the
gut (such as asthma and arthritis). Strain 433 also affects disease
processes outside of the gut in the treatment of neurodegenerative
disorders described herein.
[0046] A genome sequence for strain 433 is provided in SEQ ID NO:4.
This sequence was generated using the PacBio RS II platform.
[0047] The Roseburia intestinalis bacterium deposited under
accession number NCIMB 43043 was tested in the Examples and is also
referred to herein as strain A. A 16S rRNA sequence for strain A
that was tested is provided in SEQ ID NO:5. Strain A was deposited
with the international depositary authority NCIMB, Ltd. (Ferguson
Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research
Limited (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,
Scotland) on 3 May 2018 as "Roseburia intestinalis" and was
assigned accession number NCIMB 43043.
[0048] The examples also describe a Roseburia faecis bacterium that
was tested and referred to as strain B. A 16S rRNA sequence for
strain B that was tested is provided in SEQ ID NO:6.
[0049] In certain embodiments, the bacterial strain for use in the
invention is Roseburia hominis. In certain embodiments, the
bacterial strain for use in the invention is Roseburia
intestinalis. In certain embodiments, the bacterial strain for use
in the invention is Roseburia faecis.
[0050] Bacterial strains closely related to the strain tested in
the examples are also expected to be effective for treating or
preventing neurodegenerative disease diseases and conditions
mediated by IL-17 or the Th17 pathway. 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 Roseburia
hominis. 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, 2 or 3. Preferably, the
sequence identity is to SEQ ID NO:3. Preferably, the bacterial
strain for use in the invention has the 16S rRNA sequence
represented by SEQ ID NO:3.
[0051] In other 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 Roseburia intestinalis. 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:5. Preferably, the bacterial strain for use in the
invention has the 16S rRNA sequence represented by SEQ ID NO:5.
[0052] In other 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 Roseburia faecis. 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:6. Preferably, the bacterial strain for use in the invention has
the 16S rRNA sequence represented by SEQ ID NO:6.
[0053] Bacterial strains that are biotypes of the bacterium
deposited under accession number 42383 or NCIMB 43043 are also
expected to be effective for treating or preventing
neurodegenerative disorders. A biotype is a closely related strain
that has the same or very similar physiological and biochemical
characteristics.
[0054] Strains that are biotypes of the bacterium deposited under
accession number NCIIB 42383 or NCIMB 43043 and that are suitable
for use in the invention may be identified by sequencing other
nucleotide sequences for the bacterium deposited under accession
number NCIIB 42383 or NCIMB 43043. 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).
Other suitable sequences for use in identifying biotype strains may
include hsp60 or repetitive sequences such as BOX, ERIC,
(GTG).sub.5 (SEQ ID NO: 7), or REP or [25]. 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 NCIIB 42383 or NCIMB
43043.
[0055] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:4. In
preferred embodiments, the bacterial strain for use in the
invention has a genome 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:4 across at least 60% (e.g. at least 65%, 70%, 75%,
80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:4. For
example, the bacterial strain for use in the invention may have a
genome with at least 90% sequence identity to SEQ ID NO:4 across
70% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID
NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identity
to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequence
identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95%
sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at
least 95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID
NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 90%
of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4
across 100% of SEQ ID NO:4, or at least 98% sequence identity to
SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 98% sequence
identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 98%
sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at
least 98% sequence identity to SEQ ID NO:4 across 100% of SEQ ID
NO:4.
[0056] Alternatively, strains that are biotypes of the bacterium
deposited under accession number NCIMB 42383 or NCIMB 43043 and
that are suitable for use in the invention may be identified by
using the accession number NCIMB 42383 or NCIMB 43043 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
Roseburia hominis, Roseburia faecis and Roseburia intestinalis
strains.
[0057] In certain embodiments, strains that are biotypes of the
bacterium deposited under accession number NCIMB 42383 or NCIMB
43043 and that are suitable for use in the invention are strains
that provide the same pattern as the bacterium deposited under
accession number NCIMB 42383 or NCIMB 43043 when analysed by
amplified ribosomal DNA restriction analysis (ARDRA), for example
when using Sau3AI restriction enzyme (for exemplary methods and
guidance see, for example, [25]).
[0058] Alternatively, biotype strains are identified as strains
that have the same carbohydrate fermentation patterns as the
bacterium deposited under accession number NCIMB 42383 or NCIMB
43043. Other Roseburia strains that are useful in the compositions
and methods of the invention, such as biotypes of the bacteria
deposited under accession number NCIMB 42383 or NCIMB 43043, 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 with neuroblastoma
cells and then assessing cytokine levels and levels of
neuroprotection or neuroproliferation. In particular, bacterial
strains that have similar growth patterns, metabolic type and/or
surface antigens to the bacterium deposited under accession number
NCIMB 42383 or NCIMB 43043 may be useful in the invention. A useful
strain will have comparable immune modulatory activity to the NCIMB
42383 or NCIMB 43043 strain. In particular, a biotype strain will
elicit comparable effects on the neurodegenerative disease 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.
[0059] A particularly preferred strain of the invention is the
Roseburia hominis strain deposited under accession number NCIMB
42383. This is the exemplary 433 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
Roseburia hominis strain deposited under accession number NCIMB
42383, or a derivative thereof. The invention also provides a
composition comprising a cell of the Roseburia hominis strain
deposited under accession number NCIMB 42383, or a derivative
thereof. The invention also provides a biologically pure culture of
the Roseburia hominis strain deposited under accession number NCIMB
42383. The invention also provides a cell of the Roseburia hominis
strain deposited under accession number NCIMB 42383, or a
derivative thereof, for use in therapy, in particular for the
diseases described herein.
[0060] A particularly preferred strain of the invention is the
Roseburia intestinalis strain deposited under accession number
NCIMB 43043. This is the exemplary strain A 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
Roseburia intestinalis strain deposited under accession number
NCIMB 43043, or a derivative thereof. The invention also provides a
composition comprising a cell of the Roseburia intestinalis strain
deposited under accession number NCIMB 43043, or a derivative
thereof. The invention also provides a biologically pure culture of
the Roseburia intestinalis strain deposited under accession number
NCIMB 43043. The invention also provides a cell of the Roseburia
intestinalis strain deposited under accession number NCIMB 43043,
or a derivative thereof, for use in therapy, in particular for the
diseases described herein.
[0061] A derivative of the strain deposited under accession number
NCIMB 42383 or NCIMB 43043 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 42383 or NCIMB 43043 strain. In particular, a
derivative strain will elicit comparable effects on the
neurodegenerative disease 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 42383 or NCIMB
43043 strain will generally be a biotype of the NCIMB 42383 or
NCIMB 43043 strain.
[0062] References to cells of the Roseburia hominis strain
deposited under accession number NCIMB 42383 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 42383, and
such cells are encompassed by the invention.
[0063] References to cells of the Roseburia intestinalis strain
deposited under accession number NCIMB 43043 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 43043, and
such cells are encompassed by the invention.
[0064] In preferred embodiments, the bacterial strains in the
compositions of the invention are viable and capable of partially
or totally colonising the intestine.
[0065] The invention further provides a cell, such as an isolated
cell, of the Enterococcus gallinarum strain deposited under
accession number NCIMB 42761, or a derivative thereof. The
invention also provides a composition comprising a cell of the
Roseburia intestinalis strain deposited under accession number
NCIMB 42761, or a derivative thereof. The invention also provides a
biologically pure culture of the Roseburia intestinalis strain
deposited under accession number NCIMB 43043. The invention also
provides a cell of the Roseburia intestinalis strain deposited
under accession number NCIMB 43043, or a derivative thereof, for
use in therapy, in particular for the diseases described herein. A
derivative of the strain deposited under accession number NCIMB
43043 may be a daughter strain (progeny) or a strain cultured
(subcloned) from the original.
[0066] 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 42761 strain. In
particular, a derivative strain will elicit comparable effects on
the neurodegenerative disease models 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 43043 strain will generally be a biotype of the NCIMB
43043 strain.
[0067] References to cells of the Roseburia intestinalis strain
deposited under accession number NCIMB 43043 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strain deposited under accession number NCIMB 43043, and
such cells are encompassed by the invention. Thus, in some
embodiments, reference to cells of the Roseburia intestinalis
strain deposited under accession number NCIMB 43043 refers only to
the strain A deposited under NCIMB 43043 and does not refer to a
bacterial strain that was not deposited under NCIMB 43043.
[0068] In preferred embodiments, the bacterial strain in the
compositions of the invention is viable and capable of partially or
totally colonising the intestine.
[0069] Therapeutic Uses
[0070] As demonstrated in the examples, the bacterial compositions
of the invention are effective for treating neurodegenerative
disorders. In particular, treatment with compositions of the
invention increase neuroproliferation and act as a neuroprotectant
against agents that destroy dopaminergic neurons. Therefore, the
compositions of the invention may be useful for treating or
preventing neurodegenerative disorders that are the result of
neuron death.
[0071] Compositions of the invention can decrease the activation of
the NF.kappa.B promoter, which activates cytokine production, for
example IL-1.beta., IL-1.alpha., IL-18, TNF.alpha. and IL-6.
Treating cells with mutant .alpha.-synuclein is a model for
familial Parkinson's. A point mutation at position 53 from adenine
to threonine leads to .alpha.-synuclein mis-folding. The
incorrectly folded .alpha.-synuclein subsequently aggregates into
insoluble fibrils which form Lewy bodies. Therefore, the
compositions of the invention may be useful for treating or
preventing neurodegenerative disorders that are the result of
neuroinflammation, protein misfolding and/or environmental
exposure. Compositions of the invention can be used for treatment
of familial Parkinson's. Activation of the NF.kappa.B promoter is
mediated through the TLR4 ligand. TL4 is known to mediate cell
death in the mouse model MPTP, which simulates Parkinson's disease.
Compositions of the invention can be used to inhibit the ability of
TLR4 signalling to activate the NF.kappa.B promoter. Of particular
relevance for PD, both TLR2 and TLR4 were found to be upregulated
in brains of PD patients [26]. Moreover .alpha.-syn has been
described as a ligand for TLR2 [27] and we have demonstrated that
.alpha.-syn is also a ligand for TLR4 using HEK-TLR4 cells.
[0072] Compositions of the invention decrease the secretion of
pro-inflammatory cytokines such as IL-6, which can be induced by
lipopolysaccharide (LPS). Treatment of cells with LPS simulates
Parkinson's caused by environmental factors. Compositions of the
invention can be used to decrease IL-6 secretion. Compositions of
the invention can be used for treatment of environmental
Parkinson's.
[0073] Examples of neurodegenerative diseases to be treated by
compositions of the invention include: Parkinson's disease,
including progressive supranuclear palsy, progressive supranuclear
palsy, Steele-Richardson-Olszewski syndrome, normal pressure
hydrocephalus, vascular or arteriosclerotic parkinsonism and
drug-induced parkinsonism; Alzheimer's disease, including Benson's
syndrome; multiple sclerosis; Huntington's disease; amyotrophic
lateral sclerosis; Lou Gehrig's disease; motor neurone disease;
prion disease; spinocerebellar ataxia; spinal muscular atrophy;
dementia, including Lewy body, vascular and frontotemporal
dementia; primary progressive aphasia; mild cognitive impairment;
HIV-related cognitive impairment and corticobasal degeneration. A
further neurodegenerative diseases to be treated by compositions of
the invention is progressive inflammatory neuropathy.
[0074] In certain embodiments, the compositions of the invention
can be effective for treating neurodegenerative disorders that
occur in elderly patients. The examples show that compositions of
the invention can treat Parkinson's disease which is predominantly
diagnosed in patients aged over 65 years old. In preferred
embodiments, the compositions of the invention are for treating
patients 65 years or older. In other certain embodiments, the
patients are between 40 to 65 years old. In other embodiments, the
patients are older than 40 years. In certain embodiments, the
compositions of the invention are for use in treating a disease
associated with old age, for example, a disease diagnosed after 50
years of age.
[0075] In certain embodiments, the compositions of the invention
are for use in treating a neurodegenerative disorder mediated or
characterised by the accumulation of protein, in particular
mis-folded protein.
[0076] In certain embodiments, the compositions of the invention
are for use in treating a neurodegenerative disorder associated
with grey matter neuronal loss. In certain embodiments, the
compositions of the invention are for treating a neurodegenerative
disorder that is not associated with white matter lesions.
[0077] In certain embodiments, the compositions of the invention
are for use in treating a neurodegenerative disorder associated
with permanent symptoms.
[0078] In certain embodiments, the compositions of the invention
are for use in treating a neurodegenerative disorder that is not an
auto-immune disorder. In certain embodiments, the compositions of
the invention are for use in treating a neurodegenerative disorder
that is not multiple sclerosis.
[0079] In certain embodiments, the compositions of the invention
are for use in reducing neuron death, in particular, in the
treatment of neurodegenerative disorders. In certain embodiments,
the compositions of the invention are for use in protecting
neurons, in particular in the treatment of neurodegenerative
disorders.
[0080] The neuroprotective properties of the compositions of the
invention, as shown in the examples, mean that the compositions may
be particularly effective for preventing or delaying onset or
progression of neurodegenerative disorders. In certain embodiments,
the compositions of the invention are for use in delaying onset or
progression of a neurodegenerative disorders.
[0081] Modulation of the Microbiota-Gut-Brain Axis
[0082] 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 [17], [20],
[28].
[0083] 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.
[0084] As demonstrated in the examples, the compositions of the
present invention can modulate the microbiota-gut-brain axis and
reduce cell death associated with neurodegenerative disorders.
Accordingly, the compositions of the invention may be useful for
treating or preventing neurodegenerative disorders, in particular
those disorders and conditions associated with dysfunction of the
microbiota-gut-brain axis.
[0085] 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: Parkinson's disease,
including progressive supranuclear palsy, progressive supranuclear
palsy, Steele-Richardson-Olszewski syndrome, normal pressure
hydrocephalus, vascular or arteriosclerotic parkinsonism and
drug-induced parkinsonism; Alzheimer's disease, including Benson's
syndrome; multiple sclerosis; Huntington's disease; amyotrophic
lateral sclerosis; Lou Gehrig's disease; motor neurone disease;
prion disease; spinocerebellar ataxia; spinal muscular atrophy;
dementia; including Lewy body; vascular and frontotemporal
dementia; primary progressive aphasia; mild cognitive impairment;
HIV-related cognitive impairment and corticobasal degeneration.
[0086] 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 Levodopa, dopamine agonists, MAO-B
inhibitors, COMT inhibitors, Glutamate antagonists, and/or
anticholinergics), and/or treating or preventing the tissue damage
and symptoms associated with dysfunction of the
microbiota-gut-brain axis.
[0087] 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.
[0088] 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. In certain embodiments, the compositions
of the invention modulate the dopaminergic system.
[0089] 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.
[0090] Neurodegenerative Diseases
[0091] Tauopathies are neurodegenerative diseases associated with
the pathological aggregation of tau protein in neurofibrillary or
gliofibrillary tangles in the human brain. Alzheimer's disease is
an example of a tauopathology. Synucleinopathies (also called
.alpha.-Synucleinopathies) are neurodegenerative diseases
characterised by the abnormal accumulation of aggregates of
.alpha.-synuclein in neurons, nerve fibres or glial cells.
Parkinson's disease is an example of a synucleinopathology.
[0092] There is clinical and pathological overlap between these two
pathologies. Parkinson's disease patients frequently have dementia
and Alzheimer's disease patients often manifest parkinsonism [29].
For example, progressive supranuclear palsy (also known as
Steele-Richardson-Olszewski syndrome) has a tauopathology, but also
leads to prominent parkinsonism [30]. Mutations in LRRK2 known to
cause parkinsonism are associated with the accumulation of
synuclein, tau, neither, or both proteins [31].
[0093] Lewy body disease (LBD) is a neurodegenerative disease that
is one of the most common causes of dementia in the elderly. LBD
exemplifies the existence of a continuum between tau- and
synuclein-pathologies. LBD shares clinical and pathological
features with Parkinson disease, Parkinson disease dementia and
Alzheimer disease [29].
[0094] In particular embodiments, the compositions of the invention
may be useful for treating or preventing tauopathies and/or
synucleinopathies. In particular embodiments, the compositions of
the invention may be useful for treating or preventing tauopathies.
In particular embodiments, the compositions of the invention may be
useful for treating or preventing synucleinopathies. In certain
embodiments, the compositions of the invention may be useful for
treating or preventing a disease or condition selected from the
group consisting of: Parkinson's disease, including progressive
supranuclear palsy, progressive supranuclear palsy,
Steele-Richardson-Olszewski syndrome, normal pressure
hydrocephalus, vascular or arteriosclerotic parkinsonism and
drug-induced parkinsonism; Alzheimer's disease, including Benson's
syndrome; and dementia; including Lewy body; vascular and
frontotemporal dementia.
[0095] In preferred embodiments, the compositions of the invention
may be useful for treating or preventing Parkinson's disease,
including progressive supranuclear palsy, progressive supranuclear
palsy, Steele-Richardson-Olszewski syndrome, normal pressure
hydrocephalus, vascular or arteriosclerotic parkinsonism and
drug-induced parkinsonism. In preferred embodiments, the
compositions of the invention may be useful for treating or
preventing Alzheimer's disease, including Benson's syndrome. In
further preferred embodiments, the compositions of the invention
may be useful for treating or preventing dementia; including Lewy
body; vascular and frontotemporal dementia.
[0096] Parkinson's Disease
[0097] 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.
Parkinson's disease is a neurodegenerative diseases 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.
[0098] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing Parkinson's disease.
Compositions comprising a bacterial strain of the genus Roseburia
may improve motor and cognitive functions in models of Parkinson's
disease. Treatment with Roseburia 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.
[0099] In preferred embodiments, the invention provides a
composition comprising a bacterial strain of the species Roseburia
hominis for use in a method of treating or preventing Parkinson's
disease. Compositions using Roseburia hominis may be particularly
effective for treating Parkinson's disease. 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.
[0100] 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.
[0101] In other preferred embodiments, the compositions of the
invention prevent, reduce or alleviate cognitive dysfunctions
comorbid with Parkinson's disease.
[0102] 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.
[0103] 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.
[0104] 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).
[0105] 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.
[0106] Alzheimer's Disease and Dementia
[0107] 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.
[0108] Alzheimer's disease and dementia are also characterised by
neuronal loss, so the neuroprotective and neuroproliferative
effects shown in the examples for the compositions of the invention
indicate that they may be useful for treating or preventing these
conditions.
[0109] 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 VIDD or schizophrenia).
[0110] In addition to the primary symptom, subjects with
neurodegenerative disorders display behavioural and psychiatric
symptoms including agitation, aggression, depression, anxiety,
apathy, psychosis and sleep-wake cycle disturbances.
[0111] Neurodegenerative disorders 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 neurodegenerative disorders in a
subject. In preferred embodiments, the neurodegenerative disorder
is Alzheimer's disease. In other embodiments, the neurodegenerative
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.
[0112] In preferred embodiments, the compositions of the invention
prevent, reduce or alleviate one or more of the symptoms of
neurodegenerative 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 neurodegenerative 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
neurodegenerative disorders selected from agitation, aggression,
depression, anxiety, apathy, psychosis and sleep-wake cycle
disturbances.
[0113] 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 neurodegenerative
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 neurodegenerative disorder to
undertake everyday activities.
[0114] 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 improves 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.
[0115] In some embodiments, the compositions of the invention
improve the symptoms of neurodegenerative disorders according to a
symptomatic or diagnostic test. In certain embodiments, the tests
for assessing symptomatic improvement of Alzheimer's disease (and
other neurodegenerative 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 neurodegenerative
disorders.
[0116] 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).
[0117] 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).
[0118] 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).
[0119] In certain embodiments, the tests assessing behavioural and
psychiatric symptoms of neurodegenerative 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).
[0120] In some embodiments, the compositions of the invention are
particularly effective at preventing, reducing or alleviating
neurodegenerative disorders when used in combination with another
therapy for treating neurodegenerative disorders. In certain
embodiments, such therapies include acetylcholinesterase inhibitors
including donepezil (Aricept.RTM.), galantamine (Razadyne.RTM.) and
rivastigmine (Exelon.RTM.), and memantine.
[0121] Neurochemical Factors, Neuropeptides and Neurotransmitters
and the Microbiota-Gut-Brain Axis
[0122] 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 neurodegenerative diseases. The experiments performed by
the inventors indicate that administration of Roseburia species,
and in particular Roseburia hominis, can modulate levels of indole
and kynurenine. Dysregulation of these metabolites can lead to
neurodegenerative diseases, such as Parkinson's disease.
[0123] In certain embodiments, the compositions of the invention
modulate the levels of brain monoamines and metabolites thereof. In
preferred embodiments the metabolite is kynurenine. In certain
embodiments, the compositions of the invention modulate kynurenine,
which is the main route of tryptophan metabolism, which serves as a
route to nicotinamide adenine dinucleotide (NAD+) production.
Kynurenine can be metabolized to neuroactive compounds such as
kynurenic acid (KYNA) and 3-hydroxy-1-kynurenine (3-OH-1-KYN), and
in further steps to quinolinic acid (QUIN). Dysregulation of the
kynurenine pathway can lead to activation of the immune system and
the accumulation of potentially neurotoxic compounds. Alterations
in the kynurenine metabolism may be involved in the development of
Parkinson's diseases. Kynurenine levels have been demonstrated to
be decreased in the frontal cortex, putamen and substantia nigra
pars compacta of patients with PD [32]. Therefore, in certain
embodiments the compositions of the invention are for use in
increasing the levels of kynurenine in the treatment of Parkinson's
disease.
[0124] In certain embodiments of the invention the compositions of
the invention can increase the levels of kynurenin. Increased
levels of kynurenine have been shown to attenuated IPP+-induced
neuronal cell death in vitro in a human dopaminergic neuroblastoma
cell line [33]. In certain embodiments kynurenine and kynurenic
acid, can activate GI aryl hydrocarbon receptor (Ahr) and GPR35
receptors. Activation of Ahr receptor induces IL-22 production,
which can inhibit local inflammation. Activation of GPR35 inducing
the production of inositol triphosphate and Ca2+ mobilization.
[0125] In certain embodiments, the compositions of the invention
modulate the levels of indole. In preferred embodiments the
metabolite is kynurenine. In certain embodiments, the compositions
of the invention modulate kynurenine, which is the main route of
tryptophan metabolism.
[0126] 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.
[0127] 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.
[0128] Immune Response
[0129] 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 decrease IL-6 production and secretion. In certain
embodiments, the compositions of the invention decrease the
activation of the NF.kappa.B promoter. In certain embodiments, the
compositions of the invention are able to modulate the activation
of IL-6 production by the potent pro-inflammatory endotoxin
lipopolysaccharide (LPS). In certain embodiments, the compositions
of the invention are able to modulate the activation of the
NF.kappa.B promoter by LPS and .alpha.-synuclein mutant proteins
such as A53T. Increased circulating levels of cytokines are closely
associated with various neurodegenerative disorders, including
Parkinson's, dementia and Alzheimer's. In certain embodiments, the
compositions of the invention are for use in reducing IL-6 levels
and/or NF.kappa.B levels in the treatment of a neurodegenerative
disorder.
[0130] The signalling of the microbiota-gut-brain axis is modulated
by levels of 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.
[0131] Histone acetylation and deacetylation are important
epigenetic regulators of gene expression. An imbalance in histone
acetylation and deacetylation can result in apoptosis.
Dysregulation of such histone acetyltransferases has been
implicated in the pathogenesis associated with age-associated
neurodegenerative diseases, such as Parkinson's disease,
Huntington's disease, Alzheimer's disease, amyotrophic lateral
sclerosis and cognitive decline [34]. Accordingly, in certain
embodiments, the compositions of the invention can modulate histone
deacetylatase activity. In certain embodiments, the compositions of
the invention can reduce histone deacetylatase activity. In certain
embodiments, the compositions of the invention can reduce histone
acetylatase activity.
[0132] Patients with neurodegenerative diseases, including
Parkinson's disease, Huntington's disease, Alzheimer's disease and
amyotrophic lateral sclerosis, exhibit high levels of lipid
peroxidation. Lipid are vulnerable to oxidation by reactive oxygen
species, and the brain is rich in polyunsaturated fatty acids.
Accordingly, in certain embodiments, the compositions of the
invention can modulate lipid peroxidation. In certain embodiments,
the compositions of the invention can reduce lipid peroxidation.
Reducing the oxidative damage caused by reactive oxygen species can
be used to target early the stages neurodegenerative diseases.
Accordingly, in certain embodiments, the compositions of the
invention are for use in treating early stage neurodegeneration.
Also accordingly, in certain embodiments, the compositions of the
invention are for use in preventing the development of a
neurodegenerative disorder. In such embodiments, the compositions
of the invention may be for use in a patient that has been
identified as at risk of developing a neurodegenerative
disorder.
[0133] The signalling of the microbiota-gut-brain axis is modulated
by levels of 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.
[0134] 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.
[0135] Brain Injury
[0136] The examples demonstrate that the compositions of the
invention are neuroprotective and have HDAC inhibitory activity.
HDAC2 is a crucial target for functional recovery from stroke [35]
and HDAC inhibition can prevent white matter injury [36], so the
compositions of the invention may be useful in the treatment of
brain injury.
[0137] In certain embodiments, the compositions of the invention
are for use in treating brain injury. In some embodiments, the
brain injury is a traumatic brain injury. In some embodiments, the
brain injury is an acquired brain injury. In some embodiments, the
compositions of the invention are for use in treating brain injury
resulting from trauma. In some embodiments, the compositions of the
invention are for use in treating brain injury resulting from a
tumour. In some embodiments, the compositions of the invention are
for use in treating brain injury resulting from a stroke. In some
embodiments, the compositions of the invention are for use in
treating brain injury resulting from a brain haemorrhage. In some
embodiments, the compositions of the invention are for use in
treating brain injury resulting from encephalitis. In some
embodiments, the compositions of the invention are for use in
treating brain injury resulting from cerebral hypoxia. In some
embodiments, the compositions of the invention are for use in
treating brain injury resulting from cerebral anoxia.
[0138] In preferred embodiments, the compositions of the invention
are for use in treating stroke. The effects shown in the examples
are particularly relevant to the treatment of stroke. Stroke occurs
when blood flow to at least a part of the brain is interrupted.
Without an adequate supply of blood to provide oxygen and nutrients
to the brain tissue and to remove waste products from the brain
tissue, brain cells rapidly begin to die. The symptoms of stroke
are dependent on the region of the brain which is affected by the
inadequate blood flow. Symptoms include paralysis, numbness or
weakness of the muscles, loss of balance, dizziness, sudden severe
headaches, speech impairment, loss of memory, loss of reasoning
ability, sudden confusion, vision impairment, coma or even death. A
stroke is also referred to as a brain attack or a cerebrovascular
accident (CVA). The symptoms of stroke may be brief if adequate
blood flow is restored within a short period of time. However, if
inadequate blood flow continues for a significant period of time,
the symptoms can be permanent.
[0139] In some embodiments, the stroke is cerebral ischemia.
Cerebral ischemia results when there is insufficient blood flow to
the tissues of the brain to meet metabolic demand. In some
embodiments, the cerebral ischemia is focal cerebral ischemia, i.e.
confined to a specific region of the brain. In some embodiments the
cerebral ischemia is global cerebral ischemia, i.e. encompassing a
wide area of the brain tissue. Focal cerebral ischemia commonly
occurs when a cerebral vessel has become blocked, either partially
or completely, reducing the flow of blood to a specific region of
the brain. In some embodiments the focal cerebral ischemia is
ischemic stroke. In some embodiments, the ischemic stroke is
thrombotic, i.e. caused by a thrombus or blood clot, which develops
in a cerebral vessel and restricts or blocks blood flow. In some
embodiments the ischemic stroke is a thrombotic stroke. In some
embodiments, the ischemic stroke is embolic, i.e. caused by an
embolus, or an unattached mass that travels through the bloodstream
and restricts or blocks blood flow at a site distant from its point
of origin. In some embodiments the ischemic stroke is an embolic
stroke. Global cerebral ischemia commonly occurs when blood flow to
the brain as a whole is blocked or reduced. In some embodiments the
global cerebral ischemia is caused by hypoperfusion, i.e. due to
shock. In some embodiments the global cerebral ischemia is a result
of a cardiac arrest.
[0140] In some embodiments the subject diagnosed with brain injury
has suffered cerebral ischemia. In some embodiments, the subject
diagnosed with brain injury has suffered focal cerebral ischemia.
In some embodiments, the subject diagnosed with brain injury has
suffered an ischemic stroke. In some embodiments, the subject
diagnosed with brain injury has suffered a thrombotic stroke. In
some embodiments, the subject diagnosed with brain injury has
suffered an embolic stroke. In some embodiments, the subject
diagnosed with brain injury has suffered global cerebral ischemia.
In some embodiments, the subject diagnosed with brain injury has
suffered hypoperfusion. In some embodiments, the subject diagnosed
with brain injury has suffered a cardiac arrest.
[0141] In some embodiments, the compositions of the invention are
for use in treating cerebral ischemia. In some embodiments, the
compositions of the invention are for use in treating focal
cerebral ischemia. In some embodiments, the compositions of the
invention are for use treating ischemic stroke. In some
embodiments, the compositions of the invention are for use in
treating thrombotic stroke. In some embodiments, the compositions
of the invention are for use in treating embolic stroke. In some
embodiments, the compositions of the invention are for use in
treating global cerebral ischemia. In some embodiments, the
compositions of the invention are for use in treating
hypoperfusion.
[0142] In some embodiments, the stroke is hemorrhagic stroke.
Hemorrhagic stroke is caused by bleeding into or around the brain
resulting in swelling, pressure and damage to the cells and tissues
of the brain. Hemorrhagic stroke is commonly a result of a weakened
blood vessel that ruptures and bleeds into the surrounding brain.
In some embodiments, the hemorrhagic stroke is an intracerebral
hemorrhage, i.e. caused by bleeding within the brain tissue itself.
In some embodiments the intracerebral hemorrhage is caused by an
intraparenchymal hemorrhage. In some embodiments the intracerebral
hemorrhage is caused by an intraventricular hemorrhage. In some
embodiments the hemorrhagic stroke is a subarachnoid hemorrhage
i.e. bleeding that occurs outside of the brain tissue but still
within the skull. In some embodiments, the hemorrhagic stroke is a
result of cerebral amyloid angiopathy. In some embodiments, the
hemorrhagic stroke is a result of a brain aneurysm. In some
embodiments, the hemorrhagic stroke is a result of cerebral
arteriovenous malformation (AVM).
[0143] In some embodiments the subject diagnosed with brain injury
has suffered hemorrhagic stroke. In some embodiments, the subject
diagnosed with brain injury has suffered an intracerebral
hemorrhage. In some embodiments, the subject diagnosed with brain
injury has suffered an intraparenchymal hemorrhage. In some
embodiments, the subject diagnosed with brain injury has suffered
an intraventricular hemorrhage. In some embodiments, the subject
diagnosed with brain injury has suffered a subarachnoid hemorrhage.
In some embodiments, the subject diagnosed with brain injury has
suffered cerebral amyloid angiopathy. In some embodiments, the
subject diagnosed with brain injury has suffered a brain aneurysm.
In some embodiments, the subject diagnosed with brain injury has
suffered cerebral AVM.
[0144] In some embodiments, the compositions of the invention are
for use in treating hemorrhagic stroke. In some embodiments, the
compositions of the invention are for use in treating an
intracerebral hemorrhage. In some embodiments, the compositions of
the invention are for use in treating an intraparenchymal
hemorrhage. In some embodiments, the compositions of the invention
are for use in treating an intraventricular hemorrhage. In some
embodiments, the compositions of the invention are for use in
treating a subarachnoid hemorrhage. In some embodiments, the
compositions of the invention are for use in treating a cerebral
amyloid angiopathy. In some embodiments, the compositions of the
invention are for use in treating a brain aneurysm. In some
embodiments, the compositions of the invention are for use in
treating cerebral AVM.
[0145] Restoration of adequate blood flow to the brain after a
period of interruption, though effective in alleviating the
symptoms associated with stroke, can paradoxically result in
further damage to the brain tissue. During the period of
interruption, the affected tissue suffers from a lack of oxygen and
nutrients, and the sudden restoration of blood flow can result in
inflammation and oxidative damage through the induction of
oxidative stress. This is known as reperfusion injury, and is well
documented not only following stroke, but also following a heart
attack or other tissue damage when blood supply returns to the
tissue after a period of ischemia or lack of oxygen. In some
embodiments the subject diagnosed with brain injury has suffered
from reperfusion injury as a result of stroke. In some embodiments,
the compositions of the invention are for use in treating
reperfusion injury as a result of stroke.
[0146] A transient ischemic attack (TIA), often referred to as a
mini-stroke, is a recognised warning sign for a more serious
stroke. Subjects who have suffered one or more TIAs are therefore
at greater risk of stroke. In some embodiments the subject
diagnosed with brain injury has suffered a TIA. In some
embodiments, the compositions of the invention are for use in
treating a TIA. In some embodiments, the compositions of the
invention are for use in treating brain injury in a subject who has
suffered a TIA.
[0147] High blood pressure, high blood cholesterol, a familial
history of stroke, heart disease, diabetes, brain aneurysms,
arteriovenous malformations, sickle cell disease, vasculitis,
bleeding disorders, use of nonsteroidal anti-inflammatory drugs
(NSAIDs), smoking tobacco, drinking large amounts of alcohol,
illegal drug use, obesity, lack of physical activity and an
unhealthy diet are all considered to be risk factors for stroke. In
particular, lowering blood pressure has been conclusively shown to
prevent both ischemic and hemorrhagic strokes [37, 38]. In some
embodiments, the compositions of the invention are for use in
treating brain injury in a subject who has at least one risk factor
for stroke. In some embodiments the subject has two risk factors
for stroke. In some embodiments the subject has three risk factors
for stroke. In some embodiments the subject has four risk factors
for stroke. In some embodiments the subject has more than four risk
factors for stroke. In some embodiments the subject has high blood
pressure. In some embodiments the subject has high blood
cholesterol. In some embodiments the subject has a familial history
of stroke. In some embodiments the subject has heart disease. In
some embodiments the subject has diabetes. In some embodiments the
subject has a brain aneurysm. In some embodiments the subject has
arteriovenous malformations. In some embodiments the subject has
vasculitis. In some embodiments the subject has sickle cell
disease. In some embodiments the subject has a bleeding disorder.
In some embodiments the subject has a history of use of
nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments
the subject smokes tobacco. In some embodiments the subject drinks
large amounts of alcohol. In some embodiments the subject uses
illegal drugs. In some embodiments the subject is obese. In some
embodiments the subject is overweight. In some embodiments the
subject has a lack of physical activity. In some embodiments the
subject has an unhealthy diet.
[0148] The examples indicate that the compositions of the invention
may be useful for treating brain injury and aiding recovery when
administered before the injury event occurs. Therefore, the
compositions of the invention may be particularly useful for
treating brain injury when administered to subjects at risk of
brain injury, such as stroke.
[0149] In certain embodiments, the compositions of the invention
are for use in reducing the damage caused by a potential brain
injury, preferably a stroke. The compositions may reduce the damage
caused when they are administered before the potential brain injury
occurs, in particular when administered to a patient identified as
at risk of a brain injury.
[0150] The examples indicate that the compositions of the invention
may be useful for treating brain injury and aiding recovery when
administered after the injury event occurs. Therefore, the
compositions of the invention may be particularly useful for
treating brain injury when administered to subjects following a
brain injury, such as stroke.
[0151] In some embodiments, the compositions of the invention treat
brain injury by reducing motoric damage. In some embodiments, the
compositions of the invention treat brain injury by improving motor
function. In some embodiments, the compositions of the invention
treat brain injury by improving muscle strength. In some
embodiments, the compositions of the invention treat brain injury
by improving memory. In some embodiments, the compositions of the
invention treat brain injury by improving social recognition. In
some embodiments, the compositions of the invention treat brain
injury by improving neurological function.
[0152] Treatment of brain injury may refer to, for example, an
alleviation of the severity of symptoms. Treatment of brain injury
may also refer to reducing the neurological impairments following
stroke. Compositions of the invention for use in treating stroke
may be provided to the subject in advance of the onset of stroke,
for example in a patient identified as being at risk of stroke.
Compositions of the invention for use in treating stroke may be
provided after a stroke has occurred, for example, during recovery.
Compositions of the invention for use in treating stroke may be
provided during the acute phase of recovery (i.e. up to one week
after stroke). Compositions of the invention for use in treating
stroke may be provided during the subacute phase of recovery (i.e.
from one week up to three months after stroke). Compositions of the
invention for use in treating stroke may be provided during the
chronic phase of recovery (from three months after stroke).
[0153] In certain embodiments, the compositions of the invention
are for use in combination with a secondary active agent. In
certain embodiments, the compositions of the invention are for use
in combination with aspirin or tissue plasminogen activator (tPA).
Other secondary agents include other antiplatelets (such as
clopidogrel), anticoagulants (such as heparins, warfarin, apixaban,
dabigatran, edoxaban or rivaroxaban), antihypertensives (such as
diuretics, ACE inhibitors, calcium channel blockers, beta-blockers
or alpha-blockers) or statins. The compositions of the invention
may improve the patient's response to the secondary active
agent.
[0154] In certain embodiments, the compositions of the invention
reduce the effect of ischemia on tissues. In certain embodiments,
the compositions of the invention reduce the amount of damage to
tissues caused by ischemia. In certain embodiments, the tissues
damaged by ischemia are the cerebral tissues. In certain
embodiments, the compositions of the invention reduce necrosis or
the number of necrotic cells. In certain embodiments, the
compositions of the invention reduce apoptosis or the number of
apoptotic cells. In certain embodiments, the compositions of the
invention reduce the number of necrotic and apoptotic cells. In
certain embodiments, the compositions of the invention prevent cell
death by necrosis and/or apoptosis. In certain embodiments, the
compositions of the invention prevent cell death by necrosis and/or
apoptosis caused by ischemia. In certain embodiments, the
compositions of the invention improve the recovery of the tissue
damaged by ischemia. In certain embodiments, the compositions of
the invention improve the speed of clearance of necrotic cells
and/or apoptotic cells. In certain embodiments, the compositions of
the invention improve the efficacy of the clearance of necrotic
cells and/or apoptotic cells. In certain embodiments, the
compositions of the invention improve the replacement and/or
regeneration of cells within tissues. In certain embodiments, the
compositions of the invention improve the replacement and/or
regeneration of cells within tissues damaged by ischemia. In
certain embodiments, the compositions of the invention improve the
overall histology of the tissue (for example upon a biopsy).
[0155] Modes of Administration
[0156] 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.
[0157] In certain embodiments, the compositions of the invention
may be administered as a foam, as a spray or a gel.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] The compositions of the invention may be administered to a
patient that has been diagnosed with a neurodegenerative disease,
or that has been identified as being at risk of a neurodegenerative
disease. The compositions may also be administered as a
prophylactic measure to prevent the development of
neurodegenerative disease in a healthy patient.
[0164] 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 Roseburia, and in particular Roseburia hominis.
[0165] The compositions of the invention may be administered as a
food product, such as a nutritional supplement.
[0166] 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.
[0167] Compositions
[0168] 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.
[0169] 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 [39-41].
[0170] Alternatively, the composition of the invention may comprise
a live, active bacterial culture.
[0171] 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.
[0172] In some embodiments, the composition comprises a mixture of
live bacterial strains and bacterial strains that have been
killed.
[0173] 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 [42] and [43].
[0174] The composition may be administered orally and may be in the
form of a tablet, capsule or powder. Encapsulated products are
preferred because Roseburia 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.
[0175] The composition may be formulated as a probiotic.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] In certain embodiments, the compositions of the invention
are used in combination with another therapeutic compound for
treating or preventing the neurodegenerative disorder. In some
embodiments, the compositions of the invention are administered
with nutritional supplements that modulate neuroprotection or
neuroproliferation. 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.
[0182] In certain embodiments, the compositions of the invention
are for use in enhancing the effect of a second agent on a
neurodegenerative disease. The immune modulatory effects of the
compositions of the invention may make the brain more susceptible
to conventional therapies such as Levodopa, dopamine agonists,
MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, or
anticholinergics, which are exemplary secondary agents to be
administered in combination (sequentially or contemporaneously)
with the compositions of the invention.
[0183] The compositions of the invention may comprise
pharmaceutically acceptable excipients or carriers. Examples of
such suitable excipients may be found in the reference [44].
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art and are described, for example, in
reference [45]. Examples of suitable carriers include lactose,
starch, glucose, methyl cellulose, magnesium stearate, mannitol,
sorbitol and the like. Examples of suitable diluents include
ethanol, glycerol and water. The choice of pharmaceutical carrier,
excipient or diluent can be selected with regard to the intended
route of administration and standard pharmaceutical practice. The
pharmaceutical compositions may comprise as, or in addition to, the
carrier, excipient or diluent any suitable binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural
sugars such as glucose, anhydrous lactose, free-flow lactose,
beta-lactose, corn sweeteners, natural and synthetic gums, such as
acacia, tragacanth or sodium alginate, carboxymethyl cellulose and
polyethylene glycol. Examples of suitable lubricants include sodium
oleate, sodium stearate, magnesium stearate, sodium benzoate,
sodium acetate, sodium chloride and the like. Preservatives,
stabilizers, dyes and even flavouring agents may be provided in the
pharmaceutical composition. Examples of preservatives include
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
Antioxidants and suspending agents may be also used.
[0184] 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.
[0185] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the genus Roseburia 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
Roseburia, 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. In some
embodiments, the compositions of the invention comprise one or more
bacterial strains of the species Roseburia hominis 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
Roseburia hominis, 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.
[0186] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Roseburia
intestinalis 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 Roseburia intestinalis, 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.
[0187] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Roseburia
faecis 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 Roseburia faecis, 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.
[0188] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Roseburia
hominis and do not contain bacteria from any other Roseburia
species, or which comprise only de minimis or biologically
irrelevant amounts of bacteria from another Roseburia species.
Thus, in some embodiments, the invention provides a composition
comprising one or more bacterial strains of the species Roseburia
hominis, which does not contain bacteria from any other Roseburia
species or which comprises only de minimis or biologically
irrelevant amounts of bacteria from another Roseburia species, for
use in therapy.
[0189] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Roseburia
intestinalis and do not contain bacteria from any other Roseburia
species, or which comprise only de minimis or biologically
irrelevant amounts of bacteria from another Roseburia species.
Thus, in some embodiments, the invention provides a composition
comprising one or more bacterial strains of the species Roseburia
intestinalis, which does not contain bacteria from any other
Roseburia species or which comprises only de minimis or
biologically irrelevant amounts of bacteria from another Roseburia
species, for use in therapy.
[0190] In some embodiments, the compositions of the invention
comprise one or more bacterial strains of the species Roseburia
faecis and do not contain bacteria from any other Roseburia
species, or which comprise only de minimis or biologically
irrelevant amounts of bacteria from another Roseburia species.
Thus, in some embodiments, the invention provides a composition
comprising one or more bacterial strains of the species Roseburia
faecis, which does not contain bacteria from any other Roseburia
species or which comprises only de minimis or biologically
irrelevant amounts of bacteria from another Roseburia species, for
use in therapy.
[0191] 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.
[0192] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the genus Roseburia, 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.
[0193] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the species Roseburia
hominis, 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.
[0194] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the species Roseburia
intestinalis, 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.
[0195] In some embodiments, the invention provides a composition
comprising a single bacterial strain of the species Roseburia
faecis, 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.
[0196] 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.
[0197] In some embodiments, the composition comprises a microbial
consortium. For example, in some embodiments, the composition
comprises the Roseburia bacterial strain as part of a microbial
consortium. For example, in some embodiments, the Roseburia
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 Roseburia 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.
[0198] In some embodiments, the composition of the invention
additionally comprises a bacterial strain that has the same safety
and therapeutic efficacy characteristics as strain MRx0001, but
which is not MRx0001, or which is not a Roseburia hominis.
[0199] In some embodiments, the composition of the invention
additionally comprises a bacterial strain that has the same safety
and therapeutic efficacy characteristics as strain A, but which is
not strain A, or which is not a Roseburia intestinalis.
[0200] 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.
[0201] 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.
[0202] In some embodiments, the bacterial strain for use in the
invention is obtained from human infant 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 infant 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 infant faeces
and being used in a composition of the invention.
[0203] As mentioned above, in some embodiments, the one or more
Roseburia 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.
[0204] The compositions for use in accordance with the invention
may or may not require marketing approval.
[0205] 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.
[0206] In some cases, the lyophilised bacterial strain is
reconstituted prior to administration. In some cases, the
reconstitution is by use of a diluent described herein.
[0207] The compositions of the invention can comprise
pharmaceutically acceptable excipients, diluents or carriers.
[0208] In certain embodiments, the invention provides a
pharmaceutical composition comprising: a bacterial strain of the
invention; and a pharmaceutically acceptable excipient, carrier or
diluent; wherein the bacterial strain is in an amount sufficient to
treat a neurodegenerative disorder when administered to a subject
in need thereof.
[0209] In certain embodiments, the invention provides
pharmaceutical composition comprising: a bacterial strain of the
invention; and a pharmaceutically acceptable excipient, carrier or
diluent; wherein the bacterial strain is in an amount sufficient to
treat or prevent a neurodegenerative disorder.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising a diluent selected from the
group consisting of ethanol, glycerol and water.
[0215] 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.
[0216] In certain embodiments, the invention provides the above
pharmaceutical composition, further comprising at least one of a
preservative, an antioxidant and a stabilizer.
[0217] 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.
[0218] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein said bacterial strain is
lyophilised.
[0219] 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.
[0220] 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.
[0221] 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").
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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 [46]). In some embodiments, the
formulation is an intrinsically enteric capsule (for example,
Vcaps.RTM. from Capsugel).
[0227] 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.
[0228] Culturing Methods
[0229] The bacterial strains for use in the present invention can
be cultured using standard microbiology techniques as detailed in,
for example, references [47-49].
[0230] 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).
[0231] Bacterial Strains for Use in Vaccine Compositions
[0232] The inventors have identified that the bacterial strains of
the invention are useful for treating or preventing
neurodegenerative disorders. This is likely to be a result of the
effect that the bacterial strains of the invention have on the host
immune system. Therefore, the compositions of the invention may
also be useful for preventing neurodegenerative disorders, when
administered as vaccine compositions. In 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.
[0233] General
[0234] 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 [50] and [51-57], etc.
[0235] 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.
[0236] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0237] 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.
[0238] 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. [58]. 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
[59].
[0239] 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. 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
Example 1--Efficacy of Bacterial Inocula to Reduce IL-6
Secretion
[0240] Summary
[0241] Activation of proinflammatory cytokines has been associated
with neuron damage in neurodegenerative disease. Lipopolysaccharide
(LPS) is a known stimulator of the proinflammatory cytokine IL-6.
Human glioblastoma astrocytoma cells were treated with compositions
comprising bacterial strains according to the invention in
combination with LPS to observe their ability to modulate the
levels of IL-6.
[0242] Material and Methods
[0243] Bacterial Strain
[0244] Roseburia hominis MRx0001
[0245] Cell Line
[0246] MG U373 is a human glioblastoma astrocytoma derived from a
malignant tumour and were purchased from Sigma-Aldrich (cat n.
08061901-1VL). MG U373 human glioblastoma astrocytoma cells were
grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10%
FBS, 1% Pen Strep, 4 mM L-Glut, 1.times. MEM Non essential Amino
Acid solution and 1.times. Sodium Piruvate.
[0247] Method
[0248] Once grown the MG U373 cells were plated on 24-well plate at
100,000 cells/well. The cells were treated with LPS (1 ug/mL) alone
or with 10% of bacteria supernatant from MRx0001 for 24 h. A
control was also performed where the cells were incubated in
untreated media. The cells were treated with 10% of bacteria
supernatant from MRx0001 or YCFA alone for 24 h. A control was also
performed where the cells were incubated in untreated media.
Afterwards the cell free supernatants were collected, centrifuged
at 10,000 g for 3 min at 4.degree. C. IL-6 was measured using the
Human IL-6 ELISA Kit from Peprotech (cat n. #900-K16) according to
manufacturer instruction.
[0249] Results
[0250] The results of these experiments are shown in FIG. 1 and
FIG. 9. Treatment of neuroblastoma cells with LPS and the bacteria
strain led to a decrease in the level of IL-6 secreted to the same
level as the control cells that were untreated with LPS (FIG. 1).
Treatment of neuroblastoma cells with Roseburia hominis led to a
decrease in the level of IL-6 secreted below the levels observed
with the cells treated with the control (FIG. 9).
Example 2--Efficacy of Bacterial Inocula to Reduce NF.kappa.B
Activation
[0251] Summary
[0252] Activation of the NF.kappa.B promoter leads to the
production of proinflammatory cytokines including IL-10, IL-la,
IL-18, TNF.alpha. and IL-6. The NF.kappa.B promoter can be
activated by .alpha.-synuclein and LPS by stimulating the TLR4
ligand. Mutations in .alpha.-synuclein, such as .alpha.-synuclein
A53T, are implicated in familial Parkinson's. Treatment of neuronal
cells with LPS simulates Parkinson's caused by environmental
factors. The ability of compositions comprising bacterial strains
according to the invention to inhibit the activation of the
NF.kappa.B promoter was investigated.
[0253] Material and Methods
[0254] Bacterial Strain
[0255] Roseburia hominis MRx0001
[0256] Cell Line
[0257] Human Hek blue TLR4 were purchased from InvivoGen (cat n.
hkb-htlr4). Human Hek blue TLR4 were grown in DMEM high glucose
(Sigma Aldrich, cat n. D-6171) supplemented with 10% FBS, 1% Pen
Strep, 4 mM L-Glut, Normocin and 1.times. HEK Blue selection
solution.
[0258] Method
[0259] Once grown the Human Hek blue cells were plated in 96 well
plate at 25,000 cells/well in 4 replicates. One set of cells were
treated with .alpha.-synuclein A53T (1 ug/mL) alone or with 10% of
bacteria supernatant from MRx0001 for 22 h. The second set of cells
were treated with LPS (10 ng/mL, from Salmonella enterica serotype
Typhimurium, Sigma Aldrich, cat n. L6143) alone or with 10% of
bacteria supernatant from MRx0001 for 22 h. The cells were
subsequently spun down and 20 ul of the supernatant was mixed with
200 ul of Quanti Blue reagent (InvivoGen, cat n. rep-qb2),
incubated for 2 h and absorbance read at 655 nm.
[0260] Results
[0261] The results of these experiments are shown in FIGS. 2 and 3.
FIG. 2 shows that the activation of the NF.kappa.B promoter by
.alpha.-synuclein is inhibited by MRx0001. FIG. 3 shows that the
activation of the NF.kappa.B promoter by LPS is inhibited by
MRx0001.
Example 3--Efficacy of Bacterial Inocula to Alter Antioxidant
Capacity
[0262] Summary
[0263] The ability of compositions comprising bacterial strains
according to the invention to alter the antioxidant capacity. The
antioxidant capacity of the bacterial strain was established using
the well-known ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid))
assay.
[0264] Bacterial Strain
[0265] Roseburia hominis MRx0001
[0266] Method
[0267] Bacterial cells (10.sup.6 or greater) were collected and
centrifuged. They were resuspended in assay buffer (using three
times the pellet volume). The suspension was sonicated on ice for 5
minutes and then spun down at 12,000.times.g for 10 minutes. The
supernatant was removed and measured using the ABTS assay kit
produced by Sigma Aldrich (code CS0790), in accordance with
manufacturer's instructions.
[0268] Results
[0269] The results of these experiments are shown in FIG. 4. FIG. 4
shows that MRx0001 has an antioxidant capacity of approximately 1
mM compared to Trolox.
Example 4--Efficacy of Bacterial Inocula to Alter Lipid
Peroxidation Levels
[0270] Summary
[0271] The ability of compositions comprising bacterial strains
according to the invention to alter lipid peroxidation levels was
investigated. The thiobarbituric reactive substances assay (TBARs)
was used to measure the by-products of lipid peroxidation.
[0272] Material and Methods
[0273] Bacterial Strain
[0274] Roseburia hominis MRx0001
[0275] Method
[0276] Bacterial cells (10.sup.6 or greater) were collected and
centrifuged, a wash step was performed with isotonic saline before
the pellet was re-suspensed in potassium chloride assay buffer. The
suspension was sonicated on ice for 10 minutes and then spun down
at 10,000.times.g for 10 minutes. The supernatant was removed and
the level of lipid peroxidation evaluated using the thiobarbituric
reactive substances assay.
[0277] Results
[0278] The results of the experiments are shown in FIG. 5. FIG. 5
shows that MRx0001 is able to inhibit lipid peroxidation by
approximately 18%, which is a higher antioxidant capacity than the
positive control, butylated hydroxytoluene (1% w/v).
Example 5--Efficacy of Bacterial Inocula on Histone Deacetylatase
Activity
[0279] Summary
[0280] The ability of compositions comprising bacterial strains
according to the invention to alter histone deacetylatase activity
was investigated. Dysregulation of histone deacetylatase has been
implicated in the pathogenesis associated with age-associated
neurodegenerative diseases.
[0281] Material and Methods
[0282] Bacterial Strain
[0283] Roseburia hominis MRx0001
[0284] Cell Line
[0285] The cell line HT-29 was used because histone deacetylase is
present.
[0286] Method
[0287] Cell free supernatants of stationary phase bacterial
cultures were isolated by centrifugation and filtering in a 0.22 uM
filter. HT-29 cells were used 3 days' post confluence and stepped
down in 1 mL DTS 24 hours prior to commencement of the experiment.
The HT-29 cells were challenged with 10% cell free supernatant
diluted in DTS and this was left to incubate for 48 hours. Nuclease
proteins were then extracted using the Sigma Aldrich Nuclease
extraction kit and samples were snap frozen prior to HDAC activity
measurement. HDAC activity was assessed fluorometrically using the
Sigma Aldrich (UK) kit.
[0288] Results
[0289] The results of the experiments are shown in FIG. 6. FIG. 6
shows that MRx0001 is able reduce the levels of histone deacetylase
activity.
Example 6--Level of Indole Production in Bacteria
[0290] Summary
[0291] The ability of the bacteria of the invention to produce
indole was investigated. Indole has been implicated in attenuating
inflammation and oxidative stress.
[0292] Material and Methods
[0293] Bacterial Strain
[0294] Roseburia hominis MRx0001
[0295] ATCC 11775 is a bacterial reference strain that is known to
produce indole.
[0296] Method
[0297] Intact bacterial cells in stationary phase were incubated
with 6 mM Tryptophan for 48 hours. Bacterial species which possess
the enzyme tryptophanase will utilise tryptophan as a substrate to
produce indole. Following the 48 hour incubation period, the
supernatant was removed and added to Kovac's reagent for
quantification of indole. Standards, stock solutions and reagents
were prepared using standardised methods validated in-house.
[0298] Results
[0299] The results of the experiments are shown in FIG. 7. FIG. 7
shows that MRx0001 has the capacity to produce indole from
tryptophan, at concentrations of approximately 0.25 mM.
Example 7--Level of Kynurenine Production in Bacteria
[0300] Summary
[0301] The ability of the bacteria of the invention to produce
kynurenine was investigated. Dysregulation of the kynurenine
pathway can lead to activation of the immune system and the
accumulation of potentially neurotoxic compounds. Alterations in
the kynurenine metabolism may be involved in the development of
Parkinson's diseases.
[0302] Bacterial Strain
[0303] Roseburia hominis MRx0001
[0304] DSM 17136 is a strain of Bacteroides copricola that is known
to produce kynurenine.
[0305] Method
[0306] Cell free supernatants of stationary phase bacterial
cultures were isolated by centrifugation and filtering in a 0.22 uM
filter and frozen until use. Kynurenine standards, stock solutions
and reagents were prepared using standardised methods validated
in-house. Sample were treated with trichloroacetic acid and
centrifuged at 10,000.times.g for 10 minutes at 4.degree. C. The
supernatant was collected and dispensed into a 96 well plate.
Ehrlich's reagent was used for kynurenine detection and added at a
ratio of 1:1.
[0307] Results
[0308] The results of the experiments are shown in FIG. 8. FIG. 8
shows that MRx0001 has the capacity to produce kynurenine at a
concentration of approximately 50 .mu.M.
Example 8--Stability Testing
[0309] 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 9--Efficacy of Bacterial Inocula to Reduce IL-6
Secretion
[0310] Summary
[0311] Activation of proinflammatory cytokines has been associated
with neuron damage in neurodegenerative disease. Lipopolysaccharide
(LPS) is a known stimulator of the proinflammatory cytokine IL-6.
Human glioblastoma astrocytoma cells were treated with compositions
comprising bacterial strains according to the invention in
combination with LPS to observe their ability to modulate the
levels of IL-6.
[0312] Material and Methods
[0313] Bacterial Strain
[0314] Roseburia intestinalis Strain A
[0315] Roseburia faecis Strain B
[0316] Cell Line
[0317] MG U373 is a human glioblastoma astrocytoma derived from a
malignant tumour and were purchased from Sigma-Aldrich (cat n.
08061901-1VL). MG U373 human glioblastoma astrocytoma cells were
grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10%
FBS, 1% Pen Strep, 4 mM L-Glut, 1.times. MEM Non essential Amino
Acid solution and 1.times. Sodium Piruvate. Method
[0318] Once grown the MG U373 cells were plated on 24-well plate at
100,000 cells/well. The cells were treated with the following
conditions for 24 h: [0319] LPS (1 ug/mL) [0320] LPS with 10% of
bacteria supernatant from Strain A [0321] LPS with 10% of bacteria
supernatant from Strain B [0322] LPS with YCFA media [0323]
YCFA
[0324] A control was also performed where the cells were incubated
in untreated media.
[0325] Afterwards the cell free supernatants were collected,
centrifuged at 10,000 g for 3 min at 4.degree. C. IL-6 was measured
using the Human IL-6 ELISA Kit from Peprotech (cat n. #900-K16)
according to manufacturer instruction.
[0326] Results
[0327] The results of these experiments are shown in FIGS. 10 and
12. Treatment of neuroblastoma cells with LPS and the bacteria
strain A and B led to a decrease in the level of IL-6 secreted to
the same level as the control cells that were untreated with LPS
(FIGS. 10A and 12A).
[0328] Treatment of neuroblastoma cells with Roseburia intestinalis
and Roseburia faecis led to a decrease in the level of IL-6
secreted below the levels observed with the cells treated with the
control (FIGS. 10B and 12B).
Example 10--Efficacy of Bacterial Inocula to Reduce NF.kappa.B
Activation
[0329] Summary
[0330] Activation of the NF.kappa.B promoter leads to the
production of proinflammatory cytokines including IL-1.beta.,
IL-1.alpha., IL-18, TNF.alpha. and IL-6. The NF.kappa.B promoter
can be activated by .alpha.-synuclein and LPS by stimulating the
TLR4 ligand. Mutations in .alpha.-synuclein, such as
.alpha.-synuclein A53T, are implicated in familial Parkinson's.
Treatment of neuronal cells with LPS simulates Parkinson's caused
by environmental factors. The ability of compositions comprising
bacterial strains according to the invention to inhibit the
activation of the NF.kappa.B promoter was investigated.
[0331] Material and Methods
[0332] Bacterial Strain
[0333] Roseburia intestinalis Strain A
[0334] Roseburia faecis Strain B
[0335] Cell Line
[0336] Human Hek blue TLR4 were purchased from InvivoGen (cat n.
hkb-htlr4). Human Hek blue TLR4 were grown in DMEM high glucose
(Sigma Aldrich, cat n. D-6171) supplemented with 10% FBS, 1% Pen
Strep, 4 mM L-Glut, Normocin and 1.times. HEK Blue selection
solution.
[0337] Method
[0338] Once grown the Human Hek blue cells were plated in 96 well
plate at 25,000 cells/well in 4 replicates. One set of cells were
treated with .alpha.-synuclein A53T (1 ug/mL) alone or with 10% of
bacteria supernatant from Strain A or Strain B for 22 h. The second
set of cells were treated with LPS (10 ng/mL, from Salmonella
enterica serotype Typhimurium, Sigma Aldrich, cat n. L6143) alone
or with 10% of bacteria supernatant from Strain A or Strain B for
22 h. The cells were subsequently spun down and 20 ul of the
supernatant was mixed with 200 ul of Quanti Blue reagent
(InvivoGen, cat n. rep-qb2), incubated for 2 h and absorbance read
at 655 nm.
[0339] Results
[0340] The results of these experiments are shown in FIGS. 11 and
13.
TABLE-US-00001 Sequences (Roseburia hominis strain A2-181 16S
ribosomal RNA gene, partial sequence - AY804148) SEQ ID NO: 1 1
taaaggttga tcctggctca ggatgaacgc tggaggcgtg cttaacacat gcaagtcgaa
61 cgaagcactt taattgattt cttcggaatg aagtttttgt gactgagtgg
cggacgggtg 121 agtaacgcgt gggtaacctc gctcatacag ggggataaca
gttggaaacg actgctaata 181 ccgcataagc gcacaggatt gcatgatcca
gtgtgaaaaa ctccggtggt atgagatgga 241 cccgcgtctg attagccagt
tggcggggta acggcccacc aaagcgacga tcagtagccg 301 acctgagagg
gtgaccggcc acattgggac tgagacacgg cccaaactcc tacgggaggc 361
agcagtgggt aatattgcac aatgggggaa accctgatgc agcgacgccg agtgagcgaa
421 gaagtatttc ggtatgtaaa gctctatcag caggaagaag aatgacggta
cctgactaaa 481 aagcaccggc taaatacgtg ccagcagccg cggtaatacg
tatggtgcaa gcgttatccg 541 gatttactgg gtgtaaaggg agcgcaggcg
gtacggcaag tctgatgtga aatcccgggg 601 ctcaaccccg gtactgcatt
ggaaactgtc ggactagggt gtctgagggg taagtggaat 661 tcctagtgta
gcggtgaaat gcgtagatat taggaggaac accagtggcg aaggcggctt 721
actggacgat tactgacgct gaggctcgaa agcgtgggga gcaaacagga ttagataccc
781 tggtagtcca cgccgtaaac gatgaatact aggtgtcggg gagcattgct
cttcggtgcc 841 gcagcaaacg caataagtat tccacctggg gagtacgttc
gcaagaatga aactcaaagg 901 aattgacggg gacccgcaca agcggtggag
catgtggttt aattcgaagc aacgcgaaga 961 accttaccaa gtcttgacat
cccactgaca aagtatgtaa tgtactttct cttcggagca 1021 gtggtgacag
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1081
cgcaacgagc gcaaccccta ttcttagtag ccagcggttt ggccgggcac tctagggaga
1141 ctgccaggga taacctggag gaaggtgggg atgacgtcaa atcatcatgc
cccttatgac 1201 ttgggctaca cacgtgctac aatggcgtaa acaaagggaa
gcaatcccgc gagggggagc 1261 aaatctcaaa aataacgtct cagttcggac
tgtagtctgc aactcgacta cacgaagctg 1321 gaatcgctag taatcgcgaa
tcagaatgtc gcggtgaata cgttcccggg tcttgtacac 1381 accgcccgtc
acaccatggg agttggtaat gcccgaagtc agtgacccaa ccgcaaggag 1441
ggagctgccg aagcaggact gataactggg gtgaagtcgt aacaagt (Roseburia
hominis A2-183 16S rRNA gene, type strain A2-183T - AJ270482) SEQ
ID NO: 2 1 gatcctggct caggatgaac gctggcggcg tgcttaacac atgcaagtcg
aacgaagcac 61 tttaattgat ttcttcggaa tgaagttttt gtgactgagt
ggcggacggg tgagtaacgc 121 gtgggtaacc tgcctcatac agggggataa
cagttggaaa cgactgctaa taccgcataa 181 gcgcacagga ttgcatgatc
cagtgtgaaa aactccggtg gtatgagatg gacccgcgtc 241 tgattagcca
gttggcgggg taacggccca ccaaagcgac gatcagtagc cgacctgaga 301
gggtgaccgg ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtgg
361 ggaatattgc acaatggggg aaaccctgat gcagcgacgc cgcgtgagcg
aagaagtatt 421 tcggtatgta aagctctatc agcagggaag aagaatgcgg
tacctgacta agaagcaccg 481 gctaaatacg tgccagcagc cgcggtaata
cgtatggtgc aagcgttatc cggatttact 541 gggtgtaaag ggagcgcagg
cggtacggca agtctgatgt gaaatcccgg ggctcaaccc 601 cggtactgca
ttggaaactg tcggactaga gtgtcggagg ggtaagtgga attcctagtg 661
tagcggtgaa atgcgtagat attaggagga acaccagtgg cgaaggcggc ttactggacg
721 attactgacg ctgaggctcg aaagcgtggg gagcaaacag gattagatac
cctggtagtc 781 cacgccgtaa acgatgaata ctaggtgtcg gggagcattg
ctcttcggtg ccgcagcaaa 841 cgcaataagt attccacctg gggagtacgt
tcgcaagaat gaaactcaaa ggaattgacg 901 gggacccgca caagcggtgg
agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 961 aagtcttgac
atcccactga cagagtatgt aatgtacttt ctcttcggag cagtggtgac 1021
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga
1081 gcgcaacccc tattcttagt agccagcggt tcggccgggc actctaggga
gactgccagg 1141 gataacctgg aggaaggtgg ggatgacgtc aaatcatcat
gccccttatg acttgggcta 1201 cacacgtgct acaatggcgt aaacaaaggg
aagcaatccc gcgaggggga gcaaatctca 1261 aaaataacgt ctcagttcgg
actgtagtct gcaactcgac tacacgaagc tggaatcgct 1321 agtaatcgcg
aatcagaatg tcgcggtgaa tacgttcccg ggtcttgtac acaccgcccg 1381
tcacaccatg ggagttggta atgcccgaag tcagtgaccc aaccgcaagg agggagctgc
1441 cgaaggcagg actgataact ggggtgaagt cgtaacaagg gtacg (consensus
16S rRNA sequence for Roseburia hominis strain 433) SEQ ID NO: 3
AAGAGTTTGGGHCAGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCACTTTAAT-
TGA
TTTCTTCGGAATGAAGTTTTTGTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGG-
GGG
ATAACAGTTGGAAACGACTGCTAATACCGCATAAGCGCACAGGATTGCATGATCCAGTGTGAAAAACTCCGGTG-
GTA
TGAGATGGACCCGCGTCTGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTG-
AGA
GGGTGACCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAA-
TGG
GGGAAACCCTGATGCAGCGACGCCGCGTGAGCGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGA-
AGA
ATGACGGTACCTGACTAAGAAGCACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTT-
ATC
CGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTACGGCAAGTCTGATGTGAAATCCCGGGGCTCAACCCCGGT-
ACT
GCATTGGAAACTGTCGGACTAGAGTGTCGGAGGGGTAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATA-
TTA
GGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATTACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAAC-
AGG
ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGAGCATTGCTCTTCGGTGCCGCA-
GCA
AACGCAATAAGTATNCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACA-
AGC
GGTGGAGCNTGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCCACTGACAGAGTATG-
TAA
TGTACTTTCTCTTCGGAGCAGTGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT-
AAG
TCCCGCAACGAGCGCAACCCCTATTCTTAGTAGCCAGCGGTTTGGCCGGGCACTCTAGGGAGACTGCCAGGGAT-
AAC
CTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACTTGGGCTACACACGTGCTACAATGGCGT-
AAA
CAAAGGGAAGCAATCCCGCGAGGGGGAGCAAATCTCAAAAATAACGTCTCAGTTCGGACTGTAGTCTGCAACTC-
GAC
TACACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACAC-
CGC
CCGTCACACCATGGGAGTTGGTAATGCCCGAAGTCAGTGACCCAACCGCAAGGAGGGAGCTGCCGAAGGCAGGA-
CTG ATAACTGGGGTGAAGTCTACRSAGGGTAGCCGTRMMC. (Consensus 16S rRNA
sequence, Strain A Roseburia intestinalis) SEQ ID NO: 5 gctccctcct
tgcggttggg tcactgactt cgggcattac caactcccat ggtgtgacgg 60
gcggtgtgta caagacccgg gaacgtattc accgcgacat tctgattcgc gattactagc
120 gattccagct tcgtgcagtc gagttgcaga ctgcagtccg aactgagacg
ttatttttga 180 gatttgctcc ccctcgcagg ctcgcttccc tttgtttacg
ccattgtagc acgtgtgtag 240 cccaagtcat aaggggcatg atgatttgac
gtcatcccca ccttcctcca ggttatccct 300 ggcagtctcc ctagagtgcc
cggcttaccc gctggctact aagaataggg gttgcgctcg 360 ttgcgggact
taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420
accgatgctc cgaagagaaa acacattaca tgttctgtca tcgggatgtc aagacttggt
480 aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc
gggtccccgt 540 caattccttt gagtttcatt cttgcgaacg tactccccag
gtggaatact tattgcgttt 600 gctgcggcac cgaagagcaa tgctccccga
cacctagtat tcatcgttta cggcgtggac 660 taccagggta tctaatcctg
tttgctcccc acgctttcga gcctcagcgt cagtaatcgt 720 ccagtaagcc
gccttcgcca ctggtgttcc tcctaatatc tacgcatttc accgctacac 780
taggaattcc acttacccct ccgacactct agtccgacag tttccaatgc agtaccgggg
840 ttgagccccg ggctttcaca tcagacttgc cgtaccgcct gcgctccctt
tacacccagt 900 aaatccggat aacgcttgca ccatacgtat taccgcggct
gctggcacgt atttagccgg 960 tgcttcttag tcaggtaccg tcatttcttc
ttccctgnct gatagagctt tacataccga 1020 aatacttctt cgctcacgcg
gcgtcgctgc atcagggttt cccccattgt gcaatattcc 1080 ccactgctgc
ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg ccggtcaccc 1140
tctcaggtcg gctactgatc gtcgctttgg taggccgtta ccccaccaac tggctaatca
1200 gacgcgggtc catctcatac caccggagtt tttcacacca ggtcatgcga
ccctgtgcgc 1260 ttatgcggta ttagcagtcg tttccaactg ttatccccct
gtatgaggca ggttacccac 1320 gcgttactca cccgtccgcc actcagtcac
aaaatcttca ttccgaagaa atcaaataaa 1380 gtgcttcgtt cgactgca 1398
(Consensus 16S rRNA sequence, Strain B Roseburia faecis) SEQ ID NO:
6 agctccctcc ttgcggttgg gtcactgact tcggacattt ccaactccca tggtgtgacg
60 ggcggtgtgt acaagacccg ggaacgtatt caccgcagca ttctgatctg
cgattactag 120 cgattccagc ttcgtgtagt cgggttgcag actacagtcc
gaactgagac gttatttttg 180 agatttgctc ggcctcacgg ctttgcttcc
ctttgtttac gccattgtag cacgtgtgta 240 gcccaagtca taaggggcat
gatgatttga cgtcatcccc gccttcctcc aggttatccc 300 tggcagtctc
cctagagtgc ccggccgaac cgctggctac taaggacagg ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt
420 caccgatgct ccgaagagaa agtacattac atactctgtc atcgggatgt
caagacttgg 480 taaggttctt cgcgttgctt cgaattaaac cacatgctcc
accgcttgtg cgggtccccg 540 tcaattcctt tgagtttcat tcttgcgaac
gtactcccca ggtggaatac ttattgcgtt 600 tgctgcggca ccgaagagca
atgctccccg acacctagta ttcatcgttt acggcgtgga 660 ctaccagggt
atctaatcct gtttgctccc cacgctttcg agcctcagcg tcagttatcg 720
tccagtaagc cgccttcgcc actggtgttc ctcctaatat ctacgcattt caccgctaca
780 ctaggaattc cacttacccc tccgacactc tagtacgaca gtttccaatg
cagtaccggg 840 gttgagcccc gggctttcac atcagacttg ccgcaccgcc
tgcgctccct ttacacccag 900 taaatccgga taacgcttgc accatacgta
ttaccgcggc tgctggcacg tatttagccg 960
gtgcttctta gtcaggtacc gtcattcttc ttccctgctg atagagcttt acataccgaa
1020 atacttcttc gctcacgcgg cgtcgctgca tcagggtttc ccccattgtg
caatattccc 1080 cactgctgcc tcccgtagga gtttgggccg tgtctcagtc
ccaatgtggc cggtcaccct 1140 ctcaggtcgg ctactgatcg tcgctttggt
aggccgttac cctgccaact ggctaatcag 1200 acgcgggtcc atctcatacc
accggagttt ttcacaccgg atcatgcgat cctgtgcgct 1260 tatgcggtat
tagcagtcgt ttccaactgt tatccccctg tatgaggcag gttacccacg 1320
cgttactcac ccgtccgcca ctcagtcaca aaatcttcat ttccgaagaa aatcaaatag
1380 agtgcttcgt ccgactgcag 1400
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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=US20220096565A1).
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=US20220096565A1).
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