EA037000B1 - Compositions comprising a bacterial strain of the genus megasphera and uses thereof - Google Patents

Abstract

The invention provides compositions comprising bacterial strains for treating and preventing neurodegenerative disorders.

Description

Technology area

This invention belongs to the field of compositions containing bacterial strains isolated from the digestive tract of mammals, and the use of such compositions in the treatment of diseases.

State of the art

The human gut is believed to be sterile in utero, but it is exposed to a wide variety of maternal and environmental microorganisms immediately after birth. Thus, a dynamic period of microbial colonization and succession occurs, which are influenced by factors such as mode of delivery, environment, nutrition and host genotype, all of which affect the composition of the gut microbiota, especially during early life. Subsequently, the microbiota stabilizes and becomes similar to that inherent in an adult organism [1]. The human intestinal microbiota contains more than 500-1000 different phylotypes, essentially belonging to two main bacterial types, Bacteroidetes and Firmicutes [2]. Successful symbiotic relationships resulting from bacterial colonization of the human intestine have resulted in a wide variety of metabolic, structural, protective, and other beneficial functions. The increased metabolic activity of the colonized intestine ensures that otherwise indigestible food components are destroyed with the release of by-products, providing an important source of nutrients for the host. Likewise, the immunological importance of gut microbiota is generally recognized and exemplified in microbial-free animals that have a compromised immune system that is functionally restored after administration of commensal bacteria [3-5].

Significant changes in the composition of the microbiota have been described in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, the levels of bacteria XIVa of the Clostridium cluster decrease in patients with IBD, while the number of E. coli increases, suggesting a shift in the balance of symbionts and pathobions in the intestine [6-9].

Given the potential beneficial effect that certain bacterial strains can have on the intestines of an animal, different strains have been proposed for use in the treatment of various diseases (see, for example, [10-13]). In addition, certain strains, including mainly Lactobacillus and Bifidobacterium, have been proposed for use in the treatment of a variety of inflammatory and autoimmune diseases that are not directly related to the gut (see [14] and [15] for information). At the same time, the relationship between different diseases and different bacterial strains, as well as the exact effects of certain bacterial strains on the gut and at the systemic level and on any specific types of diseases, are not well characterized, especially in the case of neurodegenerative disorders.

Recently, there has been an increased interest in the art regarding changes in the gut microbiome that may play a pathophysiological role in diseases of the human brain [16]. The results of preclinical and clinical studies strongly suggest a link between brain development and microbiota [17]. A growing body of evidence from the preclinical literature has demonstrated bidirectional signaling between the brain and the microbiome, including neurocrine and endocrine signaling systems. Indeed, increased levels of Clostridium species in the microbiome have been associated with brain abnormalities [18], and an imbalance between the types of Bacteroidetes and Firmicutes has also been observed in disorders of brain development [19]. Suggestions that altered levels of gut commensals, including those of the genera Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus, and the Alcaligenaceae family play a role in immune-mediated central nervous system (CNS) disorders, have been evaluated in studies suggesting that patients have no microbial change and healthy subjects [19]. There have also been suggestions that probiotic administration may be effective in treating neurological disorders. At the same time, these studies did not lead to the conclusion that probiotic compositions themselves can lead to the achievement of therapeutic effects in relation to the treatment of neurodegeneration, and did not show any beneficial effects against any specific bacteria [20, 21] ... This indicates that the practical effect of the link between the microbiome and human brain disease is currently poorly characterized. Accordingly, more direct analytical studies are required to identify the therapeutic effects of microbiome alteration on neurodegenerative disorders.

There is a demand in the art for new treatments for neurodegenerative disorders. There is also a requirement for the potential effects of gut bacteria to be characterized, and therefore new therapies with gut bacteria can be developed.

- 1 037000

Brief description of the essence of the invention

The present inventors have developed new therapies for the treatment and prevention of neurodegenerative disorders. The present inventors have found that bacterial strains of the genus Megasphaera can be effective in the treatment of neurodegenerative diseases. As described in the examples, the administration of compositions containing Megasphaera massiliensis may result in protection from reactive oxygen species, thereby acting as a neuroprotective agent. The inventors have also found that treatment with Megasphaera massiliensis can reduce the activation of pro-inflammatory molecules such as NFkB and IL-6 by LPS and mutant α-synuclein. The inventors have found that treatment with Megasphaera massiliensis can lead to a decrease in histone deacetylation and lipid peroxidation activity in vitro, which can contribute to a decrease in cell death and apoptosis. The present inventors have also found that Megasphaera massiliensis can produce indole, which can reduce inflammation and oxidative stress. In addition, the present inventors have demonstrated that treatment with Megasphaera massiliensis can lead to increased levels of kynurenine.

The inventors have also found that Megasphaera massiliensis produces certain organic acids, including hexanoic acid, valeric acid and 4-hydroxyphenylacetic acid. The inventors have also found that Megasphaera massiliensis can lead to an increase in the activation of the pro-inflammatory cytokine IL-8, which can facilitate the activation of neuronal myelination. We have also found that treatment with a combination of Megasphaera massiliensis and retinoic acid can lead to increased secretion of brain neurotrophic factor (BDNF), which can facilitate the activation of neurogenesis and neuritogenesis and / or prevent cell death. The inventors have also found that treatment with Megasphaera massiliensis, which can produce valeric acid, can lead to a decrease in histone deacetylation, which can contribute to a decrease in cell death and apoptosis. In addition, the present inventors have also found that Megasphaera massiliensis can produce hexanoic acid, which can be neuroprotective or neuropreparative, for example, by activating neurite outgrowth. The inventors have found that Megasphaera massiliensis, which can produce hexanoic acid, increases the expression of MAP2 (a protein associated with microtubules 2), which is considered necessary for microtubule formation in neuritogenesis. Thus, the present inventors also found that Megasphaera massiliensis, which can produce hexanoic acid, can be used to activate neurite outgrowth. Megasphaera massiliensis and other bacteria that produce organic acids such as hexanoic acid, valeric acid and 4-hydroxyphenylacetic acid may thus be useful in the treatment of neurodegenerative disorders.

In a first embodiment, the present invention provides a composition comprising a bacterial strain of the genus Megasphaera for use in treatment, for example, for use in a method for treating or preventing a neurodegenerative disease.

In certain embodiments, the present invention provides a composition comprising a bacterial strain of the genus Megasphaera 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, Steele-Richardson syndrome Olypevsky, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism and drug parkinsonism; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron diseases; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body dementia, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration.

In preferred embodiments, the present invention provides a composition comprising a bacterial strain of the genus Megasphaera for use in the treatment or prevention of Parkinson's disease, such as environmental Parkinson's disease, Familial Parkinson's disease, or Parkinson's disease associated with a general inflammatory status. The inventors have found that treatment with Megasphaera strains can result in the activation of pro-inflammatory molecules such as NFkB and IL-6 by LPS and mutant α-synuclein in in vitro models of Parkinson's disease as a result of environmental factors and familial Parkinson's disease. In preferred embodiments, the present invention provides a composition comprising a bacterial strain of the species Megasphaera massiliensis for use in the treatment of Parkinson's disease. Compositions in which Megasphaera massiliensis is used may be particularly effective in the treatment of Parkinson's disease.

In some embodiments, the compositions of this invention are intended to

- 2,037,000 use in a method for the treatment or prevention of an early-onset neurodegenerative disease. In some embodiments, the compositions of this invention are for use in a method for preventing or delaying the onset of progression of a neurodegenerative disease.

In preferred embodiments of the present invention, the bacterial strain in the composition is Megasphaera massiliensis. Closely related strains can also be used that have a 16S rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16S rRNA sequence of the bacterial strain Megasphaera massiliensis. Preferably, the bacterial strain has a 16S rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1 or 2. Preferably, sequence identity is determined with respect to SEQ ID NO. : 2. Preferably, the bacterial strain for use in the present invention has the 16S rRNA sequence represented by SEQ ID NO: 2.

In certain embodiments, the composition of this invention is for oral administration. Oral administration of the strains of this invention may be effective for neurodegenerative disorders. In addition, oral administration is convenient for patients and physicians and facilitates intestinal delivery and / or partial or complete colonization of the intestine.

In certain embodiments, a composition of this invention comprises one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, a composition of the invention comprises a bacterial strain that has been lyophilized. Lyophilization is an efficient and convenient technique for producing stable compositions that allow delivery of bacteria.

In certain embodiments, the present invention provides a food product comprising the composition described above.

In certain embodiments, the invention provides a vaccine composition comprising a composition as described above.

In addition, the present invention provides a method for treating or preventing neurodegenerative disorders, comprising administering a composition containing a bacterial strain of the genus Megasphaera.

In developing the above invention, the inventors of the present invention have discovered and characterized a bacterial strain that is particularly useful for treatment. As shown, the Megasphaera massiliensis strain of this invention is effective for the treatment of diseases described herein, such as neurodegenerative diseases. Thus, in another aspect, the present invention provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof. The present invention also provides compositions containing such cells or biologically pure cultures of such cells. This invention also provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof, for use in the treatment, in particular, of the diseases described herein.

In certain embodiments of the present invention, the composition is for use in treating brain damage. The neuroprotective activity of the compositions of this invention and their ability to reduce levels of histone deacetylase (HDAC) activity may render them useful in the treatment of brain damage. In preferred embodiments, the compositions of this invention are for use in the treatment of stroke, for example, treatment of brain damage resulting from stroke.

Brief description of graphic materials

FIG. 1: the viability of neuroblastoma cells.

FIG. 2: decreased secretion of IL-6.

FIG. 3: secretion of IL-8.

FIG. 4: Inhibition of IL-6 and IL-8 secretion by α-synuclein.

FIG. 5: Inhibition of α-synuclein-induced activation of the NFkB promoter.

FIG. 6: Inhibition of LPS-induced activation of the NFkB promoter.

FIG. 7: change in antioxidant capacity.

FIG. 8: change in total antioxidant capacity (lipid oxidation).

FIG. 9: Change in the activity of histone deacetylase (HDAC).

FIG. 10: Indole production level.

FIG. 11: The level of kynurenine production.

FIG. 12: Mean dopamine (DA) levels (Fig. 12A), DOPAC levels (Fig. 12B) and HVA levels (Fig. 12C) in the striatum. Data are depicted as mean + SEM.

FIG. 13: activation of neurite outgrowth: light microscopy and MAP2 gene expression (Fig. 13A), im-3 037000 munofluorescence microscopy using phalloidin (Fig. 13B).

FIG. 14: Change in ROS levels in (a) U373 cells and (b) SHSY-5Y cells.

FIG. 15: neuroprotection - cell viability. FIG. 15 shows the same data as in FIG. one.

FIG. 16: Strain-induced changes in histone deacetylase activity in whole cells and cell lysates (Fig. 16A), acid-induced changes in histone deacetylase activity (Fig. 16B), production of metabolites by strains (Fig. 16C).

FIG. 17: HDAC1 inhibition (Fig. 17A), HDAC2 inhibition (Fig. 17B), HDAC3 inhibition (Fig. 17C).

FIG. 18: HDAC class I inhibition (FIG. 18A); inhibition of HDAC1 (Fig. 18B); inhibition of HDAC2 (Fig. 18C); inhibition of HDAC3 (Fig. 18D).

FIG. 19: BDNF production level.

FIG. 20: Production levels of metabolites - neurotransmitters in the brain.

FIG. 21: Production levels of metabolites - organic acids in the supernatant.

FIG. 22: Effects on intestinal barrier function.

FIG. 23: Production of neurotransmitters in the brain.

FIG. 24: Changes in the expression of receptors in the hippocampus - A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor and D) mineralocorticoid receptor.

FIG. 25: Changes in hippocampal expression A) corticotropin-releasing hormone (CRH), B) BDNF expression and C) TLR4.

FIG. 26: A) Changes in the expression of corticotropin-releasing hormone receptor 1 (CRFR1) and B) expression of corticotropin-releasing hormone receptor 2 (CRFR2) in the hippocampus.

FIG. 27: Changes in the expression in the hippocampus of A) tumor necrosis factor, B) interleukin 1b and C) IL-6.

FIG. 28: A) changes in the expression in the hippocampus of integrin alpha M (CD11b) and B) changes in the expression in the hippocampus of serotonin receptor 1A (5-HT1A receptor).

FIG. 29: A) changes in the expression of subunit 2A of glutamate ionotropic receptor type NMDA in the hippocampus (Grin2A) and B) expression of subunit 2B of glutamate ionotropic receptor type NMDA (Grin2B) in the hippocampus.

FIG. 30: Changes in the expression in the hippocampus of A) gamma-aminobutyric acid receptor 2 (GABA A2), B) gamma-aminobutyric acid receptor 1 (GABA BR1) and C) dopamine receptor 1 (DRD1).

FIG. 31: Changes in amygdala receptor expression - A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor, and D) mineralocorticoid receptor.

FIG. 32: Changes in the expression in the amygdala of A) brain neurotrophic factor (BDNF), B) Toll-like receptor 4 (TLR-4), C) corticotropin-releasing hormone receptor 1 (CRFR1) and D) corticotropin-releasing hormone receptor 2 (CRFR2).

FIG. 33: Changes in amygdala expression of A) integrin alpha M (CD11b), B) interleukin 6 (IL6), C) subunit 2A of the NMDA glutamate ionotropic receptor (Grin2A) and D) subunit 2B of the NMDA glutamate ionotropic receptor (Grin2B).

FIG. 34: Changes in amygdala expression of A) the alpha 2 subunit of the GABA-A receptor (GABRA2), B) the subunit of the GABA-A type B receptor (GABBR1) and C) the dopamine receptor 1 (DRD1).

FIG. 35: Altered expression in the prefrontal cortex of A) oxytocin receptor, B) brain neurotrophic factor (BDNF), C) mineralocorticoid receptor, and D) glucocorticoid receptor.

FIG. 36: Changes in expression in the prefrontal cortex A) Toll-like receptor 4 (TLR-4), B) corticotropin-releasing hormone receptor 1 (CRFR1), C) corticotropin-releasing hormone receptor 2 (CRFR2) and D) alpha M integrin ( CD11b).

FIG. 37: Changes in expression in the prefrontal cortex A) interleukin 6 (IL-6), B) subunit 2A of the glutamate ionotropic receptor type NMDA (Grin2A), C) subunit 2B of glutamate ionotropic receptor type NMDA (Grin2B) and D) subunit alpha 2 of the GABA receptor -A (GABRA2).

FIG. 38: Altered expression in the prefrontal cortex of A) the GABAA receptor type 1 subunit (GABBR1) and B) dopamine receptor 1 (DRD1).

FIG. 39: Changes in colon expression of A) tryptophanhydrolase 1 (Tph1) and B) indolamine2,3-dioxygenase-1 (IDO1).

FIG. 40: Changes in ileal expression of A) tryptophanhydrolase 1 (Tph1) and B) indolamine-2,3-dioxygenase-1 (IDO1).

FIG. 41: Changes in circulating tryptophan metabolites A) kynurenine, B) tryptophan and C) kynurenine / tryptophan metabolic rate.

FIG. 42: Effect on interferon production in splenocytes from mice fed MRx0029.

- 4 037000

FIG. 43: Effect on the production of interleukin 1β from splenocytes.

FIG. 44: Effect on the production of interleukin 6 from splenocytes.

FIG. 45: Effect on the production of tumor necrosis factor from splenocytes.

FIG. 46: Effect on the production of interleukin 10 from splenocytes.

FIG. 47: Influence on the production of the chemoattractant CXCL1 from splenocytes.

FIG. 48: Changes in the levels of short-chain fatty acids in the cecum.

FIG. 49: MRx0029 and MRX005-induced changes in the expression levels of genes for actin, villin, ocludin, TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7 and NSE.

FIG. 50: SHSY5Y cell differentiation induced by MRx0005 and MRx0029. (A-C) Illustrative images of phalloidin and MAP2 immunolabeled cells. (D-F) Images A-C when merged with DAPI images. (G-I) β3-tubulin immunolabeled cells. (J-L) Images when merged with images with DAPI. Magnification x630. Western blotting of the effects of MRx0005 and MRx0029 treatments on SHSY5Y cells. Western blotting membranes were probed with antibodies to MAP2 (M) and b3 tubulin (N). Actin was used as a load control. Bottom panels: illustrative blots based on one of six separate experiments; top panels: relative densitometric intensity.

Detailed description of the essence of the invention

Bacterial strains.

The compositions of this invention comprise a bacterial strain of the genus Megasphaera. These examples demonstrate that bacteria of this genus are useful in the treatment or prevention of neurodegenerative disorders. Preferred bacterial strains are Megasphaera massiliensis.

Examples of Megasphaera species for use in the invention include Megasphaera elsdenii, Megasphaera cerevisiae, Megasphaera massiliensis, Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis, and Megasphaera micronuciformis. A further example of a Megasphaera species for use in the present invention is Megasphaera hexanoica. Megasphaera are obligatory anaerobic lactate-fermenting gastrointestinal microorganisms of ruminant and non-ruminant mammals, including humans.

The type M. massiliensis strain is NP3 (= CSUR P245 = DSM 26228) [22]. GenBank accession number of 16S rRNA gene sequences of M. massiliensis NP3 strain is JX424772.1 (disclosed herein as SEQ ID NO: 1).

The Megasphaera massiliensis bacterium studied in the Examples section is referred to herein as strain MRx0029. The sequence of the 16S rRNA of the MRx0029 strain that was examined is set forth in SEQ ID NO: 2.

Strain MRx0029 was deposited with the permission of the international depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) 4D Pharma Research Ltd. (Life Sciences Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS, Scotland) July 13, 2017 as Megasphaera massiliensis MRx0029 and assigned accession number NCIMB 42787.

Bacterial strains closely related to the strain studied in the examples are also expected to be effective in the treatment or prevention of neurodegenerative disorders. 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 the Megasphaera massiliensis bacterial strain. Preferably, the bacterial strain for use in the present invention has a 16S rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1 or 2. Preferably, the sequence identity is determined by in relation to SEQ ID NO: 2. Preferably, the bacterial strain for use in the present invention has the 16S rRNA sequence represented by SEQ ID NO: 2.

Bacterial strains, which are biotypes of MRx0029 or NP3 strains, are also expected to be effective in treating or preventing neurodegenerative disorders. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.

Strains that are biotypes of strains MRx0029 or NP3 and which are suitable for use in this invention can be identified by sequencing other nucleotide sequences of strains MRx0029 or NP3. For example, essentially the entire genome can be sequenced, and the biotypic strain for use in the present invention can have at least 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity within at least 80% its entire genome (eg, within at least 85, 90, 95, or 99%, or within its entire genome). Other suitable sequences for use in identifying biotypic strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG) ₅ or REP or [23]. Biotypic strains can have sequences

- 5,037,000 with at least 95, 96, 97, 98, 99, 99.5 or 99.9% sequence identity with the corresponding sequence of strains MRx0029 or NP3.

Alternatively, strains that are biotypes of strains MRx0029 or NP3 and which are suitable for use in the present invention can be identified using strains MRx0029 or NP3 and fragment restriction analysis and / or PCR, for example, using fluorescent amplified fragment length polymorphism (FAFLP) and PCR fingerprinting of repeated DNA elements (rep), or protein profiling, or partial sequencing of 16S or 23S rDNA. In preferred embodiments, such techniques can be used to identify other strains of Megasphaera massiliensis.

In certain embodiments, strains that are biotypes of strains MRx0029 or NP3 and that are suitable for use in this invention are strains that provide the same pattern as strains MRx0029 or NP3 when analyzed by restriction analysis of amplified ribosomal DNA (ARDRA), for example, when using the restriction enzyme Sau3AI (for illustrative methods and guidance see, for example, [24]). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as MRx0029 or NP3 strains.

Other Megasphaera strains that are useful in the compositions and methods of this invention, such as the biotypes of the MRx0029 or NP3 strains, can be identified using any suitable method or strategy, including the assays described in the examples. For example, strains for use in the present invention can 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 MRx0029 or NP3 strains may be useful in the invention. A suitable strain will have comparable immunomodulatory activity against strains MRx0029 or NP3. In particular, a biotypic strain will produce comparable effects on neurodegenerative disease models and comparable effects on cytokine levels as those demonstrated in the Examples section, which can be identified using the culture and administration protocols described in the Examples section.

A particularly preferred strain according to the invention is Megasphaera massiliensis MRx0029. It is an illustrative strain tested in the examples and has been shown to be effective in the treatment of diseases. Thus, the present invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis MRx0029 strain or a derivative thereof. The present invention also provides a composition comprising a cell of the Megasphaera massiliensis MRx0029 strain or a derivative thereof. This invention also provides a biologically pure culture of the Megasphaera massiliensis MRx0029 strain. This invention also provides a cell of the strain Megasphaera massiliensis MRx0029 or a derivative thereof, for use in the treatment, in particular, of the diseases described herein.

A particularly preferred strain of this invention is the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. This is an exemplary MRx0029 strain studied in the examples and has been shown to be effective in the treatment of diseases. Thus, the present invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof. The present invention also provides a composition comprising a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof. This invention also provides a biologically pure culture of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. The present invention also provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof, for use in the treatment, in particular, of diseases, described in this document.

The derivative of the strain of the present invention may be a daughter strain (progeny) or a strain cultured (subcloned) from the parent strain. A derivative of the strain according to this invention can be modified, for example, at the genetic level, without eliminating the biological activity. In particular, the derived strain of this invention is therapeutically active. The derived strain will have comparable therapeutic activity against the MRx0029 strain. In particular, the derived strain will produce comparable effects on neurodegenerative disease models and comparable effects on cytokine levels as those shown in the Examples section, which can be identified using the culture and administration protocols described in the Examples section. The derivative of the MRx0029 strain will generally be the biotype of the MRx0029 strain.

References to cells of strain Megasphaera massiliensis MRx0029 encompass any cell that has the same safety and therapeutic efficacy characteristics as the strain

- 6 037000

MRx0029, and such cells are encompassed by this invention.

In preferred embodiments, the bacterial strains in the compositions of this invention are viable and capable of partially or completely colonizing the intestine.

The present inventors have found that Megasphaera massiliensis strains result in decreased activation of inflammatory cytokines such as IL-6 and increased activation of the inflammatory cytokine IL-8. IL-8 has been associated with the formation of myelin sheaths [ ²⁵ ]. Chronic IL-6-induced inflammation can ultimately lead to cell death. Thus, the bacterial strains of this invention are particularly useful in the treatment or prevention of neurodegenerative disorders. In some embodiments, the bacterial strains are useful in the treatment of pathological conditions characterized by increased IL-6 activation. In some embodiments, the compositions of this invention are for use in the treatment or prevention of neurodegenerative diseases characterized by demyelination. Many neurodegenerative diseases are characterized by demyelination. Demyelination interferes with the transmission of action potentials in neurons, disrupting effective communication in the nervous system. It has been shown that IL-8 has a positive effect on the formation and repair of myelin sheaths. Thus, the compositions of this invention are particularly effective in the treatment or prevention of neurodegenerative disorders characterized by demyelination, such as multiple sclerosis.

The present inventors have found that the Megasphaera massiliensis strains of this invention result in amelioration of symptoms of neurodegenerative diseases in disease models. For example, the present inventors have found that Megasphaera massiliensis strains promote neurite outgrowth in vitro, and thus can be used in activating neuronal repair to treat or prevent neurodegenerative diseases. Thus, the bacterial strains of this invention are intended for the treatment or prevention of neurodegenerative diseases.

The present inventors have also found that the bacterial strains of the present invention lead to an increase in BDNF activation. BDNF is a neurotrophic factor that has been shown to enhance neuronal differentiation and survival. Thus, the composition of this invention can be used in a method for increasing the survival of nerve cells in the treatment or prevention of neurodegenerative diseases.

An additional bacterium that can be used in the compositions of this invention is the species Parabacteroides distasonis. These examples demonstrate that both Parabacteroides distasonis and Megasphaera massiliensis have neuroprotective activity, but produce different metabolites and may have different mechanisms of action and specific neuroprotective activity. Thus, these types can be especially effective when used in combination. In preferred embodiments, the composition comprises a strain of the species Parabacteroides distasonis and a strain of the species Megasphaera massiliensis.

Parabacteroides distasonis, deposited under accession number NCIMB 42382, was examined in the Examples section and is also referred to herein as strain MRx0005. MRX0005, MRX005, MRx005, and MRx0005 are used interchangeably in this document. The sequence of the 16S rRNA of the MRx0005 strain that was examined is proposed in SEQ ID NO: 17. The MRx0005 strain was deposited with permission from the International Depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12 March 2015 as Parabacteroides sp 755 and assigned accession number NCIMB 42382. GT Biologies Ltd. subsequently changed its name to 4D Pharma Research Limited.

In preferred embodiments, the invention provides a composition comprising a strain deposited with NCIMB under accession number NCIMB 42787, or a derivative or biotype thereof, and a strain deposited with NCIMB under accession number NCIMB 42382, or a derivative or biotype thereof, preferably for use in treating a neurodegenerative disease such as Parkinson's disease.

Therapeutic uses.

As demonstrated in the examples, the bacterial compositions of this invention are effective in the treatment of neurodegenerative disorders. In particular, treatment with the compositions of this invention results in increased neuroproliferation or neuritogenesis and acts as a neuroprotective agent against agents that result in the destruction of dopaminergic neurons. Thus, the compositions of this invention may be useful for treating or preventing neurodegenerative disorders that result from neuronal death.

The compositions of this invention can result in decreased activation of the NFkB promoter, which results in activation of the production of cytokines, eg, IL-1e, IL-1a, IL-18, TNFa, and IL-6. Treatment of cells with mutant α-synuclein is a model of familial Parkinson's disease.

- 7 037000

A point mutation at position 53 from adenine to threonine results in α-synuclein misfolding. Incorrectly folded α-synuclein subsequently leads to aggregation into insoluble fibrils that form Lewy bodies. Thus, the compositions of this invention may be useful for treating or preventing neurodegenerative disorders that result from neuroinflammation, protein malfold, and / or environmental exposure. The compositions of this invention can be used to treat familial Parkinson's disease. The activation of the NFkB promoter is mediated by the TLR4 ligand. TLR4 is known to mediate cell death in the MRTP mouse model that mimics Parkinson's disease. The compositions of this invention can be used to inhibit the ability of TLR4 signaling to activate the NFkB promoter. Of particular note, in the case of PD, both TLR2 and TLR4 were found to be activated in the brains of PD patients []. In addition, α-syn has been described as a TLR2 ligand [], and it was demonstrated using HEK-TLR4 cells that α-syn is also a TLR4 ligand [ ²⁸ ].

The compositions of this invention lead to a decrease in the secretion of pro-inflammatory cytokines such as IL-6, which can be induced by lipopolysaccharide (LPS). Treatment of cells with LPS mimics environmental-induced Parkinson's disease. The compositions of this invention can be used to reduce the secretion of IL-6. The compositions of this invention can be used to treat Parkinson's disease as a result of environmental factors.

Examples of neurodegenerative diseases to be treated with the compositions of this invention include: Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism, and drug parkinsonism; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron diseases; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body dementia, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. An additional disease to be treated with the compositions of this invention is progressive inflammatory neuropathy.

In certain embodiments, the compositions of this invention are intended for use in ameliorating neuronal death, particularly in the treatment of neurodegenerative disorders. In certain embodiments, the compositions of this invention are for use in protecting neurons, particularly in the treatment of neurodegenerative disorders.

In certain embodiments, the compositions of this invention are for use in reducing or preventing the loss of dopaminergic cells in the substantia nigra. In certain embodiments, the compositions of this invention are intended for use in reducing or preventing degeneration of dopaminergic neurons in the compact portion of the substantia nigra. In certain embodiments, the compositions of this invention are intended for use in reducing or preventing degeneration of dopaminergic neurons in the compact portion of the substantia nigra and subsequent loss of their projection nerve fibers in the striatum. In certain embodiments, the compositions of this invention are for use in reducing or preventing loss of nigrostrial dopaminergic neurons.

In certain embodiments, the compositions of this invention are intended for use in increasing dopamine levels. In certain embodiments, the compositions of this invention are intended for use in increasing the levels of DOPAC (3,4-Dihydroxyphenylacetic acid). In certain embodiments, the compositions of this invention are intended for use in increasing dopamine and DOPAC levels. In certain embodiments, the striatum levels of dopamine and / or DOPAC are increased. Dopamine and DOPAC levels can be measured using any suitable method known in the art, such as a radioenzymatic method, for example, in blood plasma or CSF (for example, described in [29]), or a reverse phase HPLC method, optionally with electrochemical detection, for example, in blood plasma or CSF (for example, described in [30]).

The neuroprotective properties of the compositions of this invention, shown in the Examples section, mean that the compositions can be particularly effective in preventing or delaying the onset or progression of neurodegenerative disorders. In certain embodiments, the compositions of this invention are intended for use in delaying the onset of progression of neurodegenerative disorders.

The compositions of this invention can lead to increased secretion of IL-8. IL-8 as it was

- 8 037000 has been shown to play a role in neuronal myelination. In some embodiments, the implementation of the compositions of this invention can be used to increase the secretion of IL-8.

The therapeutic compositions of this invention can lead to increased activation of BDNF. BDNF acts on specific neurons in the central nervous system to maintain the survival of existing neurons and to promote the growth and development of new neurons and synapses. BDNF is active in the hippocampus, cerebral cortex, and basal forebrain and is also important for long-term memory. The compositions of this invention can thus be used to increase the secretion of BDNF. Thus, the compositions of this invention can be used in the treatment of neurodegenerative diseases associated with impairment of long-term memory. The compositions of the present invention can be used to improve long-term memory, in particular to improve long-term memory that is impaired as a result of a neurodegenerative disease.

In certain embodiments, the compositions of this invention result in an increase in mitochondrial metabolic activity in neurons.

Modulation of the microbiota-gut-brain axis.

The interaction between the gut and the brain (the microbiota-gut-brain axis) occurs via a bi-directional neurohumoral interaction system. Recent evidence suggests that the microbiota that resides in the gut can modulate brain development and generate behavioral phenotypes through the microbiota-gut-brain axis. Indeed, a number of reviews suggest a role for the microbiota-gut-brain axis in maintaining the functionality of the central nervous system and suggest a dysfunction of the microbiota-gut-brain axis in the development of disorders and pathological states of the central nervous system [16], [19], [31] ...

The bidirectional interaction between the brain and the gut (i.e., the gut-brain axis) includes the central nervous system, neuroendocrine, and neuroimmune systems, including the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic and parasympathetic divisions of the autonomic nervous system (ANS ), including the enteric nervous system (ENS) and vagus nerve, as well as gut microbiota.

As demonstrated in the examples, the compositions of this invention can lead to modulation of the microbiota-gut-brain axis and a decrease in cell death associated with neurodegenerative disorders. Accordingly, the compositions of this invention may be useful for treating or preventing neurodegenerative disorders, in particular those disorders and pathological conditions that are associated with dysfunction of the microbiota-gut-brain axis.

In certain embodiments, the compositions of this 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, Steele-Richardson-Olszewski syndrome, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism and drug parkinsonism; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron diseases; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body dementia, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration.

The compositions of this invention may be particularly useful for the treatment or prevention of chronic disease, treatment or prevention of disease in patients who do not respond to other drugs (such as treatment with levodopa, dopamine receptor agonists, MAO-B inhibitors, COMT inhibitors, glutamate antagonists and / or anticholinergic drugs), and / or treatment or prevention of tissue damage and symptoms associated with dysfunction of the microbiota-gut-brain axis.

In certain embodiments, the compositions of this invention result in CNS modulation. In some embodiments, the compositions of this invention result in modulation of the autonomic nervous system (ANS). In some embodiments, the compositions of this invention result in modulation of the enteric nervous system (ENS). In some embodiments, the compositions of this invention result in modulation of the hypothalamic-pituitary-adrenal axis (HPA). In some embodiments, the compositions of this invention result in modulation of the neuroendocrine pathway. In some embodiments, the implementation of the compositions of this invention result in modulation of the neuroimmune pathway. In some embodiments, the compositions of the invention result in modulation of the CNS, ANS, ENS, HPA axis, and / or neuroendocrine and neuroimmune pathways. In certain embodiments, the compositions of this invention result in modulation of commensal metabolite levels and / or permeability in the gastrointestinal tract of a subject.

- 9 037000

Signal transmission along the microbiota-gut-brain axis is modulated by the nervous systems. Accordingly, in some embodiments, the compositions of this invention result in modulation of signal transmission in the nervous systems. In certain embodiments, the compositions of this invention result in modulation of signal transmission in the central nervous system. In some embodiments, the implementation of the compositions according to this invention result in modulation of signal transmission in sensory neurons. In other embodiments, the compositions of this invention result in modulation of signal transmission in motor neurons. In some embodiments, the implementation of the compositions of this invention result in modulation of signal transmission in the ANS. In some embodiments, the ANS is the parasympathetic nervous system. In preferred embodiments, the compositions of this invention result in modulation of signal transmission involving the vagus nerve. In other embodiments, the ANS is the sympathetic nervous system. In other embodiments, the compositions of this invention result in modulation of signal transmission in the enteric nervous system. In certain embodiments, signaling involving the ANS and ENS neurons occurs directly in response to the contents of the lumen of the gastrointestinal tract. In other embodiments, signaling involving the ANS and ENS neurons occurs indirectly in response to neurochemical compounds produced by intraluminal bacteria. In other embodiments, signaling involving ANS and ENS neurons occurs in response to neurochemicals produced by intraluminal bacteria or enteroendocrine cells. In certain preferred embodiments, the ENS neurons result in the activation of the afferent pathways of the vagus nerve, which affect the functions of the central nervous system. In some embodiments, the implementation of the compositions according to this invention lead to the regulation of activity of enterochromaffinocytes.

Neurodegenerative diseases.

Parksinson's disease.

Parkinson's disease is a common neurodegenerative disease characterized from a neuropathological point of view by the degeneration of heterogeneous populations of nerve cells (dopamine-producing cells). The clinical diagnosis of Parkinson's disease requires bradykinesia and at least one of the following symptoms: resting tremor; muscle rigidity and impaired postural reflex. Other signs and symptoms that may be present or develop during the progression of the disease are disorders of the autonomic nervous system (sialorrhea, seborrhea, constipation, urinary disorders, impaired sexual function, orthostatic hypotension, hyperhidrosis), sleep disturbances, and impaired sense of smell or temperature perception. Parkinson's disease is a neurodegenerative disease that can develop or continue as a result of dysfunction of the microbiota-intestinal-brain axis. Thus, in preferred embodiments, the compositions of this invention are for use in the treatment or prevention of Parkinson's disease in a subject.

In further preferred embodiments, the present invention provides a composition comprising a bacterial strain of the genus Megasphaera for use in a method for treating or preventing Parkinson's disease. Compositions containing a bacterial strain of the genus Megasphaera can lead to improved motor and cognitive functions in models of Parkinson's disease. Treatment with Megasphaera strains can lead to modulation of signal transmission in the central, autonomic and enteric nervous systems; can lead to modulation of the pathway involving the HPA axis; can lead to modulation of neuroendocrine and / or neuroimmune pathways; and may result in levels of commensal metabolites, inflammatory markers, and / or permeability in the subject's gastrointestinal tract, all of which are associated with the neuropathology of Parkinson's disease. In preferred embodiments, the present invention provides a composition comprising a bacterial strain of the species Megasphaera massiliensis for use in a method for treating or preventing Parkinson's disease. Compositions in which Megasphaera massiliensis is used may be particularly effective in the treatment of Parkinson's disease.

In preferred embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of one or more symptoms of Parkinson's disease in a subject. In preferred embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of one or more of the underlying symptoms of Parkinson's disease in a subject. In certain embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of bradykinesia in a subject. In certain embodiments of the implementation of the composition according to this invention lead to the prevention, reduction or weakening of tremor at rest; muscle rigidity and / or impaired postural reflex in the subject. In certain embodiments, the compositions of this invention result in the prevention, reduction or amelioration of one or more symptoms associated with the progression of Parkinson's disease selected from disorders of the autonomic nervous system

- 10 037000 (sialorrhea, seborrhea, constipation, urinary disorders, sexual dysfunction, orthostatic hypotension, hyperhidrosis), sleep disturbances and disturbances in the sense of smell or temperature perception.

In preferred embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of the symptoms of depression associated with Parkinson's disease. In certain embodiments, the compositions of this invention result in improved verbal memory and / or executive functions. In certain embodiments, the compositions of this invention result in normalization of attention, working memory, fluency, and / or anxiety.

In other preferred embodiments, the implementation of the compositions according to this invention lead to the prevention, reduction or amelioration of cognitive dysfunctions associated with Parkinson's disease.

In certain embodiments of the implementation of the composition according to this invention lead to the prevention, reduction or amelioration of the progression of Parkinson's disease. In certain embodiments, the compositions of this invention result in the prevention, reduction, or mitigation of later complications. In certain embodiments, the compositions of this invention result in the prevention, reduction, or mitigation of late motor fluctuations. In certain embodiments, the compositions of this invention result in the prevention, reduction, or mitigation of neuronal loss. In certain embodiments, the compositions of the invention result in the normalization of the symptoms of Parkinson's disease dementia (PDD). In certain embodiments of the implementation of the composition according to this invention lead to the prevention, weakening or normalization of impairment of executive function, attention and / or working memory. In certain embodiments, the compositions of this invention result in an improvement in dopaminergic neurotransmission. In certain embodiments, the compositions of this invention result in the prevention, reduction, or weakening of impaired dopaminergic neurotransmission.

In some embodiments, the implementation of the compositions of this invention results in the normalization of symptoms of Parkinson's disease in accordance with a symptomatic or diagnostic scale. In certain embodiments, tests for assessing symptomatic improvement in motor function of Parkinson's disease are a unified Parkinson's disease rating scale. Specifically, UPDRS II takes into account activity in daily life, while UPDRS III takes into account locomotor activity.

In some embodiments, the implementation of the compositions of this invention result in the normalization of symptoms associated with PDD in accordance with 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); Delis-Kaplan Executive Function System (D-KEFS - Delis-Kaplan Executive Function System) test; verbal-digital interference test; the Hamilton Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating Scale (HAM-A Hamilton Anxiety Rating Scale; anxiety) and the Unified Parkinson's Disease Rating Scale (UPDRS).

In some embodiments, the compositions of this invention result in an improvement in the Clinical Global Impression Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of this invention display a beneficial effect on the overall social and occupational impairment of a subject with Parkinson's disease.

In certain embodiments, the compositions of this invention are for use in the treatment or prevention of neurological disorders such as Parkinson's disease in a subject, said use comprising reducing or preventing loss of dopaminergic cells in the substantia nigra. In certain embodiments, the compositions of this invention are for use in the treatment or prevention of neurological disorders, such as Parkinson's disease in a subject, said use comprising degeneration of dopaminergic neurons in the substantia nigra compacta. In certain embodiments, the compositions of this invention are intended for use in the treatment or prevention of neurological disorders such as Parkinson's disease in a subject, said use comprising reducing or preventing degeneration of dopaminergic neurons in the substantia nigra compacta and the subsequent loss of their projection nerve fibers in the striped body. In certain embodiments, the compositions of this invention are for use in the treatment or prevention of neurological disorders such as Parkinson's disease in a subject, said use comprising reducing or preventing loss of nigrostrial dopaminergic neurons.

In certain embodiments, the compositions of this invention are for use in the treatment or prevention of neurological disorders, such as Parkin's disease, 11,037,000 sone, in a subject, said use comprising increasing dopamine levels. In certain embodiments, the compositions of this invention are intended for use in the treatment or prevention of neurological disorders, such as Parkinson's disease, in a subject, said use comprising increasing DOPAC levels. In certain embodiments, the compositions of this invention are for use in the treatment or prevention of neurological disorders, such as Parkinson's disease in a subject, said use comprising increasing dopamine and DOPAC levels. In certain embodiments, the striatum levels of dopamine and / or DOPAC are increased.

Alzheimer's disease and dementia.

In DSM-5, the term dementia was replaced by the terms severe neurocognitive disorder and mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class of psychiatric disorders. The most common neurocognitive disorder is Alzheimer's disease, accompanied by vascular dementia or a mixed form of the two. Other forms of neurodegenerative disorders (eg, Lewy body dementia, frontotemporal dementia, Parkinson's dementia, Creutzfeldt-Jakob disease, Huntington's disease, and Wernicke-Korsakoff syndrome) are associated with dementia.

Alzheimer's disease and dementia are also characterized by loss of neurons, therefore the neuroprotective and neuroproliferative effects shown in the examples in the case of the compositions according to this invention indicate that they may be useful for the treatment or prevention of these pathological conditions.

The DSM-5 symptomatic criteria for dementia indicate a significant decline in cognitive ability from previous levels of functioning in one or more areas selected from: learning and memory; language; executive function; integrated attention; perceptual-motor and social cognition. Cognitive deficits should impair independence in daily activities. In addition, cognitive deficits do not arise exclusively in the context of delirium and are not more appropriately explained in terms of another mental disorder (eg, MDD or schizophrenia).

In addition to the primary symptom, subjects with neurodegenerative disorders exhibit behavioral and psychiatric symptoms, including agitation, aggression, depression, apathy, psychosis, and disturbances in the sleep-wake cycle.

Neurodegenerative disorders can develop or continue as a result of dysfunction of the microbiota-gut-brain axis. Thus, in preferred embodiments, the compositions of this invention are for use in the treatment or prevention of neurodegenerative disorders in a subject. In preferred embodiments, the neurodegenerative disease is Alzheimer's disease. In other embodiments, the neurodegenerative disease is selected from vascular dementia; mixed form of Alzheimer's disease and vascular dementia; dementia with Lewy bodies; frontotemporal dementia; dementia in Parkinson's disease; Creutzfeldt-Jakob disease; Huntington's disease; and Wernicke-Korsakoff syndrome.

In preferred embodiments, the implementation of the compositions of this invention result in the prevention, reduction or amelioration of one or more symptoms of neurodegenerative disorders in a subject. In certain embodiments, the compositions of this invention result in the prevention, reduction, or weakening of cognitive function in a subject. In certain embodiments, the compositions of this invention result in an increase in the level of functioning of a subject with neurodegenerative disorders in one or more areas selected from: learning and memory; language; executive function; integrated attention; perceptual-motor and social cognition. In some embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of one or more behavioral and psychiatric symptoms associated with neurodegenerative disorders selected from arousal, aggression, depression, apathy, psychosis, and sleep-wake cycle disorders.

In certain embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of symptomatic disease by interfering with putative pathogenic mechanisms at the preclinical stage. In certain embodiments, the compositions of this invention normalize disease modification while slowing or arresting progression of symptoms. In some embodiments, slowing or stopping the progression of symptoms correlates with signs of delay in underlying neuropathological processes. In preferred embodiments, the implementation of the compositions of this invention result in the normalization of symptoms of neurodegenerative disorders, including an increase in cognitive and functional capabilities. In preferred embodiments, the compositions of this invention result in the normalization of behavioral and psychiatric symptoms of dementia (BPSD). In preferred embodiments, the composition of the invention results in normalization of the ability of a subject with a neurodegenerative disorder to perform daily activities.

In preferred embodiments, the compositions of this invention result in the normalization of both cognitive function and functioning in a subject with Alzheimer's disease. In some embodiments, the implementation of the composition of this invention results in a normalization of the cognitive outcome in a subject with Alzheimer's disease. In some embodiments, the implementation of the compositions of this invention result in a normalization of the functional end result in a subject with Alzheimer's disease. In preferred embodiments, the compositions of this invention result in a normalization of the cognitive and functional endpoint in a subject with Alzheimer's disease. In yet further preferred embodiments, the compositions of this invention result in an overall clinical response (overall endpoint) in a subject with Alzheimer's disease.

In some embodiments, the implementation of the compositions according to this invention lead to the normalization of symptoms of neurodegenerative disorders in accordance with a symptomatic or diagnostic scale. In certain embodiments, tests for assessing symptomatic normalization of Alzheimer's disease (and other neurodegenerative disorders) are selected from objective cognitive tests, tests for assessing activity in daily life, cumulative assessment of changes, health tests related to quality of life, and tests assessing behavioral and psychiatric symptoms of neurodegenerative disorders.

In certain embodiments, objective cognitive tests use the Alzheimer's Disease Assessment Scale cognitive subscale (ADAS-cog) cognitive subscale and the classic ADAS scale to assess symptomatic normalization. In certain embodiments, symptomatic cognitive normalization is assessed using a neuropsychological test battery for use in Alzheimer's disease (NTB).

In some embodiments, the cumulative assessment of the change test uses the Clinical Global Impression (CGI-I) Global Improvement scale to assess psychiatric and neurological disorders. In some embodiments, the cumulative scale is an extended clinician's Interview Based Impression of Change plus (CIBICplus). In some embodiments, the cumulative scale is an ADCS-CGIC Alzheimer's Disease Cooperative Study Unit Clinician's Global Impression of Change scale.

In certain embodiments, the health-related quality of life characteristics are Alzheimer's Disease-Related QOL (ADRQL) and Alzheimer's Disease-related QOL (QOL-AD).

In certain embodiments, tests assessing behavioral and psychiatric symptoms of neurodegenerative disorders are selected from the Behavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVEAD); Behavioral Rating Scale for Dementia (BRSD); neuropsychiatric questionnaire (NPI - Neuropsychiatric Inventory); and the Cohen-Mansfield Agitation Inventory (CMAI).

In some embodiments, the compositions of this invention are particularly effective in preventing, reducing, or ameliorating neurodegenerative disorders when used in combination with another drug to treat neurodegenerative disorders. In certain embodiments, such drugs include acetylcholinesterase inhibitors including donepezil (Aricept®), galantamine (Razadyne®) and rivastigmine (Exelon®), as well as memantine.

Multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease in which the myelin sheaths surrounding neurons in the brain and spinal cord are destroyed. The exact causes underlying MS are unknown, however, they are believed to vary between individuals. Certain forms of MS are hereditary. Environmental factors are thought to also contribute to the development of MS. In some individuals, a combination of both genetic and environmental factors can cause the onset of MS.

There is a wide variety of symptoms associated with MS. Subjects can exhibit almost any neurological symptom associated with impaired autonomic, visual, motor, or sensory control. The exact symptoms will vary depending on the lesion / demyelination site.

IL-8 plays a role in the formation of myelin sheaths. Thus, the compositions of this invention can be designed for use in remyelination of neurons in subjects with

- 13 037000

MS. The compositions of this invention can also be used to protect neurons from demyelination. In other words, the compositions of this invention can be designed for use in a method for treating or preventing multiple sclerosis by restoring or preventing loss of myelin sheaths in neurons.

In some embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of one or more symptoms of MS in a subject. In some embodiments, the implementation of the compositions of this invention result in the prevention, reduction or alleviation of fatigue in a subject. In certain embodiments, the compositions of this invention result in the prevention, reduction, or abatement of resting tremor, muscle weakness, muscle spasms, muscle stiffness, paresthesia, and / or ataxia in a subject. In certain embodiments of the implementation of the compositions according to this invention lead to the prevention, reduction or weakening of one or more symptoms associated with the progression of MS, selected from the list consisting of disorders of the autonomic nervous system: constipation, urinary disorders, sexual dysfunction, dysphagia, dysarthria, syncope , vertigo and / or dizziness; sleep disorders; and impaired sense of smell or temperature perception. In some embodiments, the implementation of the compositions of this invention result in the prevention, reduction or amelioration of one or more ocular symptoms associated with MS. In some embodiments, the ocular symptom is selected from the list consisting of vision loss, eye pain, color blindness, double vision, and / or involuntary eye movement in a subject.

In some embodiments, the compositions of this invention result in the prevention, reduction, or amelioration of dizziness, vertigo, neuropathic pain, musculoskeletal pain, cognitive dysfunction, fecal incontinence, dysphagia, dysarthria, or any combination thereof.

In some embodiments, the implementation of the compositions of this invention result in the prevention, reduction or alleviation of symptoms of depression or anxiety associated with MS.

In some embodiments, symptom normalization is determined using the 2017 McDonald criteria for the diagnosis of MS.

In certain embodiments, treatment with compositions of this invention results in a decrease in the incidence of MS or the severity of MS. In certain embodiments, the compositions of this invention are intended for use in reducing the frequency of relapses or the severity of relapses. In certain embodiments, treatment with compositions of this invention results in the prevention of decreased motor function or results in normalization of motor function associated with MS. In certain embodiments, the compositions of this invention are intended for use in preventing decreased motor function or for use in normalizing motor function in the treatment of MS. In certain embodiments, treatment with compositions of this invention results in the prevention of paralysis in MS. In certain embodiments, the compositions of this invention are for use in the prevention of paralysis in the treatment of MS.

In certain embodiments, the compositions of this invention are intended for use in the prevention of multiple sclerosis in a patient who has been identified as at risk of developing multiple sclerosis, or who has been diagnosed with early stage multiple sclerosis or relapsing-remitting multiple sclerosis. The compositions of this invention may be useful for preventing the development of MS. The compositions of this invention may be useful for preventing the progression of MS. In certain embodiments, the compositions of this invention are for use in a patient identified as having a genetic predisposition to MS, such as a major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA) -DR2, or HLA-DR4.

The compositions of this invention may be useful in the treatment or normalization of multiple sclerosis. The compositions of this invention may be particularly useful for reducing symptoms associated with multiple sclerosis. Treatment or prevention of multiple sclerosis can refer, for example, to alleviating the severity of symptoms or reducing the frequency of exacerbations or the range of triggers that pose a problem to the patient. In certain embodiments, the compositions of this invention result in slowing or arresting the progression of the disease.

In certain embodiments, the compositions of this invention are for use in the treatment of relapsing-remitting MS. In alternative embodiments, the compositions of this invention are intended for use in the treatment of progressive MS, such as progressive MS (SPMS) that develops over time after diagnosis of RRMS, primary progressive MS (PPMS), which manifests itself as gradual continuous neurological impairment and progressive relapse of MS. (PRMS), which is similar to PPMS, but with overlapping relapses.

- 14 037000

In certain embodiments, the compositions of this invention are for use in the treatment of one or more symptoms of MS selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle cramps, muscle rigidity, muscle weakness, movement disorders, pain , disorders in thinking, learning and planning, depression and anxiety, sexual dysfunctions, bladder disorders, bowel disorders, difficulty speaking and swallowing.

Neurochemical factors, neuropeptides and neurotransmitters, and the microbiota-gut axis of the brain.

As stated 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 signaling molecules. Changes in the levels of these signaling molecules lead to neurodegenerative diseases. Experiments carried out by the present inventors indicate that administration of the Megasphaera species, and in particular Megasphaera massiliensis, can modulate indole and kynurenine levels. Dysregulation of these metabolites can lead to neurodegenerative diseases such as Parkinson's disease.

In certain embodiments, the compositions of this invention result in modulation of levels of brain monoamines and their metabolites. In preferred embodiments, said metabolite is kinurerin. In certain embodiments, the compositions of the invention result in the modulation of kynurenine, which is the major pathway for tryptophan metabolism, which acts as a pathway for the production of nicotinamide adenine dinucleotide (NAD +). Kynurenine can be metabolized to neuroactive compounds such as kynureic acid (KYNA) and 3-hydroxy-1-kynurenine (3-OH-1-KYN), and in additional steps to quinolinic acid (QUIN). Dysregulation of the kinurein pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds. Changes in kinurein metabolism may be associated with the development of Parkinson's disease. It has been demonstrated that kynurenine levels are reduced in the frontal cortex, shell, and the compact substantia nigra of patients with PD [ ³² ]. Thus, in certain embodiments, the compositions of this invention are intended for use in increasing kynurenine levels in the treatment of Parkinson's disease.

In certain embodiments of the implementation of this invention, the compositions of this invention can lead to increased levels of kynurenine. Elevated kynurenine levels have been shown to attenuate MPP + -induced neuronal death in vitro in a human dopaminergic neuroblastoma cell line [ ³³ ]. In certain embodiments, kynurenine and kynurenic acid can result in activation of the aryl hydrocarbon GI receptor (Ahr) and GPR35 receptors. Activation of the Ahr receptor leads to the induction of the production of IL-22, which can lead to inhibition of local inflammation. GPR35 activation leads to the induction of inositol triphosphate production and Ca2 + mobilization.

In certain embodiments, the compositions of this invention result in modulation of indole levels. In preferred embodiments, said metabolite is kinurerin. In certain embodiments, the compositions of this invention result in modulation of kynurenine, which is the major pathway for tryptophan metabolism.

Signal transmission along the microbiota-gut-brain axis is modulated by levels of neurochemical factors, neuropeptides, and neurotransmitters. Accordingly, in certain embodiments, the compositions of this invention result in modulation of the levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain embodiments of the implementation of the composition according to this invention lead to a direct change in biochemical transformations in the CNS.

Signal transmission along the microbiota-gut-brain axis is modulated by levels of γ-aminobutyric acid (GABA). Accordingly, in preferred embodiments, the compositions of this invention result in modulation of GABA levels. GABA is an inhibitory neurotransmitter that decreases neuronal excitability. In certain embodiments, the compositions of this invention result in increased levels of GABA. In certain embodiments, the compositions of this invention result in a decrease in GABA levels. In certain embodiments, the compositions of this invention result in altered GABAergic neurotransmission. In certain embodiments, the compositions of this invention result in modulation of GABA transcription at various sites in the central nervous system. In certain embodiments, commensal-derived GABA crosses the blood-brain barrier and directly affects neurotransmission. In certain embodiments, the compositions of this invention result in a decrease in GABA in the hippocampus, amygdala, and / or blue spot. In certain embodiments, the compositions of this invention result in an increase in GABA in areas of the cerebral cortex.

- 15 037000

Immune response.

Signal transmission along the microbiota-gut-brain axis is modulated by changes in the immune response and by inflammatory factors and markers. Accordingly, in certain embodiments, the compositions of this invention can result in modulation of an immune response. In certain embodiments, the compositions of this invention result in modulation of systemic levels of circulating neuroimmune signaling molecules. In certain preferred embodiments, the compositions of this invention result in modulation of pro-inflammatory cytokine production and inflammation. In certain embodiments, the compositions of this invention result in modulation of the inflammatory process. In certain embodiments, the compositions of this invention result in a decrease in the production and secretion of IL-6. In certain embodiments, the compositions of this invention result in decreased levels of NFkB promoter activation. In certain embodiments, the compositions of this invention are capable of modulating the activation of IL-6 production by the potent pro-inflammatory endotoxin lipopolysaccharide (LPS). In certain embodiments, the compositions of this invention are capable of modulating the NFkB promoter with LPS and α-synuclein mutant proteins such as A53T. Increased circulating levels of cytokines are closely associated with various neurodegenerative disorders, including Parkinson's disease, dementia, and Alzheimer's disease. In certain embodiments, the compositions of this invention are for use in lowering IL-6 levels and / or NFkB levels in the treatment of a neurodegenerative disease.

In some embodiments, the implementation of the compositions of this invention lead to an increase in the secretion of IL-8. IL-8 has been shown to induce the formation of myelin sheaths and restore effective interaction between neurons. Thus, in some embodiments, the compositions of this invention are intended for use in inducing myelin sheath formation in the treatment of neurodegenerative diseases. In some embodiments, the implementation of the compositions of this invention are intended for use in restoring interaction between neurons. In some embodiments, the implementation of the compositions of this invention are intended for use in maintaining interaction between neurons.

Signal transmission along the microbiota-gut-brain axis is modulated by the levels of commensal metabolites. Accordingly, in certain embodiments, the compositions of this invention result in modulation of systemic levels of microbiota metabolites. In certain embodiments, the compositions of this invention result in modulation of short chain fatty acid (SCFA) levels. In certain embodiments, the SCFA level 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 this invention modulate the ability of the SCFA to cross the blood-brain barrier.

Acetylation and deacetylation of histones are important epigenetic regulators of gene expression. Impaired balance of histone acetylation and deacetylation can lead to apoptosis. Dysregulation of such histone acetyltransferases plays a role in the pathogenesis associated with age-related 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 this invention may result in modulation of histone deacetylase activity. In certain embodiments, the compositions of this invention may result in a decrease in histone deacetylase activity. In certain embodiments, the compositions of this invention can result in a decrease in histone acetylase activity.

Patients with neurodegenerative diseases, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and amitrophic lateral sclerosis, exhibit high levels of lipid peroxidation. Lipids are reactive oxygen species susceptible to oxidation, and the brain has a high content of polyunsaturated fatty acids.

Accordingly, in certain embodiments, the compositions of this invention can lead to modulation of lipid peroxidation. In certain embodiments of the implementation of the composition according to this invention can lead to a decrease in lipid peroxidation. Reduction of oxidative damage caused by reactive oxygen species can be used to target neurodegenerative diseases in early stages. Accordingly, in certain embodiments, the compositions of this invention are intended for use in the treatment of early stage neurodegeneration. In addition, accordingly, in certain embodiments, the compositions of this invention are intended for

- 16,037,000 applications in the prevention of the development of neurodegenerative diseases. In such embodiments, the implementation of the compositions according to this invention can be designed for use in a patient that has been identified as at risk of developing a neurodegenerative disease.

Signal transmission along the microbiota-gut-brain axis is modulated by levels of permeability in the gastrointestinal tract. Accordingly, in some embodiments, the implementation of the compositions of this invention result in a change in the integrity of the epithelium in the gastrointestinal tract. In certain embodiments, the compositions of this invention result in modulation of gastrointestinal permeability. In certain embodiments, the compositions of this invention result in modulation of the barrier function and integrity of the gastrointestinal tract. In certain embodiments, the compositions of this invention result in modulation of gastrointestinal motility. In certain embodiments, the compositions of this invention result in modulation of the movement of commensal metabolites and inflammatory signaling molecules into the bloodstream from the lumen of the gastrointestinal tract.

Signal transmission along the microbiota-gut-brain axis is modulated by levels of microbiome composition in the gastrointestinal tract. Accordingly, in certain embodiments, the compositions of this invention result in modulation of the composition of the gastrointestinal tract microbiome. In certain embodiments, the compositions of this invention result in the prevention of microbiome dysbiosis and a concomitant increase in toxic metabolites (eg, LPS). In certain embodiments, the compositions of this invention result in modulation of Clostridium levels in the gastrointestinal tract. In preferred embodiments, the compositions of this invention result in a decrease in the level of Clostridium in the gastrointestinal tract. In certain embodiments, the compositions of this invention result in decreased levels of Campylobacter jejuni. In certain embodiments, the compositions of this invention result in the modulation of the proliferation of harmful anaerobic bacteria and the production of neurotoxins produced by said bacteria. In certain embodiments, the compositions of this invention result in the modulation of Lactobacillus and / or Bifidobacterium levels in the microbiome. In certain embodiments, the compositions of this invention result in modulation of levels of the genera Sutterella, Prevotella, Ruminococcus and / or the Alcaligenaceae family in the microbiome. In certain embodiments, the compositions of this invention result in increased levels of Lactobacillus plantarum and / or Saccharomyces boulardii.

Brain damage.

These examples demonstrate that the compositions of this invention are neuroprotective and have HDAC inhibitory activity. HDAC2 is a major target in functional recovery from stroke [35], and inhibition of HDAC can prevent white matter damage [36], so the compositions of this invention may be useful in the treatment of brain damage.

In certain embodiments, the compositions of this invention are for use in treating brain damage. In some embodiments, the brain injury is traumatic brain injury. In some embodiments, the brain injury is an acquired brain injury. In some embodiments, the implementation of the compositions of this invention are intended for use in the treatment of brain damage resulting from trauma. In some embodiments, the implementation of the compositions of this invention are intended for use in the treatment of brain damage resulting from a tumor. In some embodiments, the compositions of this invention are for use in treating brain damage resulting from stroke. In some embodiments, the compositions of this invention are for use in treating brain damage resulting from cerebral hemorrhage. In some embodiments, the compositions of this invention are for use in treating brain damage resulting from encephalitis. In some embodiments, the compositions of this invention are for use in treating brain damage resulting from cerebral hypoxia. In some embodiments, the compositions of this invention are for use in the treatment of brain damage resulting from cerebral anoxia.

In preferred embodiments, the compositions of this invention are for use in the treatment of stroke. The effects shown in the examples are especially related to the treatment of stroke. A stroke occurs when blood flow to at least part of the brain is disrupted. Without an adequate supply of blood to provide oxygen and nutrients to the brain tissue and remove byproducts from the brain tissue, brain cells begin to die. Stroke symptoms depend on the area of the brain that is impaired as a result of insufficient blood flow. Symptoms include paralysis, muscle numbness or weakness, loss of balance, dizziness, sudden severe headache, speech impairment, memory loss, loss of reasoning ability, sudden confusion, blurred vision, coma, or even death. A stroke is also referred to as a brain attack or cerebrovascular accident (CVA). Stroke symptoms can be short-lived if sufficient blood flow is restored within a short period of time. At the same time, if insufficient blood flow continues for a significant period of time, the symptoms may be irreversible.

In some embodiments, the stroke is cerebral ischemia. Cerebral ischemia occurs when there is insufficient blood flow to the brain tissue to meet metabolic needs. In some embodiments, the cerebral ischemia is focal cerebral ischemia, i. E. limited to a specific area of the brain. In some embodiments, the cerebral ischemia is global cerebral ischemia, i. E. covering a large area of brain tissue. Focal cerebral ischemia usually occurs when a cerebral vessel becomes blocked, either partially or completely, leading to a decrease in blood flow to a specific area 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 blood clot or blood clot that develops in a vessel in the brain and restricts or blocks blood flow. In some embodiments, the ischemic stroke is 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 an area distant from its site of origin. In some embodiments, the ischemic stroke is an embolic stroke. Global cerebral ischemia occurs when blood flow to the brain is generally blocked or restricted. In some embodiments, global cerebral ischemia is caused by hypoperfusion, for example, as a result of shock. In some embodiments, the global cerebral ischemia is the result of a heart attack.

In some embodiments, the subject diagnosed with a brain injury has suffered from cerebral ischemia. In some embodiments, the implementation of the subject diagnosed with a brain injury suffered from focal cerebral ischemia. In some embodiments, the implementation of the subject, diagnosed with brain damage, suffered from ischemic stroke. In some embodiments, the subject diagnosed with a brain injury has suffered from a thrombotic stroke. In some embodiments, the subject diagnosed with a brain injury suffered from an embolic stroke. In some embodiments, the subject diagnosed with a brain injury has suffered from global cerebral ischemia. In some embodiments, the subject diagnosed with a brain injury suffered from hypoperfusion. In some embodiments, the subject diagnosed with a brain injury has suffered from a heart attack.

In some embodiments, the compositions of this invention are for use in the treatment of cerebral ischemia. In some embodiments, the compositions of this invention are for use in the treatment of focal cerebral ischemia. In some embodiments, the compositions of this invention are for use in the treatment of ischemic stroke. In some embodiments, the compositions of this invention are for use in the treatment of thrombotic stroke. In some embodiments, the compositions of this invention are for use in the treatment of embolic stroke. In some embodiments, the compositions of this invention are for use in the treatment of global cerebral ischemia. In some embodiments, the compositions of this invention are for use in the treatment of hypoperfusion.

In some embodiments, the stroke is a hemorrhagic stroke. Hemorrhagic stroke is caused by bleeding in or around the brain, causing cells and tissues in the brain to swell, compress, and break down. A hemorrhagic stroke is often the result of a weakened blood vessel that ruptures and bleeds into the surrounding brain. In some embodiments, the hemorrhagic stroke is a cerebral hemorrhage, i. E. caused by a hemorrhage in the very tissue of the brain. In some embodiments, the cerebral hemorrhage is caused by an intraparenchymal hemorrhage. In some embodiments, the cerebral hemorrhage is caused by an intraventricular hemorrhage. In some embodiments, the hemorrhagic stroke is a subarachnoid hemorrhage, i.e. hemorrhage that occurs outside of the brain tissue, but is still within the skull. In some embodiments, the hemorrhagic stroke is the result of cerebral amyloid angiopathy. In some 18,037,000 embodiments, the hemorrhagic stroke is the result of a cerebral aneurysm. In some embodiments, the hemorrhagic stroke is the result of cerebral arteriovenous malformation (AVM).

In some embodiments, the subject diagnosed with a brain injury has suffered from a hemorrhagic stroke. In some embodiments, the subject diagnosed with a brain injury has suffered from a cerebral hemorrhage. In some embodiments, the subject diagnosed with a brain injury has suffered from intraparenchymal hemorrhage. In some embodiments, the subject diagnosed with a brain injury has suffered from intraventricular hemorrhage. In some embodiments, the subject diagnosed with a brain injury has suffered from subarachnoid hemorrhage. In some embodiments, the subject diagnosed with a brain injury suffered from cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with a brain injury suffered from a cerebral aneurysm. In some embodiments, the implementation of the subject diagnosed with brain damage suffered from cerebral AVM.

In some embodiments, the compositions of this invention are for use in the treatment of hemorrhagic stroke. In some embodiments, the compositions of this invention are for use in the treatment of cerebral hemorrhage. In some embodiments, the compositions of this invention are for use in the treatment of intraparenchymal hemorrhage. In some embodiments, the compositions of this invention are for use in the treatment of intraventricular hemorrhage. In some embodiments, the compositions of this invention are for use in the treatment of subarachnoid hemorrhage. In some embodiments, the compositions of this invention are for use in the treatment of cerebral amyloid angiopathy. In some embodiments, the compositions of this invention are for use in the treatment of a cerebral aneurysm. In some embodiments, the compositions of this invention are for use in the treatment of cerebral AVM.

Restoration of sufficient blood flow to the brain after a period of suspension, despite being effective in relieving symptoms associated with stroke, may paradoxically lead to further damage to brain tissue. During the suspension period, the affected tissue suffers from a lack of oxygen and nutrients, and the sudden restoration of blood flow can lead to inflammation and oxidative damage as a result of the induction of oxidative stress. This is known as reperfusion injury, and has been scientifically proven not only after a stroke, but also after a heart attack or other tissue injury, when blood supply returns to the tissue after a period of ischemia or oxygen deprivation. In some embodiments, the subject diagnosed with brain injury suffered from reperfusion injury due to stroke. In some embodiments, the compositions of this invention are for use in treating stroke reperfusion injury.

A transient ischemic attack (TIA), often referred to as a mini-stroke, is a recognized warning sign of a more serious stroke. Subjects who have experienced one or more TIAs thus have a higher risk of stroke. In some embodiments, the implementation of the subject diagnosed with brain damage suffered from TIA. In some embodiments, the compositions of this invention are for use in the treatment of TIA. In some embodiments, the compositions of this invention are for use in treating brain damage in a subject who has suffered from TIA.

High blood pressure, high blood cholesterol, family history of stroke, heart disease, diabetes mellitus, cerebral aneurysms, arteriovenous malformations, sickle cell anemia, vasculitis, blood clotting disorders, nonsteroidal anti-inflammatory drug (NSAID) use, tobacco smoking, use high alcohol consumption, illegal drug use, obesity, physical inactivity and poor diet are all considered risk factors for stroke. In particular, it has been convincingly shown that lowering blood pressure leads to the prevention of both ischemic and hemorrhagic strokes [ ³⁷ , ³⁸ ]. In some embodiments, the compositions of this invention are for use in treating brain damage in a subject that 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 a high blood cholesterol level. In some embodiments, the subject has a family history of 19,037,000 non-stroke. In some embodiments, the subject has a heart condition. In some embodiments, the subject has diabetes mellitus. 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 anemia. In some embodiments, the subject has a bleeding disorder. In some embodiments, the subject has a history of using non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the implementation of the subject smokes tobacco. In some embodiments, the implementation of the subject consumes large amounts of alcohol. In some embodiments, the subject is using illegal drugs. In some embodiments, the implementation of the subject is obese. In some embodiments, the implementation of the subject is overweight. In some embodiments, the subject is physically inactive. In some embodiments, the subject is malnourished.

These examples indicate that the compositions of this invention may be useful for treating brain damage and promoting recovery when administered before the onset of the injury. Thus, the compositions of this invention may be particularly useful for treating brain damage when administered to subjects at risk of brain damage such as stroke.

In certain embodiments, the compositions of this invention are intended for use in reducing damage caused by potential brain injury, preferably stroke. These compositions can reduce induced damage when administered prior to potential brain damage, in particular when administered to a patient identified as at risk of brain damage.

These examples indicate that the compositions of this invention may be useful for treating brain damage and promoting recovery when administered after the onset of the injury phenomenon. Thus, the compositions of this invention may be particularly useful for treating brain injury when administered to subjects following brain injury such as stroke.

In some embodiments, the implementation of the compositions of this invention result in the treatment of brain damage by reducing damage associated with motor function. In some embodiments, the implementation of the compositions of this invention results in the treatment of brain damage as a result of normalization of motor function. In some embodiments, the compositions of this invention result in the treatment of brain damage resulting from increased muscle strength. In some embodiments, the implementation of the compositions of this invention result in the treatment of brain damage resulting from improved memory. In some embodiments, the compositions of this invention result in the treatment of brain damage as a result of increased public acceptance. In some embodiments, the implementation of the compositions of this invention results in the treatment of brain damage as a result of the normalization of neurological function.

Treatment for brain damage can relate, for example, to alleviating the severity of symptoms. Treatment of brain damage can also refer to reducing neurological impairment after a stroke. Compositions of this invention for use in treating stroke may be offered to a subject prior to the onset of stroke, for example, a patient identified as at risk of stroke. Compositions of this invention for use in the treatment of stroke may be provided after stroke, for example, during recovery. Compositions of this invention for use in the treatment of stroke may be provided during the acute recovery phase (i.e., up to and including one week after stroke). Compositions of this invention for use in treating stroke may be offered during the subacute recovery phase (ie, from one week to three months inclusively after stroke). Compositions of this invention for use in the treatment of stroke may be offered during the chronic recovery phase (from three months after stroke).

In certain embodiments, the compositions of this invention are intended to be used in combination with an auxiliary active agent. In certain embodiments, the compositions of this invention are intended for use in combination with aspirin or tissue plasminogen activator (tPA). Other adjuvants include other antiplatelet agents (such as clopidogrel), anticoagulants (such as heparins, warfarin, apixaban, dabigatran, edoxaban, or rivaroxaban), antihypertensive agents (such as diuretics, ACE inhibitors, calcium channel blockers or beta alpha blockers) or statins. The compositions of this invention can lead to an improvement in the patient's response to the auxiliary active agent.

In certain embodiments, the compositions of this invention result in a reduction in the effect of ischemia on tissues. In certain embodiments of the composition according to this

- 20 037000 of the invention lead to a decrease in the degree of damage to the tissue caused by ischemia. In certain embodiments, the tissue injured by ischemia is brain tissue. In certain embodiments, the compositions of this invention result in a decrease in necrosis or the number of necrotic cells. In certain embodiments, the compositions of this invention result in a decrease in apoptosis or in the number of apoptotic cells. In certain embodiments, the compositions of this invention result in a decrease in the number of necrotic or apoptotic cells. In certain embodiments, the compositions of this invention result in the prevention of cell death due to necrosis and / or apoptosis. In certain embodiments, the compositions of this invention result in the prevention of cell death due to necrosis and / or apoptosis caused by ischemia. In certain embodiments, the compositions of this invention result in the repair of tissue damaged by ischemia. In certain embodiments, the compositions of this invention result in an increase in the clearance rate of necrotic cells and / or apoptotic cells. In certain embodiments, the compositions of this invention result in an increase in the clearance efficiency of necrotic cells and / or apoptotic cells. In certain embodiments, the compositions of this invention result in cell replacement and / or regeneration in tissues. In certain embodiments, the compositions of this invention result in cell replacement and / or regeneration in tissues damaged by ischemia. In certain embodiments, the compositions of this invention result in an improvement in the overall histological condition of the tissue (eg, on biopsy).

Routes of administration.

Preferably, the compositions of this invention are intended for administration to the gastrointestinal tract to provide delivery and / or partial or complete colonization of the intestine with the bacterial strain of this invention. Typically, the compositions of this invention are administered orally, however, they can be administered rectally, intranasally, or via the buccal or sublingual routes.

In certain embodiments, the compositions of this invention can be administered as a foam, cream, or gel.

In certain embodiments of the embodiments, the compositions of this invention can be administered as a suppository, such as a rectal suppository, for example, in the form of cocoa butter (cocoa butter), synthetic tallow (eg, supokir, witepsol), glycerogelatin, polyethylene glycol, or soapy glycerin compositions.

In certain embodiments, the composition of this invention is administered into the gastrointestinal tract using a tube, such as a nasogastric tube, an orogastric tube, a gastric tube, a jejunostomy tube (J-tube), a percutaneous endoscopic gastrostomy (PEG), or port, such as a port a system that is implanted in the chest area that provides access to the stomach, jejunum, and other suitable access ports.

The compositions of this invention can be administered in a single dose, or they can be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of this invention are for daily administration.

In certain embodiments of the invention, treatment in accordance with this invention is accompanied by an assessment of the patient's gut microbiota. Treatment may be repeated if delivery and / or partial or complete colonization with the strain of the invention is not achieved, resulting in no efficacy observed, or treatment may be discontinued if delivery and / or partial or complete colonization is successful and efficacy is observed.

In certain embodiments, a composition of the invention can be administered to a pregnant animal, eg, a mammal, such as a human, for the purpose of preventing an inflammatory or autoimmune disease developing in its child in utero and / or after birth.

The compositions of this invention can be administered to a patient who has been diagnosed with a neurodegenerative disease or who has been identified as at risk for a neurodegenerative disease. These compositions can also be administered as a prophylactic measure in order to prevent the development of a neurodegenerative disease in a healthy patient.

The compositions of this invention can be administered to a patient who has been identified as having an abnormal gut microbiota. For example, a patient may have reduced or absent colonization of Megasphaera, and in particular Megasphaera massiliensis.

The compositions of this invention can be administered as a food product, such as a food supplement.

In general, the compositions of this invention are intended to treat humans, although they can be used to treat animals, including monogastric mammals such as poultry, pigs, cats, dogs, horses, or rabbits. The compositions of this invention may be useful for enhancing the growth and characteristics of animals. When administered to animals, may

- 21 037000 a gastric tube can be used.

Compositions.

Typically, the composition of this invention contains bacteria. In preferred embodiments of the present invention, the composition is formulated in lyophilized form. For example, a composition according to this invention may contain granules or gelatin capsules, for example, hard gelatin capsules, containing the bacterial strain of this invention.

Preferably, the composition of this invention contains lyophilized bacteria. Lyophilization of bacteria is a well-established procedure, and guidance is available, for example, in the literature [39], [41].

Alternatively, the composition of this invention may comprise a live, active bacterial culture.

In some embodiments, the bacterial strain in the composition of this invention has not been inactivated, for example, has not been thermally inactivated. In some embodiments, the bacterial strain in the composition of this invention has not been killed, for example, has not been killed by heat. In some embodiments, the implementation of the bacterial strain in the composition of this invention has not been attenuated, for example, has not been attenuated by heat. For example, in some embodiments, the bacterial strain in the composition of this invention has not been killed, inactivated, and / or attenuated. For example, in some embodiments, the bacterial strain in the composition of this invention is live. For example, in some embodiments, the bacterial strain in the composition of this invention is viable. For example, in some embodiments, the bacterial strain in the composition of this invention is capable of partially or completely colonizing the intestine. For example, in some embodiments, the bacterial strain in the composition of this invention is viable and capable of partially or completely colonizing the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.

In preferred embodiments, the implementation of the composition according to this invention is encapsulated for the purpose of providing delivery of the bacterial strain into the intestine. Encapsulation protects the composition from degradation prior to administration to the target site by, for example, rupture due to chemical or physical stimuli, such as pressure, enzymatic activity or physical disintegration, which can be activated by changes in pH. Any suitable encapsulation method can be used. Exemplary encapsulation techniques include entrapment in a porous matrix, attachment or adsorption to solid surfaces of a support, self-aggregation by flocculation or by crosslinking agents, and mechanical containment behind a microporous membrane or microcapsule. Guidance on encapsulation that may be suitable for preparing the compositions of this invention is available, for example, in literature sources [42] and [43].

The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated foods are preferred because Megasphaera are anaerobic. Other ingredients (such as, for example, vitamin C) may be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and in vivo survival. Alternatively, the probiotic composition of the present invention may be orally administered as a food or nutritional product such as a fermented milk product based on milk or whey or as a pharmaceutical product.

The specified composition can be formulated as a probiotic.

The composition according to this invention contains a therapeutically effective amount of the bacterial strain according to this invention. A therapeutically effective amount of the bacterial strain is sufficient to be beneficial when administered to a patient. A therapeutically effective amount of the bacterial strain may be sufficient to result in delivery and / or partial or complete colonization of the patient's intestine.

A suitable daily dose of bacteria, for example, for an adult, may be from about 1x10 ³ to about 1x101 'colony forming units (CFU); for example, from about 1x10 ⁷ to about 1 x 10 ¹⁰ CFU; in another example, from about 1x106 to about 1x10 ¹⁰ CFU.

In certain embodiments, said composition comprises the bacterial strain in an amount of from about 1x106 to about 1x10 ^minutes cfu / g, relative to the weight of the composition; for example, from about 1x108 to about 1x 10 ¹⁰ cfu / g. The dose can be, for example, 1, 3, 5 and 10 g.

Typically, a probiotic, such as a composition of this invention, is optionally combined with at least one suitable prebiotic compound. The prebiotic compound is usually a difficult-to-digest carbohydrate, such as an oligo- or polysaccharide, or a sugar alcohol that is not broken down or absorbed in the upper digestive tract.

Known prebiotics include commercial products such as inulin and transgalactooligosaccharides.

In certain embodiments, the prebiotic composition of this invention contains the prebiotic compound in an amount from about 1 to about 30% by weight, based on the total weight of the composition (eg, from 5 to 20% by weight). Carbohydrates can be selected from the group consisting of: fructo-oligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomaltoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosanoligosaccharides (or COS), modified kravidia and beta-glucans, aravidia and beta-glucans , D-tagatose, acacia fibers, carob, oats and citrus fibers. In one aspect, the prebiotics are short-chain fructo-oligosaccharides (for simplicity, shown herein below as FOS-c.c); indicated by FOS-c.c. are hard-to-digest carbohydrates, usually obtained from the conversion of beet sugar, and contain a sucrose molecule to which three glucose molecules are linked.

In certain embodiments, the compositions of this invention are used in combination with another therapeutic compound to treat or prevent a neurodegenerative disease. In some embodiments, the compositions of this invention are administered with nutritional supplements that result in modulation of neuroprotection or neuroproliferation. In preferred embodiments, the nutritional supplement contains or consists of health benefits of vitamins. In certain embodiments, said vitamins are vitamin B6, magnesium, dimethylglycine (vitamin B16), and vitamin C. In certain embodiments, the compositions of this invention are administered in combination with another probiotic.

In certain embodiments, the compositions of this invention are for use in enhancing the effect of the second agent on a neurodegenerative disease. The immunomodulatory effects of the compositions of this invention may result in the brain becoming more responsive to conventional drugs such as levodopa, dopamine receptor agonists, MAO-B inhibitors, COMT inhibitors, glutamate antagonists and / or anticholinergics), which are illustrative adjuncts. agents to be administered in combination (sequentially or simultaneously) with the compositions of this invention.

Compositions of this invention may contain pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients can be found in the literature [44]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in the literature [45]. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerin, and water. The choice of pharmaceutical carrier, excipient or diluent can be made based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may contain, as or as an adjunct carrier, excipient or diluent, any (any) suitable binder (s), lubricant (s), suspending (s) agent (s), coating (s) agent (s), solubilizing (solubilizing) agent (s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethyl cellulose or polyethylene glycol ... Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, colorants, and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters. Antioxidants and suspending agents can also be used.

The compositions of this invention can be formulated as a food product. For example, the food product can provide a nutritional effect in addition to the therapeutic effect of the present invention, for example, in a food supplement. Likewise, a food product can be formulated to enhance the flavor of the composition of this invention or to create a more palatable composition as a result of more similarity to a common food product rather than a pharmaceutical composition. In certain embodiments, the composition of this invention is formulated as a milk-based product. The term milk-based product means any liquid or semi-liquid milk or whey-based product having a variable fat content. The milk-based product can be cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk reconstituted with powdered milk and whey without any processing, or a processed product such as

- 23 037000 yogurt, yogurt, cheese curd, sour milk, sour whole milk, buttermilk and other fermented milk products. Another important group includes dairy beverages such as whey drinks, fermented milk products, condensed milk, milk for infant or newborn formula; flavored milk, ice cream; dairy products such as candy.

In some embodiments, the compositions of this invention comprise one or more bacterial strains of the genus Megasphaera and do not contain bacteria from any other genera, or that contain only minor or biologically insignificant amounts of bacteria from other genera. Thus, in some embodiments, the present invention provides a composition comprising one or more bacterial strains of the genus Megasphaera that do not contain bacteria from any other genera, or that contain only minor or biologically insignificant amounts of bacteria from other genera for use in treatment.

In some embodiments, the compositions of this invention comprise one or more bacterial strains of the species Megasphaera massiliensis and do not contain bacteria from any other species, or that contain only minor or biologically insignificant amounts of bacteria from another species. Thus, in some embodiments, the present invention provides a composition comprising one or more bacterial strains of the species Megasphaera massiliensis that does not contain bacteria from any other species, or that contain only minor or biologically insignificant amounts of bacteria from another species for use in treatment.

In some embodiments, the compositions of this invention comprise one or more bacterial strains of the Megasphaera massiliensis species and do not contain bacteria from any other Megasphaera species, or that contain only minor or biologically insignificant amounts of bacteria from another Megasphaera species. Thus, in some embodiments, the present invention provides a composition comprising one or more bacterial strains of the Megasphaera massiliensis species that does not contain bacteria from any other Megasphaera species, or that contain only minor or biologically insignificant amounts of bacteria from another Megasphaera species for use in treatment.

In certain embodiments, the compositions of this invention contain one bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may contain only minor or biologically insignificant amounts of other bacterial strains or species. Such compositions can be a culture that is essentially free of other types of organisms.

In some embodiments, the invention provides a composition comprising a single bacterial strain of the genus Megasphaera that does not contain bacteria from any other strains, or that contain only minor or biologically insignificant amounts of bacteria from another strain for use in treatment.

In some embodiments, the invention provides a composition comprising one bacterial strain of the species Megasphaera massiliensis that does not contain bacteria from any other strains, or that contain only minor or biologically insignificant amounts of bacteria from another strain for use in treatment.

In some embodiments, the implementation of the compositions of this invention contain more than one bacterial strain. For example, in some embodiments, the compositions of this invention comprise more than one strain 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 does not contain bacteria from any other species. In some embodiments, the compositions of this invention comprise less than 50 strains 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 does not contain bacteria from any other species. In some embodiments, the implementation of the compositions according to this invention contain 1-40, 130, 1-20, 1-19, 118, 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, 210, 2-5, 6-30, 6-15, 16 -25 or 31-50 strains within one species and optionally do not contain bacteria from other species. This invention contains any combination of the above.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises a Megasphaera bacterial strain as part of a microbial consortium. For example, in some embodiments, the Megasphaera 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 Megasphaera bacterial strain in combination with a bacterial strain from another genus. In some embodiments, the implementation of the specified microbial consortium contains two or more bacterial strains obtained from a sample of feces of one organism, for example, a human. In some embodiments, the implementation of the specified microbial consortium does not coexist in

- 24 037000 nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from stool samples from at least two different organisms. In some embodiments, the implementation of two different organisms are from the same species, for example, two different people. In some embodiments, the two different organisms are infant and adult. In some embodiments, the implementation of these two different organisms are a human and a non-human mammal.

In some embodiments, the composition of this invention further comprises a bacterial strain that has the same safety and therapeutic efficacy characteristics as the MRx0029 strain, but that is not MRx0029, or that is not Megasphaera massiliensis.

In some embodiments, implementation in which the composition of this invention contains more than one bacterial strain, species or genus, the individual bacterial strains, species or genera can be designed for separate, simultaneous or sequential administration. For example, the composition can contain all of more than one bacterial strain, species or genus, or bacterial strains, species or genera can be stored separately and can be administered separately, simultaneously or repeatedly. In some embodiments, more than one bacterial strain, species, or genus is stored separately but mixed together before use.

In some embodiments, the bacterial strain for use in the present invention is obtained from the feces of an adult. In some embodiments, implementation in which the composition of this invention contains more than one bacterial strain, all of the bacterial strains are obtained from the feces of an adult, or if other bacterial strains are present, they are present in only minor amounts. The bacteria can be cultured after being obtained from adult feces and can be used in the composition of this invention.

As mentioned above, in some embodiments, the implementation of one or more bacterial strains of Megasphaera provides only the therapeutically active agent (s) in the composition of this invention. In some embodiments, the bacterial (bacterial) strain (s) in the composition is / are only the therapeutically active (s) agent (s) in the composition of this invention.

Compositions for use in accordance with this invention may or may not require registration.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilized. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is spray dried. In certain embodiments, the invention provides the aforementioned pharmaceutical composition, wherein said bacterial strain is lyophilized or spray dried and still alive. In certain embodiments, the invention provides the aforementioned pharmaceutical composition wherein said bacterial strain is lyophilized or spray dried and viable. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilized or spray dried and is capable of partially or completely coloinizing the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilized or spray dried and is viable and capable of partially or completely colonizing the intestine.

In some cases, the lyophilized bacterial strain is diluted by administration. In some cases, dilution occurs using the diluent described in this document.

Compositions of this invention may contain pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of this invention and a pharmaceutically acceptable excipient, carrier or diluent; wherein said bacterial strain is in an amount sufficient to treat a neurodegenerative disease when administered to a subject in need thereof.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of this invention and a pharmaceutically acceptable excipient, carrier or diluent; wherein said bacterial strain is in an amount sufficient to treat or prevent a neurodegenerative disease.

In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein the content of bacterial strains ranges from approximately 25 037000 Tel'nykh 1x10 ³ to about 1x1011 colony forming units per gram weight of the composition.

In certain embodiments, the invention provides the aforementioned pharmaceutical composition, wherein said composition is administered at a dose of 1, 3, 5, or 10 g.

In certain embodiments, the invention provides the above pharmaceutical composition, said composition being administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal and sublingual administration.

In certain embodiments, the invention provides a pharmaceutical composition comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

In certain embodiments, the invention provides a pharmaceutical composition comprising a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition comprising a filler selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free flowing lactose, beta-lactose, corn sweetener, gum arabic, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

In certain embodiments, the present invention provides a pharmaceutical composition further comprising at least one of a preservative, an antioxidant, and a stabilizer.

In certain embodiments, the invention provides a pharmaceutical composition comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilized.

In certain embodiments, the invention provides the aforementioned pharmaceutical composition, wherein said 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 measured in colony forming units persists after a period of at least about 1, 3, 6 months, 1 year, 1.5, 2, 2.5 or 3 years.

In some embodiments, the implementation of the composition of this invention is provided in a sealed container containing the composition described herein. In some embodiments, the sealed container is a sachet or vial. In some embodiments, the implementation of the composition of this invention is provided in a syringe containing the composition described herein.

The composition of this invention, in some embodiments, may be provided as a pharmaceutical composition. For example, said composition can be provided as a tablet or capsule. In some embodiments, the implementation of the specified capsule is a gelatin capsule (gel-cap).

In some embodiments, the compositions of this invention are administered orally. Oral administration may include swallowing, whereby the compound enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration, which allows the compound to enter the bloodstream directly from the oral cavity.

Pharmaceutical formulations suitable for oral administration include solid compressed mass, solid microparticles, soft and liquid (including multiphase or dispersed systems) such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids (eg, aqueous solutions), emulsions or powders; lozenges (including those filled with liquid); jelly Bean; gels; fast-dispersing dosage forms; films; capsules; sprays; and buccal / mucoadhesive patches.

In some embodiments, the pharmaceutical formulation is an enteric formulation, i.e. a gastric-resistant formulation (eg, gastric pH-stable) that is suitable for delivering a composition of this invention to the intestine following oral administration. Enteric formulations may be particularly useful if the bacterium or other component of the composition is acid sensitive, for example susceptible to degradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the implementation of the specified composition is an enteric dosage form. For example, the composition can be an enteric tablet or an enteric capsule or the like. The enteric coating can be a standard enteric coating, for example, a standard coating for

- 26,037,000 tablets, capsules or the like for oral delivery. The specified composition may contain a film coating, for example, a thin film of an enteric polymer, for example, an acid-insoluble polymer.

In some embodiments, the enteric formulation is enteric in nature, eg, resistant to gastric acid, without the need for an enteric coating. Thus, in some embodiments, said composition is an enteric composition that does not contain an enteric coating. In some embodiments, the composition is a capsule made from a thermogel material. In some embodiments, said thermogel material is a cellulosic material such as methyl cellulose, hydroxymethyl cellulose, or hydroxypropyl methyl cellulose (HPMC). In some embodiments, the implementation of the specified capsule contains a shell that does not contain any film-forming polymer. In some embodiments, said capsule contains a shell and said shell contains hydroxypropyl methylcellulose and does not contain any film-forming polymer (eg, see [46]). In some embodiments, the composition is inherently an enteric capsule (eg, Vcaps® from Capsugel).

In some embodiments, the composition is a soft capsule. Soft capsules are capsules which, due to the addition of softeners such as, for example, glycerin, sorbitol, maltitol and polyethylene glycols, can be present in the capsule shell, have a certain elasticity and softness. Soft capsules can be obtained, for example, based on gelatin or starch. Gelatin soft capsules are commercially available from a variety of suppliers. Depending on the route of administration, such as, for example, orally or rectally, the soft capsules can have different shapes, they can be, for example, round, oval, oblong or torpedo-like. Soft capsules can be prepared using standard methods such as, for example, the Scherer method, the Accogel method, or the drop or blow method.

Cultivation methods.

Bacterial strains for use in this invention can be cultured using standard microbiological techniques, as detailed, for example, in the literature [47], [] and [49].

The solid or liquid medium used for cultivation can be YCFA agar or YCFA medium. YCFA medium contains (per 100 ml, approximate value): casiton (1.0 g), yeast extract (0.25 g), NaHCO ₃ (0.4 g), cysteine (0.1 g), K ₂ HpO ₄ (0.045 g), KH2PO4 (0.045 g), NaCl (0.09 g), (NH ₄ ) ₂ SO ₄ (0.09 g), MgS0 ₄ 7H ₂ 0 (0.009 g), CaCl ₂ (0.009 g) , resaruzin (0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), n-aminobenzoic acid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg).

Bacterial strains for use in vaccine compositions.

The present inventors have found that the bacterial strains of this invention are useful in the treatment or prevention of neurodegenerative disorders. This appears to be the result of the effect that the bacterial strains of this invention have on the host's immune system. Thus, the compositions of this invention may also be useful for preventing neurodegenerative disorders when administered as vaccine compositions. In certain embodiments, the bacterial strains of this invention may be killed, inactivated, or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are intended to be administered by injection, for example, by subcutaneous injection.

General instructions.

The practice of this invention will be carried out, unless otherwise indicated, using standard methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the competence of specialists in this field of technology. Such techniques are fully explained in the literature. See, for example, literature sources [50] and [51, 57] and others.

The term comprising encompasses an inclusive as well as consisting, for example, a composition containing X, may consist solely of X, or may include something additional, such as X + Y.

The term approximately in relation to the numerical value of x is optional and means, for example, x + 10%.

An expression does not inherently exclude entirely, for example, a composition that is essentially free of Y may be completely free of Y. If necessary, the expression can be essentially omitted from the definition of the present invention.

References to percent sequence identity between two nucleotide sequences means that when aligned, the indicated percentage of nucleotides are the same when compared to the two sequences. The specified alignment and the specified percent homology or sequence identity can be determined using computer programs known in the art, for example, the programs described in section 7.7.18 of the literature [58]. The preferred alignment is determined using the Smith-Waterman homology search algorithm using an affine gap search, where the gap opening penalty is 12 and the gap extension penalty is 2, the BLOSUM matrix is 62. The Smith-Waterman search algorithm is disclosed in the literature [59] ...

Unless otherwise indicated, a multi-step process or method may include additional steps at the beginning or end of the method, or may include additional intermediate steps. In addition, the steps can be combined, omitted, or performed in an alternate order as needed.

Various embodiments are described herein. It should be understood that the characteristics defined in each embodiment may be combined with other defined characteristics to provide additional embodiments. In particular, embodiments highlighted herein as suitable, typical, or preferred may be combined with each other (unless they are mutually exclusive).

Ways / modes of implementation of the invention

Example 1. The effectiveness of a bacterial inoculum when acting as a neuroprotective agent.

Short description.

Neuroblastoma cells were treated with compositions containing bacterial strains in accordance with this invention. The SH-SY5Y neuroblastoma cells used are dopamine-producing and are well established as an in vitro model for the study of neurodegenerative diseases. The ability of these bacterial strains to increase the increase in neuroproliferation was observed. Neuroblastoma cells were also treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinine (MPP), which induces irreversible symptoms of Parkinson's disease in neuroblastoma cells. Studied the ability of bacterial strains to function as neuroprotective against MPP.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029; Parabacteroides distasonis MRX0005.

Cell line.

SH-SY5Y neuroblastoma cells were purchased from ECCAC (Cat # 94030304) and grown in MEM (Sigma Aldrich, Cat # M2279) supplemented with Ham's F-12 Nutritional Formula (Sigma Aldrich, Cat # N4888).

Method.

After cultivation, SH-SY5Y neuroblastoma cells were seeded in a 96-well plate at 11000 cells / well and incubated for 2 days. The cells were then transferred to differentiation medium (which contains FBS at 1%) and 10 μM retinoic acid (Sigma Aldrich, Cat # R2625-100MG). The differentiation medium was changed every other day and the cells were harvested on the 7th day of differentiation. Cells were pretreated with or without the addition of MPP (Sigma Aldrich, Cat # D048-1G) for 8 hours. Subsequently, the cells were treated with 10% bacterial supernatant and incubated overnight. Cell viability was measured using CCK-8 reagent (Sigma Aldrich, cell counting kit - 8, cat # 96992-3000TESTS-F) and read at 450 nm.

Results.

The results of these experiments are shown in FIG. 1. Treatment of neuroblastoma cells with MRx0029 or MRX0005 resulted in an increase in neuronal proliferation. Neuroblastoma cells that were treated with MPP together with the bacterial strain had an increased cell viability compared to cells treated with MPP alone (which had a reduced viability). These data show that the bacterial strain can act as a neuroprotective agent. The protective effect was higher for cells treated with MRX0029, which restored viability to a greater extent than cells treated with quercetin positive control. These data demonstrate that these bacterial strains can act as a neuroprotective agent.

Example 2. The effectiveness of a bacterial inoculum in reducing the secretion of IL-6.

Short description.

The activation of pro-inflammatory cytokines has been associated with neuronal damage in neurodegenerative diseases. Lipopolysaccharide (LPS) is a known stimulant of the pro-inflammatory cytokine IL-6. Human glioblastoma-type astrocytoma cells were treated with compositions containing the bacterial strains of this invention in combination with LPS in order to

- 28,037,000 observing their ability to modulate IL-6 levels.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029.

Cell line.

MG U373 is a glioblastoma-type astrocytoma derived from a malignant tumor and the cells were purchased from Sigma-Aldrich (Cat # 08061901-1VL). Human glioblastoma type astrocytoma cells MG U373 were grown in MEM (Sigma Aldrich, Cat # M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mM L-Glut, 1X MEM solution of nonessential amino acids and 1X sodium pyruvate.

Method.

After cultivation, MG U373 cells were plated onto a 24-well plate at 100,000 cells / well. Cells were treated with LPS alone (1 μg / ml) or 10% bacterial supernatant from MRx0029 for 24 hours. A control was also performed if cells were incubated in untreated media. Thereafter, the cell-free supernatants were centrifuged at 10,000 g for 3 min at 4 ° C. IL-6 was measured with Peprotech Human IL-6 ELISA Kit (Cat # 900-K16) according to the manufacturer's instructions.

Results.

The results of these experiments are shown in FIG. 2. Treatment of neuroblastoma cells with LPS and the bacterial strain resulted in a decrease in the level of secreted IL-6.

Example 2B. The effectiveness of a bacterial inoculum in modulating IL-8 secretion.

Short description.

Since neuroinflammation plays a key role in neurodegenerative diseases and IL-8 has been shown to have neuro-positive effects, the effect of compositions containing bacterial strains and LPS on IL-8 activation was evaluated. Human glioblastoma-type astrocytoma cells were treated with compositions containing the bacterial strains of this invention in combination with LPS to observe their ability to modulate IL-8 levels.

Materials and methods.

Bacterial strains.

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005.

Cell line.

MG U373 is a glioblastoma-type astrocytoma derived from a malignant tumor and the cells were purchased from Sigma-Aldrich (Cat # 08061901-1VL). Human glioblastoma type astrocytoma cells MG U373 were grown in MEM (Sigma Aldrich, Cat # M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mM L-Glut, 1X MEM solution of nonessential amino acids and 1X sodium pyruvate.

Method.

After cultivation, MG U373 cells were plated onto a 24-well plate at 100,000 cells / well. Cells were treated with LPS alone (1 μg / ml) or 10% bacterial supernatant from MRX0029 for 24 hours. The cell-free supernatants were then centrifuged at 10,000 g for 3 minutes at 4 ° C. IL-8 was measured using Peprotech Human IL-8 ELISA Kit (Cat # 900-K18) according to the manufacturer's instructions.

Results.

The results of these experiments are shown in FIG. 3. Treatment of neuroblastoma cells with bacterial strains led to an increase in IL-8 secretion regardless of the presence of LPS.

Example 2C. The effectiveness of bacterial inoculum in reducing asynuclein-induced inflammation.

Short description.

Neuroinflammation plays a key role in Parkinson's disease, and α-synuclein has been shown to induce neuroinflammation in vivo. Thus, the ability of the bacterial strains of this invention to inhibit α-synuclein-induced inflammation was evaluated. Coculture of human glioblastoma-type astrocytoma cells and neuroblastoma cells were exposed to wild-type α-synuclein and mutant isoforms E46K and A53T and treated with compositions containing bacterial strains in accordance with this invention. Then, the ability of bacterial strains to inhibit α-synuclein-induced IL-6 secretion was examined.

Materials and methods.

Bacterial strains.

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005

Cell line.

MG U373 is a glioblastoma-type astrocytoma derived from a malignant tumor and the cells were purchased from Sigma-Aldrich (Cat # 08061901-1VL). Human glioblastoma type astrocytoma cells MG U373 were grown in MEM (Sigma Aldrich, Cat # M-2279) supplemented with 10% FBS, 1% Pen Strep, 4 mM L-Glut, 1X MEM solution of nonessential amino acids and 1X sodium pyruvate.

- 29 037000

SH-SY5Y is a human neuroblastoma cell line derived from neuroblastoma malignant and can be purchased from Sigma-Aldrich (Cat # 94030304-1VL). The cells were grown in 50% MEM and 50% Ham's medium F-12 nutrient mixture supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U / ml penicillin, 100 μg / ml streptomycin. Cells in cell growth media were seeded in a 96-well plate at 11,000 cells / well and placed in an incubator. After 2 days, the medium was replaced with differentiation medium (cell growth medium containing 1% FBS) and 10 μM retinoic acid. The differentiation medium was changed every other day and the cells were harvested after 7 days of differentiation.

Method.

SHSY5Y cells were plated in 12-well plates at 50,000 cells / well. The cells were grown in 50% MEM and 50% Ham's medium F-12 nutrient mixture supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U / ml penicillin, 100 μg / ml streptomycin. Cells in cell growth media were seeded in a 96-well plate at 11,000 cells / well and placed in an incubator. After 2 days, the medium was replaced with differentiation medium (cell growth medium containing 1% FBS) and 10 μM retinoic acid. The differentiation medium was changed every other day and the cells were harvested after 7 days of differentiation. U373 was plated on 12 Transwell plates (0.4 μm polyester membrane, Costar) at 50,000 cells / well for 72 hours. Cells were co-cultured for 24 hours before treatment in differentiation medium (cell growth medium containing 1% FBS without retinoic acid).

Thereafter, cells were treated with 25 μg / ml α-synuclein (DT, A53T, E46K) in the presence or absence of 10% bacterial supernatant for 48 h. Cell-free supernatants were collected, centrifuged at 10,000 g for 3 min at 4 ° C , was divided into aliquots and stored at 80 ° C. Human IL-6 and IL-8 were measured as described above.

Results.

The results of these experiments are shown in FIG. 4. Treatment of cells with wild-type α-synuclein and mutant isoforms E46K and A53T led to the induction of moderate IL-6 secretion (Fig. 4A). Α-syn-induced IL-6 secretion was inhibited in cells treated with bacterial strains (Fig. 4A). The decrease in IL-6 secretion was greatest with MRX0029.

Example 3. The effectiveness of a bacterial inoculum in reducing NFkB activation.

Short description.

Activation of the NFkB promoter leads to the production of proinflammatory cytokines IL-1β, IL1α, IL-18, TNFa and IL-6. The NFkB promoter can be activated by α-synuclein, and LPS by stimulation of the TLR4 ligand. Mutations in α-synuclein, such as α-synuclein A53T, are associated with familial Parkinson's disease. LPS processing of neurons mimics environmental-induced Parkinson's disease. Studied the ability of compositions containing bacterial strains in accordance with this invention, to lead to inhibition of activation of the NFkB promoter.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029.

Cell line.

Hek blue human TLR4 was purchased from InvivoGen (cat # hkb-htlr4). Human Hek blue TLR4 was grown in high glucose DMEM (Sigma Aldrich, Cat # D-6171) supplemented with 10% FBS, 1% Pen Strep, 4 mM L-glutamine, normocin and 1X HEK Blue selection solution.

Method.

After cultivation, human Hek blue cells were plated in 96-well plates at 25,000 cells / well in 4 replicates. One group of cells was treated only with α-synuclein A53T (1 μg / ml) or 10% bacterial supernatant from MRx0029 for 22 h. The second group of cells was treated with LPS (10 ng / ml, from Salmonella enterica serotype Typhimurium, Sigma Aldrich, Cat # L6143) alone or with 10% bacterial supernatant from MR029 for 22 hours. After that, the cells were centrifuged and 20 μl of the supernatant was mixed with 200 μl of Quanti Blue reagent (InvivoGen, cat. Rep-qb2), incubated for 2 hours and read the absorbance at 655 nm.

Results.

The results of these experiments are shown in FIG. 5 and 6. FIG. 5 demonstrates that activation of the NFkB promoter by α-synuclein is inhibited by MRx0029. Ha fig. 6 demonstrates that activation of the NFkB LPS promoter is not inhibited by MRx0029.

Example 4. The effectiveness of a bacterial inoculum in changing the antioxidant capacity.

Short description.

Studied the ability of compositions containing bacterial strains in accordance with this invention, to lead to a change in antioxidant capacity. The antioxidant capacity of the bacterial strain was determined using the well-known ABTS (2,2'-azino-bis- (3

- 30 037000 ethylbenzothiazoline-6-sulfonic acid)).

Bacterial strain.

Megasphaera massiliensis MRx0029.

Method.

Bacterial cells (10 ⁶ or more) were collected and centrifuged. They were suspended in assay buffer (using three times the pellet volume). The suspension was sonicated on ice for 5 min and then centrifuged at 12,000 x g for 10 min. The supernatant was removed and measured using an ABTS assay kit manufactured by Sigma Aldrich (code CS0790) according to the manufacturer's instructions.

Results.

The results of these experiments are shown in FIG. 7. In FIG. 7 demonstrates that MRx0029 has an antioxidant capacity of about 2 mM compared to trolox.

Example 5. The effectiveness of a bacterial inoculum in changing levels of lipid peroxidation.

Short description.

Studied the ability of compositions containing bacterial strains in accordance with this invention, to change the levels of lipid peroxidation. Thiobarbituric acid reactive assay (TBAR) was used to measure the by-products of lipid peroxidation.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029.

Method.

Bacterial cells (10 or more) were harvested and centrifuged, and the washing step was performed using isotonic saline before resuspending the pellet in analytical sodium chloride buffer. The suspension was sonicated on ice for 10 min and then centrifuged at 10,000 x g for 10 min. The supernatant was removed and the level of lipid peroxidation was assessed using the analysis of substances that react with thiobarbituric acid.

Results.

The results of these experiments are shown in FIG. 8. In FIG. 8 demonstrates that MRx029 is able to inhibit lipid peroxidation by about 20%, which represents a higher antioxidant capacity than the positive control, butylated hydroxytoluene (1% w / v).

Example 6. Efficiency of a bacterial inoculum in relation to histone deacetylase activity.

Short description.

Studied the ability of compositions containing bacterial strains in accordance with this invention, to lead to a change in the activity of histone deacetylase. Dysregulation of histone deacetylase has played a role in the pathogenesis associated with age-associated neurodegenerative diseases.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029.

Cell line.

The cell line HT-29 was used because histone deacetylase was present.

Method.

The cell-free bacterial culture supernatants in the stationary phase were isolated by centrifugation and filtered on a 0.22 μM filter. HT-29 cells were used 3 days after the onset of confluence and used in reduced amounts in 1 ml of DTS 24 hours before the onset of the experiment. HT-29 cells were exposed to 10% cell-free supernatant diluted in DTS and allowed to incubate for 48 hours. The nuclease proteins were then extracted with a Sigma Aldrich nuclease extraction kit and samples were flash frozen until HDAC activity was measured. Activity for measuring HDAC activity was assessed fluorometrically using a Sigma Aldrich kit (Great Britain).

Results.

The results of these experiments are shown in FIG. 9. In FIG. 9 demonstrates that MRx0029 is able to reduce levels of histone deacetylase activity.

Example 7. The level of production of indole in bacteria.

Short description.

Investigated the ability of bacteria according to this invention to produce indole. Indole has been implicated in reducing inflammation and oxidative stress.

- 31 037000

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRx0029.

ATCC 11775 is a bacterial reference strain known to produce indole.

Method.

Intact bacterial cells in the stationary phase were incubated with 6 mM tryprofan for 48 hours. Bacterial species that have the enzyme tryptophanase will utilize tryptophan as a substrate for indole production. After a 48 hour incubation period, the supernatant was removed and added to Kovacak reagent to quantify indole. Standards, working solutions and reagents were prepared using standardized methods validated under in-laboratory conditions.

Results.

The results of these experiments are shown in FIG. 10. In FIG. 10 demonstrates that MRx0029 has the ability to produce indole from tryptophan at concentrations of about 0.2 mM.

Example 8. The level of production of kynurenine in bacteria.

Short description.

Investigated the ability of the bacteria of this invention to produce kynurenine. Dysregulation of the kinurein pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds.

Changes in kinurein metabolism may be associated with the development of Parkinson's disease.

Bacterial strain.

Megasphaera massiliensis MRx0029.

DSM 17136 is a Bacteroides copricola strain that is known to produce kynurenine.

Method.

Cell-free supernatants of bacterial cultures in the stationary phase were isolated by centrifugation and filtered on a 0.22 μM filter, and frozen before use. Standards, kynurenine working solutions and reagents were prepared using standardized methods validated under in-laboratory conditions. The samples were treated with trichloroacetic acid and centrifuged at 10000xg for 10 min at 4 ° C. The supernatant was collected and distributed in a 96 well plate. Ehrlich's reagent was used to detect kynurenine and was added in a 1: 1 ratio.

Results.

The results of these experiments are shown in FIG. 11. In FIG. 11 demonstrates that MRx0029 has the ability to produce kynurenine at a concentration of about 40 μM.

Example 9 Dopamine, DOPAC and HVA Levels in Striatum in MPTP-Treated Bacteria of Mice.

Parksinson's disease is a common neurodegenerative disease, the distinctive clinical characteristics of which include tremor, motion retardation, stiffness, and postural instability. These symptoms are mainly associated with the degeneration of dopaminergic neurons in the compact part of the substantia nigra and the subsequent loss of their projection nerve fibers in the striatum [ ⁶⁰ ]. Mice treated with MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine) selectively lose significant amounts of nigrostrial dopaminergic neurons [ ⁶¹ ]. MPTP led to the induction of dopaminergic cell loss in the substantia nigra, mimicking the clinical state of Parkinson's disease and thus a useful model for the study of antiparkinsonian drugs.

The aim of this study was to evaluate the effects of anaerobic bacteria MRX0029 using mice with damage caused by MPTP.

male mice were assigned to 4 different treatment groups (groups A, B, E and I with n = 12 animals in each group). Treatment groups are shown in table. 1 below, and the time-temporal dynamics is outlined below.

- 32 037000

Table 1

Processing groups

Gro and Treatment Damage πηϋ Substance Level and security clear GP Dose Way Scheme Substance DotA Way Scheme AND 12 Carrier (PBS) r.o. 18 days' day (- 14) - day Carrier (0.9% saline) ip DayO IN 12 Carrier (PBS) r.o. 18 days day (* 14J - laziness MRTP 4x 20 mg / kg i Р DeiO E 12 MRx.0029 Mcgaspha era sp. 1 gly) SI / S2 2 - ID S bacterium r.o. 18 days a day (- L4) - day MRTP 4x 2 € mg / kg i.p. Day 0 ] 12 Carrier (PBS) - r.o. [8 days day (- L4) - day MRTP 4x 20 mg / kg i.p. Day 0 7- nitrpinda angry fifty μκι / κι i.p day 0 (2а gr.)

Groups A, B, E and I were treated daily for 18 days by gavage with either bacteria (MRx0029 - Group E) or vehicle (PBS). Oral treatment was started 14 days before the MPTP injury. Group I animals received daily (PBS) p.o. (oral) vehicle treatment and injection i.p. (intraperitoneal) with the reference drug 30 minutes before and 90 minutes after the first administration of MPTP on day 0. The volume used in the case of po. and vehicle treatment was 200 μl per mouse. The group E bacterial strain was derived from glycerol working solutions (gly). In the case of oral treatment, the probes for use were stored in a vial containing 70% ethanol and rinsed with distilled water before and after each use. Each treatment group had its own probe and ethanol bottle and distilled water bottle.

Tubes and probes were not changed between groups. Each syringe was flushed with N2 immediately prior to treatment.

On day 0, MPTP (20 mg / kg body weight (b.w.) 4 times, 2 h interval between treatments) was injected with i.p. animals of groups B, E and I. One group of animals (A) was exposed to blank damage as a result of administration of the MPTP vehicle (0.9% saline solution). The volume used was 10 μl per g body weight. Weighing of animals was performed prior to treatment with MPTP in order to administer doses to the animals in accordance with their actual body weight. Thereafter, the animals received daily po treatment.

Compilation of preparations for dosing and preparation of glycerine working solutions for dosing.

Bacterial strain name: MRx0D29 Megasphaera sp. Storage / stability conditions: -80 C Carrier: lx PBS Treatment doses 2] 0 ^Λ 8 bacteria Introduction: 200 μl Scab number but.

In case of processing group E (MRx0029).

1) 1 glycerol working solution was removed from the -80 ° C refrigerator and placed in anaerobic conditions (anaerobic sachet can) at 37 ° C for defrosting (this was 30-40 min).

- 33 037000

2) The fully thawed glycerol working solution was centrifuged at 6000 x g for 10 min at room temperature.

3.) The supernatant was removed without disturbing the pellet (eg using a pipette).

.) 4.22 ml of sterile pre-warmed (37 ° C) 1 x PBS was added and gently mixed with a pipette.

.) Mice were injected with a dose of 200 μl of bacterial solution. The animals were dosed within 15 minutes after resuspending the pellet using PBS.

Compilation of a group of reference drugs.

Reference drug name: 7-pitroindazole Storage / Stability Conditions: -20 ° C Carrier. Peanut butter Treatment doses: 50 μg / kg Introduction i.p (30 minutes before and 90 minutes after the 1st MRTP treatment) Series number: MKBS6671V

An appropriate amount of 7-nitroindazole was dissolved in peanut oil to achieve a final concentration of 50 mg / kg.

Materials and methods.

Animals.

Line of mice C57BL / 6J (JAX ™ mouse strain) JAN ™, wep part number 000664 The supplier: Charles River Laboratories Age at the start of the study: 10 weeks Nol: Male Number of animals: 48

Special animal keeping and randomization.

Gloves were changed in each treatment group and 70% ethanol solution was sprayed into each cage of the same group in order to minimize the risk of contamination in all animal handling situations (eg, handling, behavioral studies, cleaning and tissue sampling).

The treatments were carried out in random order and alternated daily in order to prevent the treatment of the same groups at the same time. Animals were randomized to a cage when tissue samples were taken.

Collection of tissue samples and processing.

On day 4, animals of all groups were sacrificed and the brains removed. Thus, the mice were deeply anesthetized with pentobarbital injection (600 mg / kg).

Blood (approximately 500 μl) was collected by cardiac puncture. The mice were then transcardially perfused with 0.9% saline and the brains were removed and dissected longitudinally. The left hemisphere was subdivided into striatum tissue (for HPLC), substantia nigra, and residual brain, weighed and flash frozen and stored at -80 ° C. Instruments and surfaces that were in contact with animals were to be cleaned with 70% ethanol prior to dissection of the next animal.

Biochemical analysis of dopamine, DOPAC and HVA levels by HPLC in striatum.

Striatum samples (n = 6 from each treatment group; a total of 24 samples) were mixed at a 1:10 (w / v) ratio with 0.2 M perchloric acid containing 100 μM EDTA-2Na and homogenized at 0 ° C in a glass homogenizer Pestlemicro. After settling for 30 min on ice, the homogenates were centrifuged at 10,000 rpm for 10 min in a cooled Biofuge Fresco centrifuge (Heraeus Instruments, Germany). Supernatants were carefully aspirated and mixed with 0.4 M Na-acetate buffer, pH 3 in a ratio of 1: 2 (v / v) and filtered through a 0.22 μm centrifugal filter (Merck Millipore, Germany) for 4 min at 14000 g at 4 ° C. The filtrates were stored at -80 ° C until HPLC analysis.

HPLC analysis.

Concentrations of DA, DOPAC and HVA in striatum samples were determined using liquid column chromatography with electrochemical detection [ ⁶² ; ⁶³ ]. An HPLC system (NTEC-500, Eicom Corp., Kyoto, Japan) was used containing a pulseless microjet pump, a degasser, and an amperometric detector equipped with a glassy carbon electrode operating at +0.45 V versus an Ag / AgCl reference electrode. Samples were injected using a CMA / 200 frozen microsampler (CMA / Microdialysis, Stockholm, Sweden). Chromatograms were recorded and integrated using a computerized data acquisition system (DataApex, Prague, Czech Republic). DA, DOPAC and HVA were separated on a 150x2.1 id mm column (CA5-ODS, Eicom Corp., Kyoto, Japan). The mobile phase consisted of 0.1 M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mM sodium 1-octanesulfonate and 20% (v / v) methanol. The detection limit (signal-to-noise ratio = 3) in the case of DA was estimated to be 0.5 fmol in 15 μl (0.03 nM) injected onto the column.

Results.

The introduction of bacterial strains was well tolerated by the animals. On the day the lesions were performed using MPTP and, if necessary, one day after that, red light was used to warm the animals. If the animals were kept in unfavorable conditions (they felt cold, were dehydrated, had abnormal behavior), they were supplied with wet food and, if necessary, performed subcutaneous treatment.

Striatal tissue from 6 animals per treatment was used to analyze dopamine, DOPAC and HVA levels. Data were analyzed using the Kruskal-Wallis test, followed by the use of the Dune test for multiple comparisons or one-way ANOVA followed by the use of the posterior Bonferroni test (A versus all (*), B versus all, I versus all (# )). * / # = p <0.5; ** = p <0.01; *** = p <0.001.

Healthy animals in group A had high levels of dopamine, DOPAC and HVA, while treatment with MPTP in group B resulted in a decrease, and a positive control (group I) resulted in restoration of production to some extent (Fig. 12). Group I animals tended to have higher dopamine levels than the bacteria treated group and group B. DOPAC (dopamine metabolite) levels tended to be significantly lower in group B animals compared to DOPAC levels in animals without group A lesions (Fig. 12B).

Significantly, treatment with MRx0029 (group E) was found to result in a restoration of dopamine and DOPAC production (FIGS. 12A and 12B, respectively). Treatment with MRx0029, thus, may be useful in the treatment or prevention of neurodegenerative disorders.

Example 10. The effectiveness of bacteria in changing the growth of neurites.

Short description.

Neurite growth is an important process in the development of connections between neurons. Thus, the ability of bacterial strains and organic acids to induce neurite growth was investigated by measuring the levels of transcription of the protein associated with microtubules MAP2, a specific marker of neuronal differentiation.

Bacterial strain.

Megasphaera massiliensis MRX0029.

Method.

SHSY5Y were seeded in 10 cm Petri dishes with a density of 2x106 cells. After 24 hours, cells were treated with differentiation medium (differentiation medium containing 1% FBS without RA) with 10% bacterial supernatants or YCFA +, 10 μM RA, 200 μM hexanoic acid, or 200 μM valproic acid for 17 hours. images using an EVOS XL phase contrast microscope at 40X / 0.65 magnification. Cells were harvested and total RNA was isolated according to the protocol for the RNeasy mini-kit (Qiagen). cDNA was obtained using a high performance cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as an internal control. The change rate was calculated in accordance with method 2 ( ^{- cl} ).

Immunofluorescence and confocal microscopy.

Cells were plated on 8-well slides (Marienfeld Laboratory Glassware) at 5x10 ⁴ cells / well overnight and treated with 10% bacterial supernatant for 24 hours. For differentiation, cells were treated with 10 nM retinoic acid for 5 days prior to bacterial supernatant treatment. The cells were then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT). Fixed cells were washed with PBS and permeabilized with 1% Trinone X100 in PBS for 10 min. After washing with PBS, the preparations were incubated with blocking buffer (4% BSA / PBS) for 1 h at RT before adding anti-MAP2 antibody (sc-74421, Santa Cruz Biotechnology Inc) diluted in 1% BSA / PBS for 12 h at 4 ° C. They were then washed twice with PBS, then incubated with Alexa Flour 488 conjugated anti-mouse antibody (Molecular Probes Inc) and Alexa Flour 594 conjugated phalloidin (ab176757, Abcam) for 1 h at RT. After washing 3X with PBS, preparations were embedded with VectorshieldD containing DAPI (Sigma, Aldrich). The preparations were examined using a Zeiss Axioscope microscope equipped with a 63x / 1.2 W Korr objective and filter sets suitable for the detection of the fluorochromes used. The manually set exposure time in the case of digital imaging immunolabeled with MAP-2 was kept constant, allowing comparison between different wells and treatments. The exposure time of phalloidin (F-actin) and DAPI was varied to match the visual field. Randomized fields of view were acquired using a software controlled QImaging camera

- 35 037000

Image Pro Plus. Images were saved as TIF and opened in Adobe Photoshop CC 2015.1.2 and overlays using MAP-2, DAPI and phalloidin were overlapped and merged. Illustrative images were selected to illustrate differences in the amount and position of the proteins tested.

Results.

The results are shown in FIG. 13. FIG. 13A shows illustrative microscopic images of undifferentiated SHSY-5Y cells incubated with each of the acids and bacterial supernatants. Treatment of cells with MRX0029 led to the induction of a neuron-like phenotype showing similar properties to cells treated with retinoic acid (which is used for final differentiation of neuroblastoma cells), whose cell bodies were larger and pyramidal, with neurites and processes branching towards the network with neighboring cells. FIG. 13B demonstrated that MRx0029 significantly leads to the activation of MAP2 in undifferentiated neuroblastoma cells. Phalloidin staining (an agent that binds the actin cytoskeleton) further indicated a different organization of the cytoskeletal structure in cells treated with MRx0029, further confirming the hypothesis of neuronal differentiation in the case of MRx0029 (Fig. 13B).

Example 11. The effectiveness of a bacterial inoculum in reducing oxidative levels in cells.

Prerequisites.

The formation of reactive oxygen species contributes to the pathogenesis of neurodegenerative diseases. The ability of bacterial strains to protect differentiated SHSY-5Y and U373 cells from reactive oxygen species (ROS) formed as a result of treatment with tert-butyl hydroperoxide (TBHP) was studied.

Materials and methods.

Bacterial strain.

Megasphaera massiliensis MRX0029.

Method.

SHSY-5Y cells were seeded in a black flat bottom 96 well plate at 5000 cells / well and placed in a CO2 incubator. After 24 hours, the medium was replaced with differentiation medium (cell growth medium containing 1% FBS) and 10 μM retinoic acid. The differentiation medium was changed every other day. On day 10, the differentiation medium was changed and the cells were washed with pre-warmed PBS and stained with 10 μM DCFDA molecular probe for 20 min in cell growth medium containing 1% FBS. The cells were then re-washed with pre-warmed PBS and treated with 100 μM TBHP in the presence or absence of 10% bacterial supernatant for 2 hours. The fluorescence intensity was measured with a TECAN plate reader at Ex / Em 485/530 nm.

Results.

The results of these experiments are shown in FIG. 14. In FIG. 14B demonstrates that MRX0029 is capable of inhibiting ROS production in differentiated SHSY-5Y neuroblastoma cells. MRX0029 had no effect on ROS formation in U373 astroglioblastoma cells (FIG. 14A). This demonstrates that this aspect of the antioxidant effect was neuronal specific.

Example 12. Neuroprotection.

RA-differentiated SHSY-5Y cells were treated with MPP +, an active metabolite of MPTP, a chemical compound widely used to mimic in vitro and in vivo some characteristics of PD pathology. Cell viability was measured as the respiration rate in mitochondria (Fig. 15). Both MRx0005 and MRx0029 have shown significant effects and by themselves promote an increase in mitochondrial metabolic activity in SHSY-5Y cells. MRX0029 demonstrated complete protection against MPP +, restoring cell viability to almost the same level as untreated cells and more than a positive control with quercetin. Protection of MRx0005 was approximately 20% compared to the sample treated with YCFA-MPP +, approximately the same was observed in the case of the positive control with quercetin (Fig. 15).

Example 13. Further analysis of the histone deacetylase inhibition mechanism.

Introduction.

The gut microbiota, with its immense diversity and metabolic capacity, represents a huge metabolic reservoir for the production of a wide variety of molecules with the ability to affect HDAC activity. Several studies have evaluated the HDAC inhibitory activity of microbial metabolites other than butyrate, which has been shown to lead to HDAC inhibition and is associated with improved motor function in Huntington's disease [ ⁶⁴ ]. Thus, the authors of the present invention were interested in determining which metabolites are responsible for the inhibition of HDAC and further identify the mechanisms by which inhibition is achieved.

- 36 037000

Materials and methods.

Collect bacterial cultures and cell-free supernatants.

Pure cultures of bacteria were grown under anaerobic conditions in YCFA broth until they reached their stationary growth phase. The cultures were centrifuged at 5000xg for 5 min and the cell-free supernatant (CFS) was filtered using a 0.2 μM filter (Millipore, UK). 1 ml aliquots of CFS were stored at -80 ° C until use. Sodium butyrate, hexanoic acid and valeric acid were obtained from Sigma Aldrich (UK) and suspensions were prepared in YCFA broth.

Quantification of SCFA and MCFA of Bacterial Supernatants Short chain fatty acids (SCFA) and medium chain fatty acids (MCFA) from bacterial supernatants were analyzed and quantified by MS Omics APS as follows. Samples were acidified with hydrochloric acid and deuterium-labeled internal standards were added. All samples were analyzed in random order. Analysis was performed using a high-polarity column (Zebron ™ ZB-FFAP column, GC Cap 30 mx 0.25 mm x 0.25 μm) installed in a GC (7890B, Agilent) connected to a quadropole detector (59977B, Agilent). The system was monitored using a ChemStation (Agilent). The raw materials were converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [ ⁶⁵ ].

Analysis of the specific activity of HDAC.

FIDAC specific inhibitory activity was analyzed for FIDAC1, 2, 3, 4, 5, 6, 9 using fluorogenic assay kits for each FIDAC type (BPS Bioscience, Calif.). The analyzes were performed according to the manufacturer's instructions and the analysis of each sample was performed in triplicate. Cell-free supernatants were diluted 1 in 10 and exposed to specific HDAC proteins provided in the kit to maintain consistency between methods.

Results.

The metabolites of commensal microorganisms that inhibit histone deacetylase are butyrate and valeric acid MRx0029, the supernatant of which showed inhibition of HDAC in both HT29 whole cells and HT29 cell lysates, and produced valeric acid and hexanoic acid at mean concentrations of 5.08 and 1.60 mM, respectively (Fig. 16A and C).

To investigate which metabolites were responsible for the strain-induced HDAC inhibition, different concentrations of hexanoic acid, valeric acid and sodium butyrate were measured in the case of their HDAC inhibition against HT-29 whole cells and HT-29 cell lysate. The results in FIG. 16B demonstrates significant (P <0.05) inhibition of HDAC activity by sodium butyrate on whole cells as well as cell lysate, while hexanoic acid did not show significant inhibitory activity. Valeric acid resulted in inhibition of the total HDAC activity (* (p <0.05), ** (p <0.005), *** (P <0.001), **** (p <0.0001)).

Highly effective total inhibitors of the investigated HDACs target HDAC class I.

Investigated the specific profile of inhibition of HDAC of the studied bacterial strain. HDAC Specific Inhibition Assays (BPS Bioscience,

California) was performed in the case of HDAC class I and class II. The ability of a bacterial strain to inhibit HDAC enzymes was compared with butyrate, hexanoic and valeric acid. The results demonstrate that MRX0029 is a very potent inhibitor of HDAC class 1 enzymes (HDAC1, 2 and 3). The inhibition of HDAC class II was not as significant (data not shown).

Discussion.

The strain with inhibitory activity against HDAC produced significant amounts of valeric acid and hexanoic acid, as well as significant amounts of sodium butyrate (Fig. 16C). When tested as pure compounds, valeric acid and sodium butyrate resulted in significant inhibition of HDAC (p <0.0001).

Interestingly, the results in the case of specific HDAC activity demonstrate that the investigated strain is a highly potent inhibitor of HDAC class I and in particular HDAC2 (FIGS. 17 and 18). HDAC class I (HDAC1, 2, 3 and 8) are found in the nucleus and are ubiquitously expressed in several types of human cells. HDAC 1-3 have more than 50% of the total homology, but they have different structures and cellular functions [ ⁶⁶ ]. They are mainly involved in cell survival, proliferation and differentiation, and thus their inhibition may be useful in a number of diseases [ ⁶⁷ ]; [ ⁶⁸ ]; [ ⁶⁹ ]; [ ⁷⁰ ]; [ ⁷¹ ].

Example 14. Level of BDNF secretion in SHSY-5Y cells.

Prerequisites.

Brain neurotrophic factor (BDNF) is a versatile molecule in

- 37,037,000 in the brain associated with neuronal development, neuroprotection and neurodegeneration. BDNF not only leads to protection against neurodegeneration, but also psychiatric disorders such as depression and anxiety, which are quite common among patients diagnosed with PD or

AD.

Ways.

SH-SY5-SY was seeded in 24-well plates at 60,000 cells / well and placed in an incubator. After 24 hours, the medium was replaced with differentiation medium (cell growth medium containing 1% FBS) and 10 μM retinoic acid. The differentiation medium was changed every other day and the cells were harvested on day 10 of differentiation. In the case of treatment, the differentiation medium was removed and replaced with 450 μl of complete cell growth medium and 50 μl of SN bacteria was added to the treated wells or YCFA + was added as a negative control.

Results.

The results are shown in FIG. 19, which shows that administration of MRX0029 in combination with retinoic acid results in increased secretion of BDNF from differentiated neuroblastoma cells. Compositions containing commensal bacteria and organic acids can thus be useful in therapy.

Example 15. Production of metabolites - metabolites in the brain.

Prerequisites.

Metabolites present in bacterial supernatants can directly influence the host's response to oxidative stress, cell-cell communication, and neuroprotection. Metabolites, which play a major role in neurological processes, were measured during ex vivo screening in the brain tissue of mice fed MRx0005 and MRx0029.

Ways.

Animals.

Adult male BALBc mice (Envigo, UK) were housed in groups under a 12 hour light-dark cycle; standard rodent food and water were available in unlimited quantities. All experiments were performed in accordance with European guidelines with approval by the Animal Ethics Committee of the Irish National University in Cork. The animals were 8 weeks old at the beginning of the experiment.

Study design.

The animals were allowed to adapt to the containment room for one week after arrival at the animal housing complex. They received by gastric tube (dose 200 μl) live biotherapeutic drugs at a dose of 1x109 CFU for 6 consecutive days between 15:00 and 17:00. On day 7, animals were decapitated and tissues were removed for experiment.

Collection of tissues.

Animals were sacrificed at random depending on the treatment and study conditions; sampling took place between 9:00 and 13:00. Blood from the trunk was collected in tubes with potassium-EDTA (ethylenediaminetetraacetic acid) and centrifuged for 15 min at 4000 g. Blood plasma was isolated and stored at -80 ° C for subsequent analysis. The brain was rapidly excised, dissected, and each region of the brain was flash frozen on dry ice and stored at -80 ° C for later analysis. The spleen was removed and processed immediately after culls for ex-vivo immunostimulation. Intestinal tissue (2 cm segments of the ileum and colon, adjacent to the cecum were excised, and the outermost 1 cm of cecum tissue was used) was placed in Ussing chambers for intestinal permeability analysis. The cecum was removed, weighed and stored at 80 ° C for SCFA analysis.

Analysis of monoamines.

The concentration of neurotransmitters was analyzed by HPLC against samples from the brainstem. Briefly, brainstem tissue was sonicated in 500 μl cold mobile phase supplemented with 4 ng / 40 μl N-methyl-5-HT (Sigma Chemical Co., UK) as internal standard. The mobile phase contained 0.1 M citric acid, 5.6 mM octane-1-sulfonic acid (Sigma), 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA (Alkem / Reagecon, Cork) and 9% (v / vol.) methanol (Alkem / Reagecon) and brought to pH 2.8 with 4 N sodium hydroxide (Alkem / Reagecon). Then the homogenates were centrifuged for 15 min at 22000 xg at 4 ° C and 40 μl of the supernatant was injected into the HPLC system, which consisted of an SCL 10-Avp system controller, an LECD 6A electrochemical detector (Shimadzu), an LC-10AS pump, an STO-10A oven. , auto-injector SIL-10A (with sample cooler kept at 40 ° С) and online degasser Gastorr (ISS, UK). A reverse phase column (Kinetex 2.6 µm C18 100 x 4.6 mm, Phenomenex) maintained at 30 ° C was used in separation (flow rate 0.9 ml / min). Glassy carbon working carbon in combination with an Ag / AgCl reference electrode (Shimdazu) operating at +0.8 V and the resulting chromatograms were analyzed using the computer program Class-VP 5 (Shimadzu). Neurotransmitters were identified by their characteristic retention time, determined

- 38 037000 by internal injections, which were chromatographed at regular intervals during the analysis of samples. The peak height ratios of the analyte versus the internal standard were measured and compared to the standard injection. Results were expressed as ng per g wet weight.

Analysis of metabolites.

In the case of analysis of GC metabolites, samples of bacterial supernatants were derivatized with methyl chloroformate using a slightly modified version of the protocol described in [ ⁷² ]. All samples were analyzed in random order. Analysis was performed with a GC (7890B, Agilent) coupled to a quad-pole detector (59977B, Agilent). The system was monitored using a ChemStation (Agilent). The raw materials were converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [65].

For fatty acid analysis, the samples were acidified with hydrochloric acid and deuterium-labeled internal standards were added. All samples were analyzed in random order. The analysis was performed using a highly polar column (Zebron ™ ZB-FFAP column, GC Cap. 30 mx 0.25 mm x 0.25 μm) installed in a GC (7890B, Agilent) connected to a quadropole detector (59977B, Agilent). The system was monitored using a ChemStation (Agilent). The raw materials were converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in [65].

Results - production of neurotransmitters.

The results are shown in FIG. 20, which shows that in the brains of mice that were fed MRx0029, norepinephrine levels increased (p = 0.0507), accompanied by a slight increase in serotonin and 5-HIAA. These data support the analysis of metabolites described below, suggesting that MRx00029 is the main producer of 4-hydroxyphenylacetic acid, a known antioxidant [73]. More importantly, 4-hydroxyphenylacetic acid is a synthetic intermediate between dopamine and norepinephrine and an important bioactive molecule [74]. In fact, in PD, degenerative changes extend beyond the dopaminergic system, affecting the serotonergic and noradrenergic systems equally, which in turn leads to elevated serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (norepinephrine) levels as in striatum body, and in extrastriary structures [ ⁷⁵ ]. L-DOPA primarily targets PD characteristics, while it does not target either 5-HT or norepinephrine reduction. In addition, the longer the duration of L-DOPA treatment, the more visible the range of motor and non-motor complications (eg, dyskinesia, psychiatric symptoms) is [ ⁷⁶ ]. Thus, these data demonstrate that bacteria that produce organic acids such as 4-hydroxyphenylacetic acid can be useful in treatment, in particular in the treatment of neurodegenerative diseases.

Results - production of metabolites.

Metabolites present in bacterial supernatants can directly influence the host's response to oxidative stress, cell-cell communication, and neuroprotection in specific ways. Metabolites in the supernatant of MRX0029 and MRX0005 cultures were analyzed and the results are shown in FIG. 21.

A small amount of metabolites showed a pronounced difference between the two analyzed strains. The concentration of succinic acid was particularly elevated in MRx0005. Interestingly, the sample / medium ratio for 4-hydroxyphenylacetic acid was significantly higher in MRx0029 (FIG. 21A).

Analysis of fatty acids in supernatants revealed an interesting dichotomy in the two strains: MRx0005 produced mainly acetic and propanoic acid, while MRx0029 produced butanoic, pentanoic and hexanoic acid, both in linear and branched forms (FIG. 21B). These two strains were significantly different and, in particular, the production of 4-hydroxyphenylacetic acid MRx0005 and MRx0029, respectively, was remarkable (Fig. 21A). In addition, MRx0005 appears to produce more C2 and C3 short chain fatty acids, while MRx00029 produced more C4 (butyrate) and both linear and branched medium chain fatty acids, including hexanoic acid.

Succinic acid is a Krebs cycle metabolite involved in oxidative phosphorylation. The oxidative phosphorylation complex is the main stage of synaptic transfer of proteins and vesicles to the proximal and distal regions [ ⁷⁷ ]. Its impairment has been described in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and type 1 spinocerebellar ataxia [ ⁷⁸ ]. These results are particularly interesting because succinic acid can enhance mitochondrial activity and maintain susceptible neurons in neurodegenerative disease associated with

- 39 037000 incorrect protein folding, including PD [ ⁷⁹ ]. Both BDNF and succinic acid have similar protective activity not only in neurodegeneration, but also in psychiatric disorders such as depression and anxiety, which are quite common in patients diagnosed with PD or AD.

FIG. 21B also demonstrates that MRX0029 is a butyrate (butanoic acid) producer. This may be more important, since butyrate has a known role in reducing the impermeability of the blood-brain barrier, which has a neuroprotective effect [80]. This property of MRx0029 (and other neuroprotective bacteria) may contribute to its effectiveness.

Example 16. Modulation of mRNA expression of tight junction proteins using MRx0029.

Since recent evidence suggests that intestinal dysfunction and inflammation is a non-motor symptom associated with PD, the ability of the bacterial strains of this invention to cause any dysfunction of the intestinal barrier was studied.

Monolayers of epithelial mucin-producing cells HT29-mtx [ ⁸¹ ] were used as an in vitro model to assess intestinal barrier damage and immunostimulation after treatment with MRx0005 and MRx0029. Differentiated HT29-mtx cells exposed to phorbol-12myristate-13-acetate (PMA) secreted significant amounts of IL-8; in contrast, treatment for 24 h with bacterial supernatants MRx005 and MRx0029 resulted in the induction of even more IL-8 secretion compared to untreated and YCFA-treated cells (Fig. 22A).

The ability of MRx0005 and MRx0029 to regulate epithelial permeability as a result of modification of intracellular signaling was studied, including expression and localization of proteins involved in the formation of the intestinal barrier.

RNA was isolated and quantitative RT-PCR (qRT-PCR) analysis was performed to characterize changes in tight junction protein gene expression during incubation with MRx0005 and MRx0029. Administration of MRx0029 increased the expression of occludin mRKN, villin, tight junction protein 1 and 2 (TJP1 and TJP2, respectively) 2 hours after incubation (Fig. 22B). In contrast, exposure to MRx0005 did not alter gene expression for tight junction proteins, indicating that the two strains function differently at the intestinal barrier.

The in vitro results were compared with data from a parallel ex vivo assay for the intestines of mice fed MRx0005 and MRx0029. Expression of TJP2 and occludin genes was quantified in the colon and ileum. The ex vivo data accurately reflects the in vitro data as MRx0029 was able to significantly increase the activity of TJP1 and occludin (p = 0.073) in the colon region of the mouse intestine (Fig. 22C + 22D). MRx0029 was also able to reduce colon permeability function in the same mice (Fig. 22E + 22F).

Materials and Methods — RNA extraction and qPCR analysis.

Total RNA was extracted using the RNeasy mini-kit (Qiagen, Manchester, UK) according to the manufacturer's instructions, and the RNA concentration was determined by absorbance at 260/280 nm using a spectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington, Delaware). In the case of analysis of mRNA expression, cDNA was obtained from total RNA using a high-throughput reverse transcription kit (Applied Biosystems, UK) according to the manufacturer's instructions. Reverse transcription reactions were performed in a thermal cycler (Biometra, Germany) at 25 ° C for 10 min, 37 ° C for 120 min, and 85 ° C for 5 min, and kept at 4 ° C. The resulting cDNA was amplified in duplicate using SYBR-Green PNR analysis, and the products were detected on a QuantStudio 6 flex real-time PCR device (Applied Biosystems, UK) using a standardized profile (initial denaturation at 95 ° C for 10 min, followed by 40 cycles of 15 seconds (sec) denaturation at 95 ° C and 60 sec annealing / extension at 60/65 ° C, depending on the primers A dissociation step was added after 40 cycles to obtain a melting curve. Applied Biosystems QuantStudio v1.2 Real Time PCR Software The primer sequences of actin, villin, occludin, TJP1 and TJP2 are provided in the sequence listing.

Stability study.

The composition described herein, containing at least one bacterial strain described herein, is stored in a sealed container at 25 or 4 ° C and the container is placed in an atmosphere having 30, 40, 50, 60, 70, 75, 80 , 90 or 95% relative humidity. After 1, 2, 3, 6 months, 1 year, 1.5, 2, 2.5 or 3 years, at least 50, 60, 70, 80 or 90% of the bacterial strain will be retained as measured by colony forming units defined by standard protocols.

Example 17.

Ways.

Animals.

- 40 037000

The animals used and the study design were the same as in example 15.

Bacterial strains.

755: Parabacteroides distasonis (MRX005).

Megasphaera massiliensis (MRX0029).

Collection of tissues.

Animals were sacrificed at random depending on the treatment and study conditions; sampling took place between 9.00 and 14.30. Blood from the trunk was collected in tubes with potassium-EDTA (ethylenediaminetetraacetic acid) and centrifuged for 15 min at 4000 g. Blood plasma was isolated and stored at -80 ° C for subsequent analysis. The brain was rapidly excised, dissected, and each region of the brain was flash frozen on dry ice and stored at -80 ° C for later analysis. The spleen was removed, collected in 5 ml RPMI medium (with L-glutamine and sodium bicarbonate, R8758 Sigma + 10% FBS (F7524, Sigma) + 1% Pen / Strep (P4333, Sigma)) and processed immediately after ex-vivo immunostimulation. Intestinal tissue (2-3 cm segments of the ileum and colon, next to the cecum were excised, and the outermost 1-2 cm of cecum tissue was used) was placed in Ussing chambers for intestinal permeability analysis. The cecum was removed, weighed and stored at -80 ° C for SCFA analysis.

Analysis of monoamines.

The concentration of neurotransmitters was analyzed as described in example 10.

Analysis of cytokines in the spleen.

The spleen was removed immediately in 5 ml of RPMI medium after sacrifice and immediately cultured. Spleen cells were first homogenized in the indicated RPMI medium, then incubated for 5 min with 1 ml of RBC lysis buffer (11814389001 ROCHE, Sigma). Another 10 ml of RPMI medium was added, followed by 200 G centrifugation for 5 min. The supernatant was then filtered through a 40 µm sieve. Cells were counted and plated (4,000,000 / ml medium). 2.5 h after adaptation, cells were stimulated with lipopolysaccharide (LPS-2 μg / ml) or concavalin A (ConA-2.5 μg / ml) for 24 h. After stimulation, supernatants were collected to assess cytokine release using a pro-inflammatory panel kit 1 (mouse) V-PLEX Kit (Meso Scale Discovery, Maryland, USA) for TNFa, IL-10, IL-1e, interferon γ, CXCL2 and IL6. Analyzes were performed using MESO QuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S600.

Gene expression analysis.

Total RNA was extracted using the mirVana ™ miRNA isolation kit (Ambion / Llife technologies, Paisley, UK) and treated with DNase (Turbo DNA-free, Ambion / life technologies) according to the manufacturer's recommendations. RNA was quantified using a NanoDrop ™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed using an Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer's procedure and the RNA integrity number (RIN) was calculated. RNA with RIN> 7 was used for subsequent experiments. RNA was reverse transcribed into cDNA using an Applied Biosystems high throughput cDNA kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. Briefly, Multiscribe reverse transcriptase (50 U / μL) (1) (2) (1) (10) was added as part of the master mix at RT, incubated at 25 ° C for 10 min, at 37 ° C for 2 h at 85 ° C for 5 min and stored at 4 ° C. Quantitative PNR was performed using probes (6-carboxyfluorescein - FAM) designed by Applied Biosystems for specific target genes of the mouse, while β-actin was used as an endogenous control. The amplification reactions contained 1 μl cDNA, 5 μl 2X PCR master mix (Roche), 900 nM of each primer and made up a total of 10 μl with the addition of RNase-free water. All reactions were performed in triplicate using 96-well plates in the LightCycler®480 system. The thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for contamination during amplification, template-free controls were run in triplicate for each probe used in each cycle. The values of the threshold cycle (Ct) were recorded. Data were normalized with β-actin and transformed using the 2-ΔΔCT method and presented as fold change compared to the control group.

Analysis of short-chain fatty acids in the contents of the cecum.

The contents of the cecum were mixed and vortexed with MilliQ water and incubated at room temperature for 10 minutes. Supernatants were obtained by centrifugation (10,000 g, 5 min, 4 ° C) to pellet bacteria and other solid particles and filter to 0.2 μm. It was transferred to a clear GC vial and 2-ethylbutyric acid (Sigma) was used as an internal standard. The SCFA concentration was analyzed using a Varian 3500 GC flame ionization system, fitted to a ZB-FFAP column (30 m x 0.32 mm x 0.25 mm; Phenomenex). The standard curve was constructed at various concentrations of the standard mixture containing acetate, propionate, isobutyrate, n-butyrate, isovalerate and valerate (Sigma). The peaks were integrated using Varian Star Chromatography Workstation version 6.0 software. All SCFA data were expressed as µmol / g.

Statistical analysis.

Normally distributed data were presented as mean ± SEM; nonparametric populations were presented as median with interquartile range. An unpaired two-tailed t-test was used for the analysis of parametric data, and the Mann-Whitney test was used for nonparametric data. Spearman's rank correlation coefficient was used for correlation analysis in pooled data sets. P-Value <0.05 was considered significant in all cases.

Results - production of neurotransmitters.

The results in FIG. 23 demonstrate the effect of MRx005 treatment on the concentration of neurotransmitters in the brain of mice. Most notably, MRx005 treatment results in a decrease in dopamine.

Results - gene expression.

Expression of genes for neurotransmitter receptors [serotonin receptor 1a (5-HTR1a), dopamine receptor D1, subunit B1 of the GABA receptor, GABAA receptor, NMDA2A receptor (Grin2A) and NMDA2B (Grin2b)], inflammatory markers [EL-1e, IL6, TNF, and TLR4] and endocrine markers [corticosterone-releasing factor (CRF), corticosterone-releasing factor 1 and 2 (CRFR1, CRFR2), brain neurotrophic factor (BDNF), vasopressin receptor, oxytocin receptor, glucocorticoid receptor and mineralocorticoid receptor] were analyzed in brain tissue from the hippocampus, amygdala, and prefrontal cortex.

FIG. 24-38 show changes in gene expression after treatment with MRX005 or MRX0029 in the hippocampus, amygdala and prefrontal cortex. Treatment with MRx0029 resulted in increased expression of the glucocorticoid receptor in the amygdala (Fig. 31C). FIG. 32A demonstrated that MRx005 resulted in a significant increase in BDNF expression in the amygdala, while treatment with MRx0029 resulted in a significant increase in TLR4 expression in the amygdala (FIG. 32).

Both MRx005 and MRx0029 can increase the expression of CD11b in the amygdala (Fig. 33A), while the expression of IL-6, Grin2a, and Grin2b is reduced after MRx005 treatment (Fig. 33B-D). In addition, MRx005 and MRx0029 resulted in a significant increase in GABRA2 expression and an increase in GABBR1 expression in the amygdala.

MRx005 treatment resulted in a significant increase in BDNF expression in the prefrontal cortex (Fig. 35B).

Discussion.

Administration of MRx005 and MRx0029 caused changes in gene expression, especially in the amygdala.

Results - influence on Tph1 and IDO-1 expression.

FIG. 39 demonstrated that MRx0029 can lead to a significant increase in the expression of tryptophan hydroxylase-1 (Tph1) in the colon, and treatment with MRX005 can lead to an increase in the expression of IDO-1 in the colon. Treatment with MRX005 increased the expression of Tph1 and IDO1 in the ileum (Fig. 40).

Indolamine pyrrole 2,3-dioxygenase-1 (IDO-1) is the first and rate limiting enzyme in the tryptophan / kynurenine pathway, while tryptophan hydroxylase 1 (Tph1), an isoform of the enzyme tryptophan hydrolase, is responsible for serotonin synthesis. These data suggest that MRx0029 and MRx005 may affect serotonin levels and the tryptophan / kynurenine pathway.

Results - Effects on tryptophan metabolite levels.

FIG. 41 demonstrates the effect of MRx005 treatment on circulating kynurenine and tryptophan levels.

Results - influence on the expression of cytokines from splenocytes.

Ex-vivo splenocyte analysis involves exposing splenocytes (cells isolated from the spleen, the main organ involved in immune defense) to a bacterio- or virus-mimicking attack.

MRX005 resulted in a significant decrease in interferon y levels in splenocytes after exposure to LPS (Fig. 42). In addition, MRX005 resulted in a significant reduction in interleukin 6 and tumor necrosis factor levels after exposure to LPS (Figures 44 and 45, respectively). MRx0029 treatment resulted in a decrease in interferon γ, interleukin 1β and interleukin 6 after exposure to LPS (Fig. 42, 43 and 44, respectively).

Treatment with MRx005 and MRx0029 resulted in increased levels of the chemoattractant CXCL1 (FIG. 47).

Results - Effect on the levels of short-chain fatty acids in the cecum.

Short-chain fatty acids (SCFAs) were produced when indigestible fiber from food was fermented by bacteria in the intestine. The results of MRX005 administration are shown in FIG. 48.

- 42 037000

Example 18. Additional analysis of MRX029 and MRX005-induced changes in gene expression levels.

Ways.

Cell line.

SH-SY5Y cells.

Bacterial strains.

755: Parabacteroides distasonis (MRX005).

Megasphaera massiliensis (MRX0029).

qPCR.

SHSY5Y were seeded in 10 cm Petri dishes with a density of 2x10 ⁶ cells. After 24 hours, cells were treated with differentiation medium (differentiation medium containing 1% FBS without RA) with 10% bacterial supernatants or YCFA +, 10 μM RA, 200 μM hexanoic acid, or 200 μM valproic acid for 17 hours. images with an EVOS XL phase contrast microscope at 40X70.65 magnification. Cells were harvested and total RNA was isolated according to the protocol for the RNeasy mini-kit (Qiagen). cDNA was obtained using a high performance cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as an internal control. The multiplicity of changes was calculated in accordance with method 2 ^{i- c} . The primer sequences of MAP2, DRD2, GABRB3, SYP, PINK1, PARK7 and NSE are suggested in the sequence listing.

Immunolabeling and cell imaging.

Cells were plated on 8-well slides (Marienfeld Laboratory Glassware) # 5x10 ⁴ cells / well overnight and treated with 10% bacterial supernatant for 24 hours. For differentiation, cells were treated with 10 nM RA for 5 days prior to treatment with cell-free bacterial supernatant for 24 h. After that, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT). Fixed cells were washed with PBS and permeabilized with 1% Trinone X-100 in PBS for 10 min. After washing with PBS, the preparations were incubated with blocking buffer (4% BSA / PBS) for 1 h at RT before adding anti-MAP2 antibody or P3-tubulin (sc-74421 and sc-80005, respectively, Santa Cruz Biotechnology Inc) diluted in 1% BSA / PBS for 12 h at 4 ° C. They were then washed twice with PBS, then incubated with Alexa Flour 488 conjugated anti-mouse antibody (Molecular Probes Inc) and Alexa Flour 594 conjugated phalloidin (ab176757, Abcam) for 1 h at RT. After washing 3X with PBS, preparations were stained with DAPI and embedded in Vectashield® (Vector Laboratories). The preparations were examined using an Axioskop 50 microscope (Zeiss) equipped with a 63x / 1.2 W Korr objective and filter sets suitable for the detection of the fluorochromes used. The manually set exposure time in the case of digital imaging immunolabeled with MAP-2 was kept constant, allowing comparison between different wells and treatments. The exposure time of phalloidin (F-actin) and DAPI was varied to match the visual field. The randomized fields of view were acquired using a QImaging camera controlled by the Image Pro Plus software. Images were saved as TIFF files and opened in Adobe Photoshop CC 2015.1.2. Images using MAP-2, DAPI and phalloidin were then overlaid and merged. Illustrative images were selected to illustrate differences in the amount and position of the proteins tested.

Immunoblotting.

SH-SY5Y cells were cultured under the conditions described above, treated with MRx0005 and MRx0029 for 24 h, and then lysed in RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, UK). Protein concentration was assessed using a BCA assay kit (Pierce Biotechnology, Rockford, IL), separated by SDS-PAGE, and transferred to a PVDF membrane. The membranes were then blocked with 5% non-fat dry milk or 5% BSA and incubated overnight at 4 ° C with primary antibodies (MAP2 and β3-tubulin, respectively). Blots were then incubated with the appropriate horseradish peroxidase (HRP) -conjugated secondary antibody and proteins were detected using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). In both MAP2 and P3-tubulin, β-actin acted as a control to observe the variability of protein loading in the samples.

Results and discussion.

Gene expression.

FIG. 13A (graphical inset) and 49 show MRx0029 and MRX005-induced changes in the expression levels of actin, villin, ocludin, TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7, and NSE.

Microscopy and immunoblotting.

FIG. 50 shows the change in the level of MAP2 expression in SHSY5Y cells, determined using confocal microscopy. Expression levels of MAP2 and B3-tubulin were also quantified by immunoblotting. The results shown in FIG. 50M and N,

- 43 037000 indicate that MRX029 leads to the induction of an increase in the level of expression of MAP2.

Sequences

SEQ ID NO: 1 (Megasphaera massiliensis 16S gene of ribosomal RNA, a partial sequence, strain: NP3 - JX424772.1) agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 61 gagaagagat gagaagcttg cttcttatca attcgagtgg caaacgggtg agtaacgcgt 121 aagcaacctg cccttcagat ggggacaaca gctggaaacg gctgctaata ccgaatacgt 181 tctttccgcc gcatgacggg aagaagaaag ggaggccttc gggctttcgc tggaggaggg 241 gcttgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg atcagtagcc 301 ggtctgagag gatgaacggc cacattggga ctgagacacg gcccagactc ctacgggagg 361 cagcagtggg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgaacga 421 tgacggcctt cgggttgtaa agttctgtta tatgggacga acagggcatc ggttaatacc 481 cggtgtcttt gacggtaccg taagagaaag ccacggctaa ctacgtgcca gcagccgcgg 541 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggcgc gcaggcggca 601 tcgcaagtcg gtcttaaaag tgcggggctt aaccccgtga ggggaccgaa actgtgaagc 661 tcgagtgtcg gagaggaaag cggaattcct agtgtagcgg tgaaatgcgt agatattagg 721 aggaacacca gtggcgaaag cggctttctg gacgaca act gacgctgagg cgcgaaagcc 781 aggggagcaa acgggattag ataccccggt agtcctggcc gtaaacgatg gatactaggt 841 gtaggaggta tcgactcctt ctgtgccgga gttaacgcaa taagtatccc gcctggggag 901 tacggccgca aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagtat 961 gtggtttaat tcgacgcaac gcgaagaacc ttaccaagcc ttgacattga ttgctacgga 1021 aagagatttc cggttcttct tcggaagaca agaaaacagg tggtgcacgg ctgtcgtcag 1081 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctat cttctgttgc 1141 cagcacctcg ggtggggact cagaagagac tgccgcagac aatgcggagg aaggcgggga 1201 tgacgtcaag tcatcatgcc ccttatggct tgggctacac acgtactaca atggctctta 1261 atagagggac gcgaaggagc gatccggagc aaaccccaaa aacagagtcc cagttcggat 1321 tgcaggctgc aactcgcctg catgaagcag gaatcgctag laatcgcagg Icagcatact 1381 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgaa agtcattcac 1441 acccgaagcc ggtgaggcaa ccgcaaggaa ccagccgtcg aaggtggggg cgatgattgg 1501 ggtgaagtcg taacaaggt

SEQ ID NO: 2 (consensus sequence 16S rRNA strain Megasphaera massiliensis MRx0029)

TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTA AGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATA CGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCCTTCGGGCTTTCGCTGG AGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGAC GATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGG AGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATATGGGAC GAACAGGACATCGGTTAATACCCGGTGTCTTTGACGGTACCGTAAGAGAAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAA TTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGTCGGTCTTAAAAGTGCGGG GCTTAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCG GAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAA AGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACG GGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGAGGT ATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGC CGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGT GGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGCCTTGACATTGATTGCTACGG AAAGAGATTTCCGGTTCTTCTTCGGAAGACAAG AAAACAGGTGGTGCACGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCT TCTGTTGCCAGCACCTCGGGTGGGGACTCAGAAGAGACTGCCGCAGACAATGCGGA GGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGGCTTGGGCTACACACGT ACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGC AAACCCCAAA AACAGAGTCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCG CTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGAGGCAACCGCAAG primers used for the qPCR (from SEQ ID NO: in brackets)

- 44 037000

Name

Direct sequence

Reverse sequence

ASTV

GATCAAGATCATTGCTCCTC (3) TTGTCAAGAAAGGGTGTAAC (4)

GAPDH

GGTATCGTGGAAGGACTCATG ATGCCAGTGAGCTTCCCGTTC (6) (5)

MAP2

CTCAGCACCGCTAACAGAGG CATTGGCGCTTCTCTCCTC (8) (7)

Okklyudin

AAGAGGAATTTTGACACTGG (9) GCCATGTACTCTTCACTTTC (10)

TJ1

AAGTCACACTGGTGAAATSS CTCTTGCTGCCAAAASTATST (12)

TJP2 (11)

CCCTCCCCTGGATCAGGAT (13) GCCATCAAACTCGTCCATCA (14)

Willin

CATTACCTGCTCTACGTTTG (15) AGATGGACATAAGATGAGGTG (16)

SEQ ID NO: 17 (consensus sequence 16S rRNA of Parabacteroides distasonis MRX0005 strain)

AMCCGGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTGCCTAT

CAGAGGGGGATAACCCGGCGAAAGTCGGACTAATACCGCATGAAGCAGGGATCCC

GCATGGGAATATTTGCTAAAGATTCATCGCTGATAGATAGGCATGCGTTCCATTAGG

CAGTTGGCGGGGTAACGGCCCACCAAACCGACGATGGATAGGGGTTCTGAGAGGAA

GGTCCCCCACATTGGTACTGAGACACGGACCAAACTCCTACGGGAGGCAGCAGTGA

GGAATATTGGTCAATGGGCGTGAGCCTGAACCAGCCAAGTCGCGTGAGGGATGAAG

GTTCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGTGTCCCGTT

TTGTATGTACCTTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATA

CGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTT

TTAAGTCAGCGGTGAAAGTCTGTGGCTCAACCATAGAATTGCCGTTGAAACTGGGA

GGCTTGAGTATGTTTGAGGCAGGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGA

TATCACGCAGAACCCCGATTGCGAAGGCAGCCTGCCAAGCCATTACTGACGCTGAT

GCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCAGTAAA

CGATGATCACTAGCTGTTTGCGATACACTGTAAGCGGCACAGCGAAAGCGTTAAGT

GATCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCC

CGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCG

GGTTTGAACGCATTCGGACMGAKGTGGAAACACATTTTCTAGCAATAGCCATTTGC

GAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATA

ACGAGCGCAACCCTTGCCACTAGTTACTAACAGGTAAAGCTGAGGACTCTGGTGGG

ACTGCCAGCGTAAGCTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTT

ACATCCGGGGCGACACACGTGTTACAATGGCGTGGACAAAGGGAAGCCACCTGGCG

ACAGGGAGCGAATCCCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCAACCCGAC

TCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTT

CCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGCCGGGGGTACCTGAAGTC

CGTAACCGCGAGGATCGGCCTAGGGTAAAACTGGTGACTGGGGCTAAGTCGTACGG

GG

Primers and probes used for ex vivo qPCR (with SEQ ID NO: in brackets) Ex vivo:

1 rim for the fact that the reciprocal of the sequence ι- ϊ '' ''

GAT TAC TGC TCT GGC TCC TAG GAC TCA TCG TAC 1 CC TGC TTG mqx _; (18)

V \ T GGT GAA GGT CGG TGT G GTG GAG TCA TACTGG AAC ATG TAG ^; (22): GCT GCC TTG ATG ITT ACT TTG AC GCAACCGAAGTATGAAATAACCA _ΛΓ , i (24) (25); AGC CAG AGT CCT TCA GAG A ₍₂₇₎ TCC TTA GCC ACT CCT TCT GT _}

-Probe / 56-FAWC1G GCC TCA / ZfcN / CTG TGC

ACCTTCCHIABkFQr ( ²⁰ ) / 56-FAM / TGC AAA TGG / ZEN / CAG CCC

TGG TGaiABkFQ / (23) / 56-FAM / ACC ASG TGA YENYUAA GAG TGATGACCATCCSIABkFOl 1 ²⁶ 1/56-FMVCCT ACC CCA / ZEN / ATT TCC AAT GCT CTC CTaiABkFOf (2Θ)

Additional primers used in cTCCR (with SEQ ID NO in brackets)

Gene ID Direct sequence Reverse sequence

NSE ((VTGTATCGTAAGAACGGT (30) GCCACCATTGATCACGTTGA (31)

PINK1 CCCAAGCAACTAGCCCCTC (32) GGCAGCACATCAGGGTAGTC (33)

PARK7 GTAGCCGTGATGTGGTCATTT (34) CTGTGCGCCCAGATTACCT (35)

SYP CTCGGCTTTGTGAAGGTGCT (36) GGCTTCATGGCATCAACTTCA (37)

- 45 037000

List of references

[I] Sptrr ei al. (2011) Nai Rev Mkmbial. 9 (4): 279-90.

[2] Eckbwge / aZ (2005) Sdatce. 10; 308 (5728): 1635-8.

[3] Macphersoneial. (21SH) Microbes Infect. 3 (12): 1021-35

[4] Macpherson et al. (2002) Cell Mol Life 59 (12): 2088-96.

[5] Mazmantan et al. (2005) Cell 15: 122 (1): 107-18.

[6] Frank et al. (2007) PNAS 104 (34): 13780-5.

[7] Scanlan et _a (. (2iWbUClin Microbiol. 44 (11): 3980-8.

[8] Kana ei al. (2 () 1 ()) Inflamm Bowel Dis 16 (12): 2034-42.

And Machiek et aL (2013) Gut. 63 (8): 1275-83.

[10) WO 2013/050792

[H] WO 03/046580

[12] WO 2013/008039

[13] WO 2014/167338

[14] Goldin and Gorbach (2008) Clm Infect Dis. 46 Suppl 2: 896-100.

(15j Azad et al. (2W 3) BW. 347: (647 f.

[16) Mayer et al (2014) The Journal of Neumsdence 34 (46): 15490 .15496

[17] Ciyan ami Dimm (2015) Nanvpsyehopharmaeology, 40; 241 -2.

[18] Zhou and Foster (2015) Neumpsychialric Disease and Treatment 11: 715 723.

[I9] Wangand Kasper (2014) Brain Behov Immim. 38: 1-12.

[20] US2 "WOO53t) 4

[21] US2004 / 170617

[22] Padmanabhan el al (2013) Standards in Genomic Sciences 8: 525-538

[23] Masco et al. (2 () 03) Systematic and Applied Mierobtologt '26: 557-563.

[24] Sriakova et al. (2011), /. Microbiol. Methods, 87 (1): 10-6.

[25] Kadi & al (2006) J Neumimmunol174: 133-46 '

[26] Pal R et al (2016) Neurol Res 38 (12): 1111-1122 (27] Daniele ei ul (2015) Act Signal 8 (376): ra45

[28] Ahmed et al, manuscript in preparation

[29] Baraczka et al. (1983), / Neural Transm. 58 (3-4): 299-304.

[30] Ekirup ei al. (1995) Aaa Neurol Seaiid. 92 (2): 116-21.

[311 Wang a al. (2016) J Neurogasitveniend Mold 22: 589-605.

[32] Zadori et al (2012) Jotmial of Neural Transmission, 119,2,275 283

[33] Lee et al (2008) European J. Cell Biology 87: 389-397

[34] Pirooznia and Elefant (2013) Front Cell Ncurosci. It 30.

[35] Tang, et al. (2M7} JAm Heart Assoc, 6 (10).

[36] Wang & al, (2015) PNAS 112 (9): 2583-2858

[37] Psaty et al. ¢ 2003) JAMA, 289 (19): 2534 44

[38] Lancet. (I ^ 5) 346 (8991 8992): 1647 53

[39] Miyamoto-Shinohara et ul. (2008) 7. Gen. Appl. Microbiol., 54.9 24.

[40] Cryopreservation and Freeze-Drying Protocols, cd. by Day and McLellan, Humana Press.

[41] Leslie er a /. (i995) Appl. Environ. Microbiol. 61,359.2 3597.

[42] Mitropoulou et al. (20 i 3) J Nulr Meiab. (2013) 716861.

[43] Kailasapathy et al. (2002) Gurr Issues Infest Microbiol. 3 (2): 39-48.

[44] Handbook of Pharmaceutical Excipients, 2nd Edition. (1994), Edited by A Wade and PJ Weller

[45] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985)

Claims (11)

1. The use of a composition containing a bacterial strain of the genus Megasphaera for the treatment or prevention of a neurodegenerative disease, wherein said bacterial strain has a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2. 2. The use according to claim 1, characterized in that the neurodegenerative disease is selected from the group consisting of Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olshevsky syndrome, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism and drug parkinsonism; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron diseases; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body dementia, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration. 3. Use according to claim 2, characterized in that the neurodegenerative disease is Parkinson's disease. 4. Use according to any one of claims 1 to 3, characterized in that the neurodegenerative disease - 46 037000 is a neurodegenerative disease that occurs at an early age and / or said composition is intended to prevent or delay the onset or progression of a neurodegenerative disease. 5. Use according to any of the preceding claims, characterized in that said bacterial strain is Megasphaera massiliensis. 6. Use according to any of the preceding claims, characterized in that said bacterial strain has a 16s rRNA sequence which is at least 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 2. 7. Use according to claim 1, characterized in that said composition comprises a bacterial strain of the species Megasphaera massiliensis for the treatment or prevention of Parkinson's disease. 8. Use according to any of the preceding claims, characterized in that said composition is intended for oral administration and / or said composition contains one or more pharmaceutically acceptable excipients or carriers and / or said bacterial strain is lyophilized or a food product containing said composition for use for any of the above items. 9. Use according to any one of claims 1 to 4, wherein the composition comprises a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. 10. Use according to claim 9, wherein the composition comprises a pharmaceutically acceptable carrier or excipient. 11. Use according to any one of claims 1 to 4, wherein the composition comprises a biologically pure culture of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787.