EA038133B1 - Compositions comprising bacterial strains - Google Patents

Abstract

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

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 body [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 degraded to release byproducts, 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 from 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 strains, have been proposed for use in the treatment of various 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]. Preclinical and clinical studies have largely suggested 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 [10]. Parabacteroides distasonis has been proposed for the treatment of a number of disorders, including asthma, rheumatoid arthritis, and multiple sclerosis [ ²⁰ ].

Like asthma and rheumatoid arthritis, multiple sclerosis is mediated primarily by the immune system. The immune system attacks myelin axons in the central nervous system, destroying plaque or damage from the myelin. Demyelination occurs, in particular, in the optic nerves located under the pia mater, brainstem, cerebellum, and juxtacortical and periventricular areas of the white matter.

Therefore, multiple sclerosis has a different pathogenesis compared to other neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease or dementia. For example, multiple sclerosis is usually diagnosed in patients between the ages of 20 and 30, while many other neurodegenerative diseases such as Parkinson's, Alzheimer's, and dementia are diagnosed mainly in patients over the age of 65.

Parkinson's disease, like many neurodegenerative diseases, is mediated primarily by the accumulation of misfolded protein. Parkinson's disease is a synucleinopathology that involves the accumulation of α-synuclein, which aggregates as non- 1038133 soluble fibrils in Lewy bodies in the cytoplasm of the neuron body. The accumulation of α-synuclein is toxic and leads to disruption of the functions of mitochondria, lysosomes and the endoplasmic reticulum, and also leads to disruption of microtubule transport.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen have been investigated for their effectiveness in treating a range of neurological diseases, however the clinical impact of NSAIDs on neurodegenerative diseases such as Parkinson's disease remains unclear. Although some studies have shown that chronic NSAID use has a protective effect against Parkinson's disease, other studies have not been able to confirm a significant association. A recent meta-analysis showed that the use of non-aspirin NSAIDs, in particular ibuprofen, reduced the risk of PD by 15%, while the use of aspirin showed no effect [ ²¹ ].

Currently, the practical effect of the link between the microbiome and diseases of the human brain is poorly characterized. Accordingly, more direct analytical studies are required to identify the therapeutic effect 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.

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 from the genus Parabacteroides can be effective in the treatment of neurodegenerative diseases. As described in the examples, administration of compositions containing Parabacteroides distasonis can result in protection from reactive oxygen species, thereby acting as a neuroprotective agent. The inventors have also found that treatment with Parabacteroides distasonis can reduce the activation of pro-inflammatory molecules such as NFkB and IL-6 by LPS and the mutant α-synuclein A53T. The inventors have found that treatment with Parabacteroides distasonis 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 inventors have also found that Parabacteroides distasonis can produce indole, which can reduce inflammation and oxidative stress. In addition, the present inventors have demonstrated that treatment with Parabacteroides distasonis can lead to an increase in kynurenine levels.

The inventors have also found that Parabacteroides distasonis leads 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.

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

In certain embodiments, the invention provides a composition comprising a bacterial strain of the genus Parabacteroides 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-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.

In preferred embodiments, the present invention provides a composition comprising a bacterial strain of the genus Pambacteroides 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 Parabacteroides strains can lead to the activation of pro-inflammatory molecules such as NFkB and IL-6 by LPS and mutant α-synuclein A53T 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 Parabacteroides distasonis for

- 2,038,133 applications in the treatment of Parkinson's disease. Compositions in which Parabacteroides distasonis is used may be particularly effective in the treatment of Parkinson's disease.

In some embodiments, the compositions of this invention are for use in a method for treating or preventing 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 Parabacteroides distasonis. Also can be used closely related strains that have a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of the bacterial strain Parabacteroides distasonis ... Preferably, the bacterial strain has a 16S rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 8 or 9. Preferably, the sequence identity is determined with respect to SEQ ID NO: 9. Preferably, the bacterial strain for use in the present invention has the 16S rRNA sequence represented by SEQ ID NO: 9.

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 the 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 a composition as 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 Parabacteroides.

In certain embodiments of the 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, eg, treatment of brain damage resulting from stroke.

Brief description of graphic materials

FIG. 1 - viability of neuroblastoma cells, Fig. 2A: Decreased IL-6 secretion, FIG. 2B: Decreased secretion of IL-8, FIG. 3: Inhibition of IL-6 and IL-8 secretion by α-synuclein, FIG. 4: Inhibition of α-synuclein-induced activation of the NFkB promoter, FIG. 5: Inhibition of LPS-induced activation of the NFkB promoter, FIG. 6 - change in antioxidant capacity, FIG. 7 - change in the total antioxidant capacity (lipid oxidation), Fig. 8 - change in the activity of histone deacetylase (HDAC), FIG. 9 - the level of production of indole, FIG. 10 - the level of production of kynurenine, Fig. 11 - neuroprotection - cell viability. FIG. 11 shows the same data as in FIG. 1, fig. 12 - levels of production of metabolites - neurotransmitters in the brain, FIG. 13 shows the production levels of organic acid metabolites in the supernatant, FIG. 14 - effect on intestinal barrier function, FIG. 15 - the level of production of BDNF, FIG. 16: Changes in ROS levels in (A) U373 cells, (B) SH-SY5Y cells, FIG. 17 shows the production of neurotransmitters in the brain, FIG. 18 - changes in the expression of receptors in the hippocampus A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor and D) mineralocorticoid receptor,

- 3 038133 fig. 19: Changes in hippocampal expression of A) corticotropin-releasing hormone (CRH), B) BDNF expression and C) TLR4, FIG. 20 - 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. 21: Changes in the expression in the hippocampus of A) tumor necrosis factor, B) interleukin 1b and C) IL-6, FIG. 22 - A) changes in the expression in the hippocampus of integrin α M (CD11b) and B) changes in the expression in the hippocampus of serotonin receptor 1A (5-HT1A receptor), FIG. 23 - 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. 24 - changes in the expression in the hippocampus of A) A-receptor 2 γ-aminobutyric acid (GABA A2), B) B-receptor 1 γ-aminobutyric acid (GABABR1) and C) dopamine receptor 1 (DRD1), FIG. 25: Changes in the expression of receptors in the amygdala of A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor and D) mineralocorticoid receptor, FIG. 26 - changes in the expression in the amygdala of A) brain neurotrophic factor (BDNF), B) Toll-like receptor 4 (TLR-4), C) receptor 1 corticotropin-releasing hormone (CRFR1) and D) receptor 2 corticotropin-releasing hormone (CRFR2), Fig. 27 - changes in the expression in the amygdala of A) integrin α M (CD11b), B) interleukin 6 (IL-6), C) subunit 2A of glutamate ionotropic receptor type NMDA (Grin2A) and D) subunit 2B of glutamate ionotropic receptor type NMDA (Grin2B) , fig. 28 - changes in the expression in the amygdala of A) the α 2 subunit of the AMK-A G receptor (GABRA2), B) the subunit of the GABA-A type B receptor (GABBR1) and C) the dopamine receptor 1 (DRD1), FIG. 29: Changes in the expression in the prefrontal cortex of A) oxytocin receptor, B) brain neurotrophic factor (BDNF), C) mineralocorticoid receptor and D) glucocorticoid receptor, FIG. 30 - changes in expression in the prefrontal cortex A) Toll-like receptor 4 (TLR-4), B) receptor 1 corticotropin-releasing hormone (CRFR1), C) receptor 2 corticotropin-releasing hormone (CRFR2) integrin α M (CD11b), fig. 31 - changes in the expression in the prefrontal cortex of A) interleukin 6 (IL-6), B) subunit 2A of the glutamate ionotropic receptor type NMDA (Grin2A), C) subunit 2B of the glutamate ionotropic receptor type NMDA (Grin2B) and D) subunit α 2 of the GABA receptor -A (GABRA2), fig. 32: Changes in the expression in the prefrontal cortex of A) subunit of GABAA type B receptor 1 (GABBR1) and B) dopamine receptor 1 (DRD1), FIG. 33: Changes in colon expression of A) tryptophanhydrolase 1 (Tph1) and B) indolamine2,3-dioxygenase-1 (IDO1), FIG. 34: Changes in the expression in the ileum of A) tryptophanhydrolase 1 (Tphl) and B) indoleamine 2,3-dioxygenase-1 (IDO1), FIG. 35: Changes in the levels of circulating tryptophan metabolites A) kynurenine, B) tryptophan and C) kynurenine / tryptophan metabolic rate, FIG. 36 — Effect on interferon γ production from splenocytes from mice fed MRx0005 and MRx0029, FIG. 37 shows the effect on the production of interleukin 1β from splenocytes, FIG. 38 — Influence on the production of interleukin 6 from splenocytes, FIG. 39: Influence on the production of tumor necrosis factor from splenocytes, FIG. 40 shows the effect on the production of interleukin 10 from splenocytes, FIG. 41: Effect on the production of CXCL1 chemoattractant from splenocytes, FIG. 42 shows changes in the levels of short-chain fatty acids in the caecum, FIG. 43: 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. 44 - differentiation of SHSY5Y cells 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. (J-K) 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 contain a bacterial strain of the genus Parabacteroides. These examples demonstrate that bacteria of this genus are useful in the treatment or prevention of neurodegenerative disorders. Preferred bacterial strains are Parabacteroides distasonis.

Examples of Parabacteroides species for use in the present invention include Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides merdae, and Parabacteroides johnsonii. Parabacteroides resemble Bacteroides and are gram-negative, obligate anaerobic, non-spore-forming, immotile and rod-shaped, and also have a size of 0.8-1.6x1.2-12 microns. Parabacteroides distasonis is one of the most common species in human feces. A typical P. distasonis strain is JCM 5825 ^T (= CCUG 4941 ^T = DSM 20701 ^T = ATCC 8503 ^T ). GenBank / EMBL / DDBJ accession numbers of 16S rRNA gene sequences of P. distasonis JCM 5825T, JCM 13400, JCM 13401, JCM 13402, JCM 13403 and JCM 13404 strains, as well as of P. merdae strains JCM 9497T and AB23829922 are respectively (disclosed herein as SEQ ID NO: 1-8). Illustrative strains are also described in [22].

Parabacteroides distasonis, deposited under accession number NCIMB 42382, was examined in the examples section and is also referred to herein as strain 755 or MRx0005. The sequence of the 16S rRNA of the strain 755 that was examined is set forth in SEQ ID NO: 9. Strain 755 was deposited with permission from the International Depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) GT Biologies 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.

WO 2016/203220 describes the administration of strain 755 to mice and shows that it can influence the course of the disease outside the intestine (eg asthma and arthritis). Strain 755 also affects the course of the disease outside the intestine in the treatment of neurodegenerative disorders described herein.

The genomic sequence of strain 755 is set forth in SEQ ID NO: 10. Said sequence was obtained using the PacBio RS II platform.

Bacterial strains closely related to the strain studied in the examples are also expected to be effective in treating or preventing neurodegenerative disorders. In certain embodiments, 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 the 16s rRNA sequence of the bacterial strain Parabacteroides distasonis. 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, 2, 3, 4, 5 , 6, 7, 8 or 9. Preferably, sequence identity is determined with respect to SEQ ID NO: 9. Preferably, the bacterial strain for use in the present invention has the 16s pRNA sequence represented by SEQ ID NO: 9.

Bacterial strains, which are biotypes of the bacterium deposited under accession number 42382, are also expected to be effective in the treatment or prevention of 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 the bacterium deposited under accession number NCIMB 42382 and which are suitable for use in this invention can be identified by sequencing other nucleotide sequences of the bacterium deposited under accession number NCIMB 42382. 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% of its entire genome (for example, in 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 may have sequences with at least 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity to the corresponding bacterial sequence deposited under accession number 42382 in NCIMB.

In certain embodiments, the bacterial strain for use in this invention has a genome with sequence identity to SEQ ID NO: 10. In preferred embodiments, the bacterial strain for use in this invention has a genome with at least 90% sequence identity (e.g. at least 92, 94, 95, 96, 97, 98, 99 or 100% sequence identity) relative to SEQ ID NO: 10 for at least 60% (for example, at least 65, 70, 75, 80 , 85, 95, 96, 97, 98, 99 or 100%) SEQ ID NO: 10. For example, a bacterial strain for use in the present invention may have a genome with at least 90% sequence identity to SEQ ID NO: 10 over 70% of SEQ ID NO: 10, or at least 90% sequence identity with respect to SEQ ID NO: 10 over 80% of SEQ ID NO: 10, or at least 90% sequence identity with respect to to SEQ I D NO: 10 over 90% of SEQ ID NO: 10, or at least 90% sequence identity with respect to SEQ ID NO: 10 over 100% of SEQ ID NO: 10, or at least 95% sequence identity with respect to to SEQ ID NO: 10 over 70% of SEQ ID NO: 10, or at least 95% sequence identity to SEQ ID NO: 10 over 80% of SEQ ID NO: 10, or at least 95% sequence identity with respect to SEQ ID NO: 10 over 90% of SEQ ID NO: 10, or at least 95% sequence identity with respect to SEQ ID NO: 10 over 100% of SEQ ID NO: 10, or at least 98% sequence identity to SEQ ID NO: 10 over 70% of SEQ ID NO: 10, or at least 98% sequence identity to SEQ ID NO: 10 over 80% of SEQ ID NO: 10, or at least 98% sequence identity to SEQ ID NO: 10 over 90% of SEQ ID NO: 10, or at least 98% sequence identity to SEQ ID NO: 10 over 100% of SEQ ID NO: 10.

Alternatively, strains that are biotypes of the bacterium deposited under accession number NCIMB 42382 and which are suitable for use in the present invention may be identified using deposit accession number NCIMB 42382 and fragment restriction analysis and / or PCR, for example, with 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 Parabacteroides distasonis.

In certain embodiments, the strains that are biotypes of the bacterium deposited under accession number NCIMB 42382 and which are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42382 , 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, biotypic strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42382.

Other strains of Parabacteroides that are useful in the compositions and methods of this invention, such as the biotypes of the bacterium deposited under accession number NCIMB 42382, 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 bacteria deposited under accession number NCIMB 42382 may be useful in the present invention. A suitable strain will have comparable immunomodulatory activity against strain NCIMB 42382. In particular, a biotypic strain will produce comparable effects on neurodegenerative disease models and comparable effects on cytokine levels as the effects shown in the examples section that can be identified with using the culture and administration protocols described in the examples section.

A particularly preferred strain of this invention is the Parabacteroides distasonis strain deposited under accession number NCIMB 42382. This is an illustrative strain 755 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 Parabacteroides distasonis strain deposited under accession number NCIMB 42382, or a derivative thereof. The present invention also provides a composition comprising a cell of the Parabacteroides distasonis strain deposited under accession number NCIMB 42382, or a derivative thereof. This invention also provides a biologically pure culture of the Parabacteroides distasonis strain deposited under accession number NCIMB 42382. This invention also provides a cell of the Parabacteroides distasonis strain deposited under accession number NCIMB 42382, or a derivative thereof, for use in the treatment, in particular, of the diseases described in this document.

The derivative of the strain deposited under accession number NCIMB 42382 can be a daughter strain (progeny) or a strain cultured (subcloned) from the original 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 derivative strain will have comparable immunomodulatory activity with respect to the parent strain NCIMB 42382. In particular, the derivative strain will produce comparable effects on neurodegenerative disease models and comparable effects on cytokine levels as the effects demonstrated in the examples section. which can be identified using the culture and administration protocols described in the examples section. Derivative of strain NCIMB

42382 will generally be the biotype of strain NCIMB 42382.

References to cells of the Parabacteroides distasonis strain deposited under accession number NCIMB 42382 encompass any cells that have the same safety and therapeutic efficacy characteristics as strains deposited under accession number NCIMB 42382, 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 strains of Parabacteroides distasonis lead to a decrease in the secretion of pro-inflammatory cytokines such as IL-6 and IL-8. IL-8 has been associated with myelin sheath formation. Chronic inflammation induced by IL-6 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 treating 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. MRx0029 by itself leads to increased secretion of IL-8. Thus, the compositions of this invention are particularly effective in the treatment or prevention of neurodegenerative disorders characterized by demyelination.

The present inventors have also found that the bacterial strains of this 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 Megasphaera massiliensis. 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 herein. The sequence of the 16S rRNA of the MRx0005 strain that was examined is proposed in SEQ ID NO: 9. The MRx0005 strain was deposited with permission from the International Depository NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) GT Biologies 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.

The Megasphaera massiliensis strain, deposited under accession number NCIMB 42787, is an illustrative strain MRx0029 studied in the examples and shown to be effective in the treatment of diseases. Strain NCIMB 42787 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, Aberdeen, AB25 2ZS, Scotland) July 13, 2017 as Megasphaera massiliensis MRx0029 and assigned accession number NCIMB 42787. Thus, the present invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis strain, deposited under access number NCIMB 42787, or its derivative. 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. 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 diseases, described in this document.

In some 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 bio-type 7 038133 thereof, preferably for use in treatment, preferably for use in the treatment of 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 lead to 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 leads to the activation of the production of cytokines, for example IL-1e, IL-1a, IL-18, TNFα and IL-6. Treatment of cells with mutant α-synuclein is a model of familial Parkinson's disease. 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. TL4 is known to mediate cell death in a mouse MPTP 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 have been found to be activated in the brains of PD patients []. In addition, α-syn has been described as a TLR2 ligand [] and it has been demonstrated using HEK-TLR4 cells that α-syn is also a TLR4 ligand.

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.

It has been hypothesized that chemokines have important functions in the central nervous system (CNS), in addition to their primary role in the directed migration of leukocytes. In the murine oligodendrocyte precursor cell line, the chemokine MCP-1 did not increase the proliferation of oligodendrocyte precursors. During primary myelination, MCP-1 cultures did not lead to an increase in the formation of myelin segments in this system. The inventors have found that MRx0005 leads to increased levels of MCP-1. In certain embodiments, the compositions of this invention are intended for use in increasing MCP-1 levels in the treatment of diseases.

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-induced 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 neurodegenerative disease to be treated with the compositions of this invention is progressive inflammatory neuropathy.

In certain embodiments, the compositions of this invention may be effective for treating neurodegenerative disorders that occur in elderly patients. These examples show that the compositions of this invention can lead to the treatment of Parkinson's disease, which is mainly diagnosed in patients over the age of 65 years. In preferred embodiments, the compositions of this invention are for treating patients 65 years of age or older. In certain other embodiments, the patients are between 40 and 65 years of age. In other embodiments, the patients are over 40 years of age. In certain embodiments, the compositions of this invention are for use in the treatment of a disease associated with old age, for example, a disease diagnosed over the age of 50 years.

In certain embodiments, the compositions of the present invention are intended

- 8 038133 for the treatment of a neurodegenerative disease mediated or characterized by the accumulation of a protein, in particular misfolded protein.

In certain embodiments, the compositions of this invention are for use in the treatment of a neurodegenerative disease associated with loss of neurons in the gray matter of the brain. In certain embodiments, the compositions of this invention are for the treatment of a neurodegenerative disease that is not associated with lesions in the white matter of the brain.

In certain embodiments, the compositions of this invention are intended for use in the treatment of a neurodegenerative disease associated with irreversible symptoms.

In certain embodiments, the compositions of this invention are for use in the treatment of a neurodegenerative disease that is not an autoimmune disorder. In certain embodiments, the compositions of this invention are for use in the treatment of a neurodegenerative disease other than multiple sclerosis.

In certain embodiments, the compositions of this invention are 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.

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 for use in delaying the onset of progression of neurodegenerative disorders.

The compositions of this invention can lead to an increase in the secretion of IL-8. IL-8 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.

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 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 this invention can be used to improve long-term memory, in particular to improve long-term memory, which 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 [16-27].

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 the vagus nerve, as well as the gut microbiota.

As demonstrated in the examples, the compositions of this invention can modulate the microbiota-gut-brain axis and reduce 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,

- 9 038133 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.

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 lead to modulation of the neuroimmune pathway. In some embodiments, the compositions of this 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. In certain embodiments, the compositions of this invention can be used to modulate the dopaminergic system.

Signal transmission along the microbiota-gut-brain axis is modulated by the nervous systems. Accordingly, in some embodiments, the implementation of 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 transduction in the central nervous system. In some embodiments, the implementation of the compositions of this invention result in modulation of signal transmission in sensoronic 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 implementation of 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 transduction 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 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 neurochemical compounds produced by intraluminal bacteria or enteroendocrine cells. In certain preferred embodiments, the ENS neurons lead to 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 the activity of enterochromaffinocytes.

Neurodegenerative diseases.

Taupathies are neurodegenerative diseases associated with abnormal aggregation of tau protein in neurofibrillary or gliofibrillary tangles in the human brain. Alzheimer's disease is an example of taupathology. Synucleopathies (also called α-synucleopathies) are neurodegenerative diseases characterized by abnormal accumulation of α-synuclein aggregates in neurons, nerve fibers, or glial cells. Parkinson's disease is an example of synucleinopathology.

There is a clinical and pathological parallel between these two pathologies. Patients with Parkinson's disease often have dementia, and patients with Alzheimer's disease often exhibit parkinsonism [ ²⁸ ]. For example, progressive supranuclear palsy (also known as Steele-Richardson-Olszewski syndrome) has taupathology, but also results in significant

- 10 038133 parkinsonism [ ²⁹ ]. Mutations in LRRK2, which are known to cause parkinsonism, are associated with the accumulation of synuclein, tau, neither or both of them [ ³⁰ ].

Lewy body disease (LBD) is a neurodegenerative disease that is one of the most common causes of dementia in the elderly. The LBD provides an example of the existence of a continuity between tau and synucleinopathologies. LBD shares clinical and pathological properties with Parkinson's disease, Parkinson's disease dementia, and Alzheimer's disease [28].

In certain embodiments, the compositions of this invention may be useful for treating or preventing taupathies and / or synucleinopathies. In certain embodiments, the compositions of this invention may be useful for treating or preventing taupathies. In certain embodiments, the compositions of this invention may be useful for treating or preventing synucleinopathies. 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 Olshevsky syndrome, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism, and drug parkinsonism; Alzheimer's disease, including Benson's syndrome; and dementia; including dementia with Lewy bodies, vascular and frontotemporal dementia.

In preferred embodiments, the compositions of this invention may be useful for treating or preventing Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normotensive hydrocephalus, vascular or arteriosclerotic parkinsonism, and drug-induced parkinsonism. In preferred embodiments, the compositions of this invention may be useful for treating or preventing Alzheimer's disease, including Benson's syndrome. In further preferred embodiments, the compositions of this invention may be useful for treating or preventing dementia; including dementia with Lewy bodies; vascular and frontotemporal dementia.

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 disease progression are disorders of the autonomic nervous system (sialorrhea, seborrhea, constipation, urinary disturbances, sexual dysfunction, orthostatic hypotension, hyperhidrosis), sleep disturbances, and disturbances in the 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 invention provides a composition comprising a bacterial strain of the genus Parabacteroides for use in a method for treating or preventing Parkinson's disease. Compositions containing a bacterial strain of the genus Parabacteroides can lead to improved motor and cognitive functions in models of Parkinson's disease. Treatment with Parabacteroides strains can modulate signal transduction 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 can lead to levels of commensal metabolites, inflammatory markers and / or permeability in the gastrointestinal tract of a subject, 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 Parabacteroides distasonis for use in a method for treating or preventing Parkinson's disease. Compositions in which Parabacteroides distasonis 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 implementation of the compositions according to this invention result in the prevention, reduction or amelioration of one or more of the main symptoms of Parkinson's disease in the 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 abatement of tremor at rest; muscle rigidity and / or impaired postural reflex in

- 11 038133 subjects. In certain embodiments of the implementation of the composition according to this invention lead to the prevention, reduction or weakening of one or more symptoms associated with the progression of Parkinson's disease, selected from disorders of the autonomic nervous system (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, the compositions of this invention result in the prevention, reduction, or mitigation of the progression of Parkinson's disease. In certain embodiments, the compositions of this invention result in the prevention, reduction, or amelioration 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 this 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 result 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; Hamilton Depression Rating Scale (HAM-D 17 Hamilton Depression Rating Scale; depression); the Hamilton Anxiety Rating Scale (HAM-A Hamilton Anxiety Rating Scale) 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.

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 mixed forms of the two. Other forms of neurodegenerative disorders (eg, Lewy body dementia, frontotemporal dementia, Parkinson's disease 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, so the neuroprotective and neuroproliferative effects shown in the examples in the case of the compositions of this invention indicate that they may be useful in 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

- 12 038133 multiple areas selected from learning and memory; language; executive function; integrated attention; perceptual-motor and social cognition. Cognitive deficits should impair independence in daily activities. Moreover, 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 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 of the implementation of the composition according to this invention result in the prevention, reduction or weakening of the manifestation of cognitive functions in the 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 agitation, 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 of the implementation of the composition according to this invention result in the normalization of modification of the disease, while slowing or stopping the 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 compositions of this invention result in normalization of the ability of a subject with a neurodegenerative disease to carry out 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 outcome 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 to assess 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 sub scale (ADAS-cog) and the classic ADAS subscale to assess symptomatic normalization.

- 13 038133

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, 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 evaluating 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. It is believed that environmental factors 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 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 implementation of 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 composition 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 alleviation 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 a decrease in 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 intended 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 preventing 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 in preventing the development of MS. The compositions of this invention may be useful in preventing the progression of MS. In certain embodiments, the compositions of this invention are intended 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 for treating or normalizing 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 recurrence of MS. (PRMS), which is similar to PPMS, but with overlapping relapses.

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 Parabacteroides species, and in particular Parabacteroides distasonis, 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 the levels of cerebral 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 a major pathway for tryptophan metabolism that acts as a pathway for the production of nicotinamide adenine dinucleotide (NAD +). Kynurenine can be metabolized to neuroactive compounds such as kinureic acid (KYNA) and 3-hydroxy-l-kynurenine (3-0H-l-KYN), and to

- 15 038133 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. Kynurenine levels have been demonstrated to be decreased in the frontal cortex, shell, and nigra compacta of patients with PD [31]. 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 weaken MPP + -induced neuronal death in vitro in a human dopaminergic neuroblastoma cell line [32]. 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. Activation of GPR35 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 the modulation of kynurenine pathways, which are 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.

Immune response.

Signal transmission along the microbiota-gut-brain axis is modulated by changes in the immune response and 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 decreased 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. Elevated 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 intended for use in lowering IL-6 levels and / or NFkB levels in the treatment of neurodegene.

- 16 038133 active diseases.

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 myelin sheath formation and restore effective interaction between neurons. Thus, in some embodiments, the compositions of this invention are intended for use in the induction of 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 the 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. Disruption of the balance of acetylation and deacetylation of histones 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 [33]. Accordingly, in certain embodiments, the compositions of this invention can result in modulation of histone deacetylase activity. In certain embodiments of the implementation of the composition according to this invention can lead to a decrease in the activity of histone deacetylase. In certain embodiments of the implementation of the composition according to this invention can lead to a decrease in the activity of histone deacetylase.

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. The reduction in oxidative damage caused by reactive oxygen species can be used to target neurodegenerative diseases in the early stages. Accordingly, in certain embodiments, the compositions of this invention are intended for use in the treatment of early stage neurodegeneration. Additionally, accordingly, in certain embodiments, the compositions of this invention are intended for use in preventing the development of a neurodegenerative disease. In such embodiments, the implementation of the compositions of 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

17,038,133 embodiments of the composition 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 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 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 [34], and inhibition of HDAC can prevent white matter damage [35], 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 the treatment of 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 compositions of this invention are for use in treating 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 implementation of the compositions of this invention are intended for use in the treatment of brain damage resulting from cerebral hypoxia. In some embodiments, the compositions of this invention are for use in treating 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 affected by 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 cerebral 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, then 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 in order 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, resulting in decreased 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 not

- 18 038133 a fortified mass that passes through the bloodstream and restricts or blocks blood flow in an area distant from its place 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, the implementation of 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 subject diagnosed with a brain injury has suffered from focal cerebral ischemia. In some embodiments, the subject diagnosed with a brain injury has 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 has 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 destroy. 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 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 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 has suffered from cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with a brain injury has suffered from a cerebral aneurysm. In some embodiments, the subject diagnosed with a brain injury has suffered from cerebral AVM.

In some embodiments, the implementation of the compositions of this invention are intended 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 treating a cerebral aneurysm. In some embodiments, the implementation of the compositions of this invention are intended for use in the treatment of cerebral AVM.

Restoring 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 brain tissue damage. 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 it has been scientifically proven not only after a stroke, but also after a heart attack or other tissue damage, when the blood supply returns to the tissue after a period of ischemia or lack of oxygen. In some embodiments, the subject diagnosed with a brain injury suffered from reperfusion injury from a stroke. In some embodiments, the implementation of the compositions of this invention are intended for use in the treatment of reperfusion injury due to stroke.

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 subject diagnosed with a brain injury 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, non-steroidal 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 [36, 37]. 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 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 cerebral aneurysm. In some embodiments, the subject has arteriovenous malformations. In some embodiments, the subject has vasculitis. In some embodiments, the subject has sickle cell disease. In some embodiments, the subject has a bleeding disorder. In some embodiments, the subject has a history of 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 implementation of 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 implementation of 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 prior to 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 injury, particularly 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 resulting from the 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 results in the treatment of brain damage as a result of improving 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, eg, a patient identified as at risk of stroke. Compositions of this invention for use in the treatment of stroke may be provided after stroke, eg, during recovery. Compositions of this invention for use in the treatment of stroke may be offered during the acute recovery phase (i.e., up to and including one week after stroke). Compositions of this invention for use in the treatment of stroke may be offered during the subacute recovery phase (i.e., from one week to three months after stroke). Compositions of this invention for use in the treatment of stroke can be offered during the chronic recovery phase (from three months after stroke).

In certain embodiments, the compositions of this invention are intended for use 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, β-channel blockers, α-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, the compositions of this invention result in a reduction in ischemic tissue damage. 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 as a result of 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 the replacement and / or regeneration of cells 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).

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Routes of administration.

Preferably, the compositions of this invention are intended to be administered to the gastrointestinal tract to provide delivery and / or partial or complete colonization of the intestine with the bacterial strain of the 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 may be administered as a foam, cream, or gel.

In certain embodiments, the compositions of this invention may be administered in the form of 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 a soapy glycerin composition.

In certain embodiments, the composition of this invention is administered into the gastrointestinal tract using a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J-tube), 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 the 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 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 decreased or absent colonization of Pambacteroides, and in particular Parabacteroides distasonis.

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, a gastric tube may 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 [38-40].

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, such as not 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.

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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 technique can be used. Exemplary encapsulation techniques include entrapment in a porous matrix, attachment or adsorption to solid surfaces of a carrier, self-aggregation by flocculation or by cross-linking 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 the literature [41] and [42].

The specified composition can be administered orally and can be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Parabacteroides are anaerobic. Other ingredients (such as, for example, vitamin C) can 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 this invention can 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 the bacterium, for example, for an adult, may be from about 1 x 10 ³ to about 1 x 101 'colony forming units (CFU); for example, from about ^{1 x 10 7} to about 1 x 10 ¹⁰ CFU; in another example, from about 1x10 ⁶ to about 1x10 ¹⁰ CFU.

In certain embodiments, said composition comprises a bacterial strain in an amount from about 1x10 ⁶ to about 1x10 ¹¹ CFU / g, based on the weight of the composition; for example, from about 1x 108 to about 1x 10 ¹⁰ CFU / g. The dose can be, for example, 1 g, 3 g, 5 g 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 sugar alcohol that is not degraded 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 fructo-oligosaccharides, inulin, isomaltoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosanoligosaccharides (or COS), modified polyglucans, a-cams , D-tagatose, acacia fiber, carob, oat and citrus fiber. In one aspect, the prebiotics are short-chain fructo-oligosaccharides (shown herein below as FOS-c.c for simplicity); 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 modulate neuroprotection or neuroproliferation. In preferred embodiments, the nutritional supplements comprise or consist of beneficial

- 23 038133 for vitamins health. 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 [43]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in the literature [44]. 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 binding agent (s), lubricating agent (s), suspending (s) suspending agent (s), coating agent (s), solubilizing agent (s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum acacia, 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, a food product can provide a nutritional effect in addition to the therapeutic effect of this 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 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 yogurt, curdled milk, 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 contain one or more bacterial strains of the genus Parabacteroides 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 invention provides a composition comprising one or more bacterial strains of the Parabacteroides genus 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 Parabacteroides distasonis 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 Parabacteroides distasonis that do 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 implementation of the compositions according to this invention contain one or

- 24 038133 several bacterial strains of the species Parabacteroides distasonis and do not contain bacteria from any other species of Parabacteroides, or which contain only minor or biologically insignificant amounts of bacteria from another species of Parabacteroides. Thus, in some embodiments, the invention provides a composition comprising one or more bacterial strains of the species Parabacteroides distasonis that does not contain bacteria from any other species of Parabacteroides, or that contain only minor or biologically insignificant amounts of bacteria from another species of Parabacteroides for use in treatment.

In certain embodiments, the compositions of this invention comprise one bacterial strain or species and no 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 Parabacteroides 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 a single bacterial strain of the species Parabacteroides distasonis 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 compositions of this invention comprise 1-40, 1-30, 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, 2-10, 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 bacterial strain of Parabacteroides as part of a microbial consortium. For example, in some embodiments, the Parabacteroides 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 Parabacteroides bacterial strain in combination with a bacterial strain from a different 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 occur together in 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 two different organisms are human and 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 MRX005 strain, but that is not MRx0005, or that is not Parabacteroides distasonis.

In some embodiments, implementation, in which the composition of this invention contains more than one bacterial strain, species or genus, 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, the 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, then they

- 25 038133 are present only in insignificant quantities. The bacteria can be cultured after being obtained from adult feces and can be used in the composition of this invention.

In some embodiments, the bacterial strain for use in the present invention is obtained from the feces of a child. 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 the child, or if other bacterial strains are present, they are present in only minor amounts. The bacteria can be cultured after being obtained from the feces of a child 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 Parabacteroides 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 aforementioned pharmaceutical composition, wherein said bacterial strain is lyophilized or spray dried and is capable of partially or completely colonizing the intestine. In certain embodiments, the invention provides a pharmaceutical composition as defined above, wherein said bacterial strain is lyophilized or spray dried and viable and capable of partially or completely colonizing the intestine.

In some cases, the lyophilized bacterial strain is diluted by the addition to the composition. In some cases, dilution occurs using the diluent described herein.

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 the bacterial strain is about 1 × 10 ³ to about 1x10 ^minutes colony forming units per 1 g relative to the weight of the composition.

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

In certain embodiments, the invention provides the aforementioned 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,

- 26 038133 gelatin, glucose, anhydrous lactose, free-flowing lactose, β-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 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 a bacterial strain measured in colony forming units persists after a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

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, the specified composition can be provided in the form of a tablet or capsule. In some embodiments, said capsule is a gel-cap.

In some embodiments, the implementation of the compositions of this invention is 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 the 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, eg 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 may be an enteric tablet or an enteric capsule or the like. The enteric coating may be a standard enteric coating, for example a standard tablet, capsule or the like coating. for oral delivery. The composition may contain a film coating, such as a thin film of an enteric polymer, such as 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, the 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, the implementation of the specified thermogel material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose, or hydroxypropylmethylcellulose (HPMC). In some embodiments, the implementation of the specified capsule contains a shell that does not contain any film-forming polymer. In some embodiments, the implementation of the specified capsule contains a shell and the specified shell contains hydroxypropyl methylcellulose and does not contain any film-forming polymer (eg, see [45]). In some 27,038,133 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 and have a certain elasticity and softness. Soft capsules can be prepared, for example, based on gelatin or starch. Gelatin soft capsules are commercially available from various suppliers. Depending on the route of administration, such as, for example, orally or rectally, the soft capsules can have various 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 [46-48].

The solid or liquid media used for cultivation can be YCFA agar or YCFA medium. YCFA medium contains (per 100 ml, approximate values): 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), MgSO4 7H2O (0.009 g), CaCl ₂ (0.009 g), rezaruzin ( 0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-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 can 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, eg, by subcutaneous injection.

General instructions.

The practical application 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, literary sources [49] and [50-56] and others.

The term “containing” encompasses an inclusive as well as consisting, for example, a composition containing X, may consist exclusively 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 desired, an expression in essence can be omitted from the definition of the present invention.

References to a percentage of sequence identity between two nucleotide sequences means that when aligned, the indicated percentage of nucleotides is the same when compared to the indicated two sequences. The indicated alignment and the indicated percentage of 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 [57]. 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 [58] ...

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 others.

- 28 038133 specific characteristics to provide additional options for implementation. In particular, embodiments identified herein as suitable, typical, or preferred may be combined with each other (unless they are mutually exclusive).

Ways of carrying out 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 neuroproliferation was observed. Neuroblastoma cells were also treated with the dopaminergic neurotoxin 1-methyl-4phenylpyridinine (MPP), which induces irreversible symptoms of Parkinson's disease in neuroblstoma cells. Studied the ability of bacterial strains to function as neuroprotective against MPP.

Materials and methods.

Bacterial strain 755: Parabacteroides distasonis, Megasphaera massiliensis MRx0029.

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).

Way.

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. The 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 MRx0005 or MRx0029 resulted in increased 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 demonstrate that these bacterial strains can act as a neuroprotective agent.

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

Short description.

The activation of proinflammatory 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 to observe their ability to modulate IL-6 levels.

Materials and methods.

Bacterial strain 755: Parabacteroides distasonis.

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 IX sodium pyruvate.

Way.

After cultivation, MG U373 cells were plated in a 24-well plate at 100,000 cells / well. Cells were treated with LPS alone (1 μg / ml) or 10% bacterial supernatant from MRx0005 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 using 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. 2A. Treatment of neuroblastoma cells with LPS and the bacterial strain resulted in a decrease in the level of secreted IL-6.

- 29 038133

Example 2B. Efficiency 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.

Way.

After cultivation, MG U373 cells were plated in 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. 2B.

Example 2C. Efficacy of a bacterial inoculum in reducing α-synuclein-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 the present 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 investigated.

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.

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 on 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.

Way.

SHSY5Y cells were seeded 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 on 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 cocultured 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 10000 g for 3 min at 4 ° C , divided into aliquots and stored at

- 30 038133

-80 ° C. Human IL-6 and IL-8 were measured as described above.

Results.

The results of these experiments are shown in FIG. 3. Treatment of cells with wild-type α-synuclein and mutant isoforms E46K and A53T led to the induction of moderate secretion of IL-6. Α-syn-induced IL-6 secretion was inhibited in cells treated with bacterial strains.

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

Short description.

Activation of the NFkB promoter results in the production of proinflammatory cytokines IL-1e, 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 inhibit the activation of the NFkB promoter.

Materials and methods.

Bacterial strain 755: Parabacteroides distasonis.

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.

Way.

After cultivation, human Hek blue cells were plated in 96-well plates at 25,000 cells / well in 4 repetitions. One group of cells was treated only with α-synuclein A53T (1 μg / ml) or 10% bacterial supernatant from MRx0005 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 MRx0005 for 22 hours. Then 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. 4 and 5. FIG. 4 demonstrates that activation of the NFkB promoter by α-synuclein is inhibited by MRx0005. Ha fig. 5 demonstrates that activation of the NFkB LPS promoter is not inhibited by MRx0005.

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 (3ethylbenzothiazoline-6-sulfonic acid)) assay.

Bacterial strain 755: Parabacteroides distasonis.

Way.

Bacterial cells (10 or more) were harvested 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 12000xg 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. 6. In FIG. 6 demonstrates that MRx0005 has an antioxidant capacity of about 0.5 mM compared to trolox.

Example 5. The effectiveness of a bacterial inoculum in changing the 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 755: Parabacteroides distasonis.

Way.

Bacterial cells (10 ⁶ or more) were harvested and centrifuged, the washing step was performed using isotonic saline until the pellet was resuspended in analytical buffer 31 038133 re sodium chloride. The suspension was sonicated on ice for 10 min and then centrifuged at 10,000xg 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. 7. FIG. 7 demonstrates that MRx0005 is able to inhibit lipid peroxidation by about 20%, which is a higher antioxidant capacity than the positive control, butylated hydroxytoluene (1% w / v).

Example 6. The effectiveness of a bacterial inoculum in relation to the activity of histone deacetylase.

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 played a role in the pathogenesis associated with age-associated neurodegenerative diseases.

Materials and methods.

Bacterial strain 755: Parabacteroides distasonis.

Cell line.

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

Way.

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 (UK).

Results.

The results of these experiments are shown in FIG. 8. In FIG. 8 demonstrates that MRx0005 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.

Materials and methods.

Bacterial strain 755: Parabacteroides distasonis.

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

Way.

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. 9. In FIG. 9 demonstrates that MRx0005 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 bacteria according to 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 755: Parabacteroides distasonis.

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

Way.

The 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

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. 10. In FIG. 10 demonstrates that MRx0005 has the ability to produce kynurenine at a concentration of about 70 μM.

Example 9. 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. 11). Both MRx0005 and MRx0029 have shown significant effects and by themselves enhance mitochondrial metabolic activity in SHSY-5Y cells. 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. 11).

Example 10. 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 the approval of 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 arriving 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, the animals were decapitated and tissues were removed for experiment.

Collection of tissues.

Animals were sacrificed at random depending on treatment and study conditions; sampling took place between 9:00 and 13:00. Blood from the trunk was collected in tubes with potassium-EDTA (ethylenediaminetetrauccinic acid) and centrifuged for 15 min at 4000 g. Blood plasma was isolated and stored at -80 ° C for subsequent analysis. The brain was quickly excised, dissected, and each region of the brain was instantly 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 using HPLC in relation to samples from the brainstem. Briefly, brainstem tissue was sonicated in 500 μl of chilled mobile phase supplemented with 4 ng / 40 μl of N-methyl-5-HT (Sigma Chemical Co., UK) as an 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 / v. ) methanol (Alkem / Reagecon) and adjusted to pH 2.8 with 4N sodium hydroxide (Alkem / Reagecon). Then the homogenates were centrifuged for 15 min at 22000xg at 4 ° C and 40 μl of the supernatant was injected into the HPLC system, which consisted of the SCL 10-Avp system controller, LECD 6A electrochemical detector (Shimadzu), LC-10AS pump, STO10A oven, SIL autoinjector -10A (with sample cooler kept at 40 ° C) and Gastorr online degasser (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 Class-VP 5 computer program (Shimadzu). Neurotransmitters were identified by their characteristic retention times as determined by internal injections, which were chromatographed at regular intervals during sample analysis. The peak height ratios of analyte versus internal standard were measured and compared to standard injection. Results were expressed as ng per g wet weight.

Analysis of metabolites.

In the case of GC metabolite analysis, bacterial supernatant samples were derivatized with methyl chloroformate using a slightly modified version of the protocol described by Smart et al. (DOI: 10.1038 / nprot.2010.108). All samples were analyzed in random order. Analysis was performed using 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 by Johnsen et. al (DOI: 10.1016 / j.chroma.2017.04.052).

In the case of fatty acid analysis, the 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 by Johnsen et al. (DOI: 10.1016 / j.chroma.2017.04.052).

Results - production of neurotransmitters.

The results are shown in FIG. 12, 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 outlined below, suggesting that MRx00029 is the main producer of 4-hydroxyphenylacetic acid, a known antioxidant (Weon et al., 2016). More importantly, 4-hydroxyphenylacetic acid is a synthetic intermediate between dopamine and norepinephrine and an important bioactive molecule (Huot et al., Parkinson's Disese 2015). 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 structures. body and extrastriatal structures (Scatton B, Javoy-Agid F, Rouquier L, Dubois B, Agid Y Brain Res. 1983 Sep 26; 275 (2): 321-8.). L-DOPA primarily targets PD characteristics, while not targeting either 5-HT or norepinephrine reduction. In addition, the longer the duration of the L-DOPA treatment, the more visible are the range of motor and non-motor complications (eg, dyskinesia, psychiatric symptoms) (Hely MA, Morris JG, Reid WG, Trafficante R., Mov Disord. 2005 Feb; 20 (2): 190-9.) Thus, these data demonstrate that bacteria that produce organic acids such as 4-hydroxyphenylacetic acid or succinic acid may 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. The metabolites in the supernatant of the MRX0029 and MRX0005 cultures were analyzed and the results are shown in FIG. 13.

An insignificant 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. 13).

Analysis of fatty acids in the 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. 14B). These two strains were significantly different and, in particular, the production of 4-hydroxyphenylacetic acid MRx0005 and MRx0029, respectively, was remarkable (Fig. 14A). 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 metabolite of the Krebs cycle involved in oxidative phosphorylation. The oxidative phosphorylation complex is a major step in synaptic transfer of proteins and vesicles to the proximal and distal regions (Budd S.L. and Nichols,

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1998). Its impairment has been described in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and type 1 spinocerebellar ataxia (Manczak M. et al. 2004; Ebadi et al., 2001). These results are especially interesting because succinic acid can lead to an increase in mitochondrial activity and support susceptible neurons in neurodegenerative disease associated with protein malfold, including PD (Ferroet al., Plos one, 2017). 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. 14B 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 [59]. This property of MRx0029 (and other neuroprotective bacteria) may contribute to its effectiveness.

Example 11. Modulation of mRNA expression of tight junction proteins.

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 (Gagnon et al., J. Microbiological Methods, 2013) were used as an in vitro model to assess intestinal barrier damage and immunostimulation after treatment with MRx0005 and MRx0029. Differentiated HT29mtx cells exposed to phorbol-12-myristate-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. 14A).

Studied the ability of MRx0005 and MRx0029 to regulate epithelial permeability as a result of modification of intracellular signaling included the 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. 14B). In contrast, exposure to MRx0005 did not alter gene expression for tight junction proteins, suggesting that these 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 intestinal region of the intestine of the mouse (Fig. 14C + 14D). MRx0029 was also able to reduce colon permeability function in the same mice (Fig. 14E + 14F).

Materials and Methods — RNA Extraction and qPCR Analysis.

Total RNA was extracted with an 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) ... For analysis of mRNA expression, cDNA was prepared from 2000 ng of total RNA using a high throughput reverse transcription kit (Thermo Fisher, Loughborough) 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. The resulting cDNA was amplified in duplicate using SYBR-Green PNR analysis, and the products were detected on a QuantStudio 7 real-time PNR device (Applied Biosystems, UK) using a standardized profile (initial denaturation at 95 ° C for 10 min , followed by 40 cycles of 10 s denaturation at 95 ° C and 30 s annealing / extension at 60 ° C). A dissociation step was added after 40 cycles in order to obtain a melting curve. Analysis was performed using Applied Biosystems QuantStudio v1.2 real-time PCR software. The primer sequences for actin, villin, occludin, TJP1 and TJP2 are suggested in the sequence listing.

Example 12. Level of BDNF secretion in SHSY-5Y cells.

Prerequisites.

Brain neurotrophic factor (BDNF) is a versatile molecule 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 plated 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 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. 15, which shows that administration of MRX0005 in combination with retinoic acid leads to increased secretion of BDNF from differentiated neuroblastoma cells.

Example 13. 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. We studied 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).

Materials and methods.

Bacterial strain Megasphaera massiliensis MRX0029.

Way.

SHSY-5Y cells were seeded in a black flat bottom 96 well plate at 5000 cells / well and placed in a CO ₂ 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 h. 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. 16. In FIG. 16b demonstrates that MRX0005 is capable of inhibiting ROS production in differentiated SHSY-5Y neuroblastoma cells. MRX0005 also reduced ROS production in astroglioblastoma cells (Fig. 16a). This shows that MRX0005 has overall antioxidant activity.

Example 14. Study of stability.

The composition described herein, containing at least one bacterial strain described herein, is stored in a sealed container at 25 ° C 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 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years at least 50%, 60%, 70%, 80% or 90% of the bacterial strain will persist as measured by colony forming units defined by standard protocols.

Example 15.

Ways.

Animals.

The animals used and the study design were the same as in example 10.

Bacterial strains 755: Parabacteroides distasonis (MRX005), Megasphaera massiliensis (MRX0029).

Collection of tissues.

Animals were sacrificed at random depending on treatment and study conditions; sampling took place between 9.00 and 14.30. Blood from the trunk was collected in tubes with potassium-EDTA (ethylenediaminetetrauccinic acid) and centrifuged for 15 min at 4000 g. Blood plasma was isolated and stored at -80 ° C for subsequent analysis. The brain was quickly excised, dissected, and each region of the brain was instantly 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 the cecum tissue was used),

- 36 038133 were 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). An additional 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 of cDNA, 5 μl of a 2X PCR master mix (Roche), 900 nM of each primer and 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 in 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 a 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 software version 6.0. 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 non-parametric 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. 17 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.

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Results - gene expression.

Expression of genes for neurotransmitter receptors [serotonin receptor 1a (5-HTRla), dopamine receptor D1, subunit B1 of the GABA receptor, GABAA receptor, NMDA2A receptor (Grin2A) and NMDA2B (Grin2b)], inflammation markers [IL-1b, IL6, CDa 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. 18-32 demonstrate 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. 25C). FIG. 26A 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. 26B).

Both MRx005 and MRx0029 can increase the expression of CD11b in the amygdala (Fig. 27A), while the expression of IL-6, Grin2a, and Grin2b is reduced after MRx005 treatment (Fig. 27B-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. 29B).

Discussion.

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

Results - influence on the expression of Tph1 and IDO-1.

FIG. 33 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 IDO-1 in the ileum, while MRX029 had no effect on the expression of these genes in the ileum (Fig. 34).

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 - Effect on tryptophan metabolite levels.

FIG. 35 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 γ levels in splenocytes after exposure to LPS (FIG. 36). In addition, MRX005 resulted in a significant reduction in interleukin 6 and tumor necrosis factor levels after exposure to LPS (FIGS. 38 and 39, respectively). MRx0029 treatment resulted in a decrease in interferon γ, interleukin 1β and interleukin 6 after exposure to LPS (FIGS. 36, 37 and 38, respectively).

Treatment with MRx005 and MRx0029 resulted in increased levels of the chemoattractant CXCL1 (FIG. 41).

Results - Effects on the levels of short-chain fatty acids in the caecum.

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. 42.

Example 16. 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.

SH-SY5Y was plated 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 phase

- 38 038133 EVOS XL in-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 fold change was calculated in accordance with method 2 ^(-AAct) . The primer sequences for 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) at 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 β3-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 with 3X 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 Qlmaging camera controlled by 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 superimposed 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 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 β3-tubulin, β-actin acted as a control to observe the variability of protein loading in the samples.

Results and discussion.

Gene expression.

FIG. 43 demonstrates MRx0029 and MRX005-induced changes in the expression levels of actin, villin, ocludin, TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7, and NSE.

Results - microscopy and immunoblotting.

FIG. 44 demonstrates the change in the level of MAP2 expression in SHSY5Y cells, determined using confocal microscopy. Expression levels of MAP2 and β3-tubulin were also quantified by immunoblotting. The results shown in FIG. 44M and N indicate that MRX029 induces an increase in MAP2 expression.

Sequences.

Additional primers used in qPCR (with SEQ ID NO in parentheses)

Gene ID NSE PINK1 Direct sequence CCCTGTATCGTAAGAACGGT (30) CCCAAGCAACTAGCCCCTC (32) Reverse sequence GCCACCATTGATCACGTTGA (31) GGCAGCACATCAGGGTAGTC (33) PARK7 GTAGCCGTGATGTGGTCATTT (34) CTGTGCGCCCAGATTACCT (35) SYP CTCGGCTTTGTGAAGGTGCT (36) GGCTTCATGGCATCAACTTCA (37)

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Sequences.

SEQ ID NO: 1 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 5825 - AB238922)

121 aacttgccta tcagaggggg ataacccggc gaaagtcgga ctaataccgc atgaagcagg

181 gatcccgcat gggaatattt gctaaagatt catcgctgat agataggcat gcgttccatt

241 aggcagttgg cggggtaacg gcccaccaaa ccgacgatgg ataggggttc tgagaggaag

301 gtcccccaca ttggtactga gacacggacc aaactcctac gggaggcagc agtgaggaat

361 attggtcaat gggcgtaagc ctgaaccagc caagtcgcgt gagggatgaa ggttctatgg

421 atcgtaaacc tcttttataa gggaataaag tgcgggacgt gtcccgtttt gtatgtacct

481 tatgaataag gatcggctaa ctccgtgcca gcagccgcgg taatacggag gatccgagcg

541 ttatccggat ttattgggtt taaagggtgc gtaggcggcc ttttaagtca gcggtgaaag

601 tctgtggctc aaccatagaa ttgccgttga aactgggggg cttgagtatg tttgaggcag

661 gcggaatgcg tggtgtagcg gtgaaatgca tagatatcac gcagaacccc gattgcgaag

721 gcagcctgcc aagccattac tgacgctgat gcacgaaagc gtggggatca aacaggatta

781 gataccctgg tagtccacgc agtaaacgat gatcactagc tgtttgcgat acactgtaag

841 cggcacagcg aaagcgttaa gtgatccacc tggggagtac gccggcaacg gtgaaactca

901 aaggaattga cgggggcccg cacaagcgga ggaacatgtg gtttaattcg atgatacgcg

961 aggaacctta cccgggtttg aacgcattcg gaccgaggtg gaaacacctt ttctagcaat

1021 agccgtttgc gaggtgctgc atggttgtcg tcagctcgtg ccgtgaggtg tcggcttaag

1081 tgccataacg agcgcaaccc ttgccactag ttactaacag gttaggctga ggactctggt

1141 gggactgcca gcgtaagctg cgaggaaggc ggggatgacg tcaaatcagc acggccctta

1201 catccggggc gacacacgtg ttacaatggc gtggacaaag ggaggccacc tggcgacagg

1261 gagcgaatcc ccaaaccacg tctcagttcg gatcggagtc tgcaacccga ctccgtgaag

1321 ctggattcgc tagtaatcgc gcatcagcca tggcgcggtg aatacgttcc cgggccttgt

1381 acacaccgcc cgtcaagcca tgggagccgg gggtacctga agtccgtaac cgaaaggatc

1441 ggcctagggt aaaactggtg actggggcta agtcgtaaca aggtaacc

SEQ ID NO: 2 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 13400 - AB238923)

121 cttacctatc agagggggat aacccggcga aagtcggact aataccgcat gaagcagggg

181 ccccgcatgg ggatatttgc taaagattca tcgctgatag ataggcatgc gttccattag

241 gcagttggcg gggtaacggc ccaccaaacc gacgatggat aggggttctg agaggaaggt

301 cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat

361 tggtcaatgg gcgtaagcct gaaccagcca agtcgcgtga gggatgaagg ttctatggat

421 cgtaaacctc ttttataagg gaataaagtg cgggacgtgt cctgttttgt atgtacctta

481 tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccgagcgtt

541 atccggattt attgggttta aagggtgcgt aggcggcctt ttaagtcagc ggtgaaagtc

601 tgtggctcaa ccatagaatt gccgttgaaa ctggggggct tgagtatgtt tgaggcaggc

661 ggaatgcgtg gtgtagcggt gaaatgctta gatatcacgc agaaccccga ttgcgaaggc

721 agcctgccaa gccatgactg acgctgatgc acgaaagcgt ggggatcaaa caggattaga

781 taccctggta gtccacgcag taaacgatga tcactagctg tttgcgatac agtgtaagcg

841 gcacagcgaa agcgttaagt gatccacctg gggagtacgc cggcaacggt gaaactcaaa

901 ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag

961 gaaccttacc cgggtttgaa cgcattcgga ccgaggtgga aacacctttt ctagcaatag

1021 ccgtttgcga ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc ggcttaagtg

1081 ccataacgag cgcaaccctt gccactagtt actaacaggt aaagctgagg actctggtgg

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1141 gactgccagc gtaagctgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca

1201 tccggggcga cacacgtgtt acaatggcgt ggacaaaggg aagccacctg gcgacaggga

1261 gcgaatcccc aaaccacgtc tcagttcgga tcggagtctg caacccgact ccgtgaagct

1321 ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac

1381 acaccgcccg tcaagccatg ggagccgggg gtacctgaag tccgtaaccg aaaggatcgg

1441 cctagggtaa aactggtgac tggggctaag tcgtaacaag gtaacc

SEQ ID NO: 3 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 13401 - AB238924)

121 cttgcctatc agagggggat aacccggcga aagtcggact aataccgcat gaagcagggg

181 ccccgcatgg ggatatttgc taaagattca tcgctgatag ataggcatgc gttccattag

241 gcagttggcg gggtaacggc ccaccaaacc gacgatggat aggggttctg agaggaaggt

301 cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat

361 tggtcaatgg gcgtaagcct gaaccagcca agtcgcgtga gggatgaagg ttctatggat

421 cgtaaacctc ttttataagg gaataaagtg tgggacgtgt cctgttttgt atgtacctta

481 tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccgagcgtt

541 atccggattt attgggttta aagggtgcgt aggcggcctt ttaagtcagc ggtgaaagtc

601 tgtggctcaa ccatagaatt gccgttgaaa ctgggaggct tgagtatgtt tgaggcaggc

661 ggaatgcgtg gtgtagcggt gaaatgctta gatatcacgc agaaccccga ttgcgaaggc

721 agcctgccaa gccatgactg acgctgatgc acgaaagcgt ggggatcaaa caggattaga

781 taccctggta gtccacgcag taaacgatga tcactagctg tttgcgatac actgtaagcg

841 gcacagcgaa agcgttaagt gatccacctg gggagtacgc cggcaacggt gaaactcaaa

901 ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag

961 gaaccttacc cgggtttgaa cgcattcgga ccgaggtgga aacacctttt ctagcaatag

1021 ccgtttgcga ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc ggcttaagtg

1081 ccataacgag cgcaaccctt gccactagtt actaacaggt gatgctgagg actctggtgg

1141 gactgccagc gtaagctgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca

1201 tccggggcga cacacgtgtt acaatggcgt ggacaaaggg atgccacctg gcgacaggga

1261 gcgaatcccc aaaccacgtc tcagttcgga tcggagtctg caacccgact ccgtgaagct

1321 ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac

1381 acaccgcccg tcaagccatg ggagccgggg gtacctgaag tccgtaaccg aaaggatcgg

1441 cctagggtaa aactggtgac tggggctaag tcgtaacaag gtaacc

SEQ ID NO: 4 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 13402 - AB238925) agagtttgat cctggctcag gatgaacgct agcgacaggc ttaacacatg caagtcgagg

161 ggcagcacag gtagcaatac cgggtggcga ccggcgcacg ggtgagtaac gcgtatgcaa

121 cttacctatc agagggggat aacccggcga aagtcggact aataccgcat gaagcagggg

181 ccccgcatgg ggatatttgc taaagattca tcgctgatag ataggcatgc gttccattag

241 gcagttggcg gggtaacggc ccaccaaacc gacgatggat aggggttctg agaggaaggt

- 41 038133

301 cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat

361 tggtcaatgg gcgtaagcct gaaccagcca agtcgcgtga gggatgaagg ttctatggat

421 cgtaaacctc ttttataagg gaataaagtg cgggacgtgt cccgttttgt atgtacctta

481 tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccgagcgtt

541 atccggattt attgggttta aagggtgcgt aggcggcctt ttaagtcagc ggtgaaagtc

601 tgtggctcaa ccatagaatt gccgttgaaa ctgggaggct tgagtatgtt tgaggcaggc

661 ggaatgcgtg gtgtagcggt gaaatgctta gatatcacgc agaaccccga ttgcgaaggc

721 agcctgccaa gccatgactg acgctgatgc acgaaagcgt ggggatcaaa caggattaga

781 taccctggta gtccacgcag taaacgatga tcactagctg tttgcgatac actgtaagcg

841 gcacagcgaa agcgttaagt gatccacctg gggagtacgc cggcaacggt gaaactcaaa

901 ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag

961 gaaccttacc cgggtttgaa cgcattcgga ccgaggtgga aacacctttt ctagcaatag

1021 ccgtttgcga ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc ggcttaagtg

1081 ccataacgag cgcaaccctt gccactagtt actaacaggt aaagctgagg actctggtgg

1141 gactgccagc gtaagctgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca

1201 tccggggcga cacacgtgtt acaatggcgt ggacaaaggg aggccacctg gcgacaggga

1261 gcgaatcccc aaaccacgtc tcagttcgga tcggagtctg caacccgact ccgtgaagct

1321 ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac

1381 acaccgcccg tcaagccatg ggagccgggg gtacctgaag tccgtaaccg aaaggatcgg

1441 cctagggtaa aactggtgac tggggctaag tcgtaacaag gtaacc

SEQ ID NO: 5 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 13403 - AB238926)

121 cttacctatc agagggggat aacccggcga aagtcggact aataccgcat gaagcagggg

181 ccccgcatgg ggatatttgc taaagattca tcgctgatag ataggcatgc gttccattag

241 gcagttggcg gggtaacggc ccaccaaacc gacgatggat aggggttctg agaggaaggt

301 cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat

361 tggtcaatgg gcgtaagcct gaaccagcca agtcgcgtga gggatgaagg ttctatggat

421 cgtaaacctc ttttataagg gaataaagtg tgggacgtgt cccgttttgt atgtacctta

481 tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccgagcgtt

541 atccggattt attgggttta aagggtgcgt aggcggcctt ttaagtcagc ggtgaaagtc

601 tgtggctcaa ccatagaatt gccgttgaaa ctgggaggct tgagtatgtt tgaggcaggc

661 ggaatgcgtg gtgtagcggt gaaatgctta gatatcacgc agaaccccga ttgcgaaggc

721 agcctgccaa gccatgactg acgctgatgc acgaaagcgt ggggatcaaa caggattaga

781 taccctggta gtccacgcag taaacgatga tcactagctg tttgcgatac attgtaagcg

841 gcacagcgaa agcgttaagt gatccacctg gggagtacgc cggcaacggt gaaactcaaa

901 ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag

961 gaaccttacc cgggtttgaa cgcattcgga ccgaggtgga aacacctttt ctagcaatag

1021 ccgtttgcga ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc ggcttaagtg

1081 ccataacgag cgcaaccctt gccactagtt actaacaggt aaagctgagg actctggtgg

1141 gactgccagc gtaagctgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca

1201 tccggggcga cacacgtgtt acaatggcgt ggacaaaggg aggccacctg gcgacaggga

1261 gcgaatcccc aaaccacgtc tcagttcgga tcggagtctg caacccgact ccgtgaagct

1321 ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac

1381 acaccgcccg tcaagccatg ggagccgggg gtacctgaag tccgtaaccg aaaggatcgg

1441 cctagggtaa aactggtgac tggggctaag tcgtaacaag gtaacc

SEQ ID NO: 6 (Parabacteroides distasonis 16S ribosomal RNA gene, partial sequence, strain: JCM 13404 - AB238927)

- 42 038133 agagtttgat cctggctcag gatgaacgct agcgacaggc ttaacacatg caagtcgagg ggcagcacag gtagcaatac cgggtggcga ccggcgcacg ggtgagtaac gcgtatgcaa

121 cttacctatc agagggggat aacccggcga aagtcggact aataccgcat gaagcagggg

181 ccccgcatgg ggatatttgc taaagattca tcgctgatag ataggcatgc gttccattag

241 gcagttggcg gggtaacggc ccaccaaacc gacgatggat aggggttctg agaggaaggt

301 cccccacatt ggtactgaga cacggaccaa actcctacgg gaggcagcag tgaggaatat

361 tggtcaatgg gcgtaagcct gaaccagcca agtcgcgtga gggatgaagg ttctatggat

421 cgtaaacctc ttttataagg gaataaagtg tgggacgtgt cccgttttgt atgtacctta

481 tgaataagga tcggctaact ccgtgccagc agccgcggta atacggagga tccgagcgtt

541 atccggattt attgggttta aagggtgcgt aggcggcctt ttaagtcagc ggtgaaagtc

601 tgtggctcaa ccatagaatt gccgttgaaa ctgggaggct tgagtatgtt tgaggcaggc

661 ggaatgcgtg gtgtagcggt gaaatgctta gatatcacgc agaaccccga ttgcgaaggc

721 agcctgccaa gccatgactg acgctgatgc acgaaagcgt ggggatcaaa caggattaga

781 taccctggta gtccacgcag taaacgatga tcactagctg tttgcgatac attgtaagcg

841 gcacagcgaa agcgttaagt gatccacctg gggagtacgc cggcaacggt gaaactcaaa

901 ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag

961 gaaccttacc cgggtttgaa cgcattcgga ccgaggtgga aacacctttt ctagcaatag

1021 ccgtttgcga ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc ggcttaagtg

1081 ccataacgag cgcaaccctt gccactagtt actaacaggt aaagctgagg actctggtgg

1141 gactgccagc gtaagctgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca

1201 tccggggcga cacacgtgtt acaatggcgt ggacaaaggg aggccacctg gcgacaggga

1261 gcgaatcccc aaaccacgtc tcagttcgga tcggagtctg caacccgact ccgtgaagct

1321 ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac

1381 acaccgcccg tcaagccatg ggagccgggg gtacctgaag tccgtaaccg aaaggatcgg

1441 cctagggtaa aactggtgac tggggctaag tcgtaacaag gtaacc

SEQ ID NO: 7 (Parabacteroides merdae 16S ribosomal RNA gene, partial sequence, strain: JCM 9497 - AB238928) agagtttgat cctggctcag gatgaacgct agcgacaggc ttaacacatg caagtcgagg

161 ggcagcatga tttgtagcaa tacagattga tggcgaccgg cgcacgggtg agtaacgcgt

121 atgcaactta cctatcagag ggggatagcc cggcgaaagt cggattaata ccccataaaa

181 caggggtccc gcatgggaat atttgttaaa gattcatcgc tgatagatag gcatgcgttc

- 43 038133

241 cattaggcag ttggcggggt aacggcccac caaaccgacg atggataggg gttctgagag

301 gaaggtcccc cacattggta ctgagacacg gaccaaactc ctacgggagg cagcagtgag

361 gaatattggt caatggccga gaggctgaac cagccaagtc gcgtgaagga agaaggatct

421 atggtttgta aacttctttt ataggggaat aaagtggagg acgtgtcctt ttttgtatgt

481 accctatgaa taagcatcgg ctaactccgt gccagcagcc gcggtaatac ggaggatgcg

541 agcgttatcc ggatttattg ggtttaaagg gtgcgtaggt ggtgatttaa gtcagcggtg

601 aaagtttgtg gctcaaccat aaaattgccg ttgaaactgg gttacttgag tgtgtttgag

661 gtaggcggaa tgcgtggtgt agcggtgaaa tgcatagata tcacgcagaa ctccgattgc

721 gaaggcagct tactaaacca taactgacac tgaagcacga aagcgtgggg atcaaacagg

781 attagatacc ctggtagtcc acgcagtaaa cgatgattac taggagtttg cgatacaatg

841 taagctctac agcgaaagcg ttaagtaatc cacctgggga gtacgccggc aacggtgaaa

901 ctcaaaggaa ttgacggggg cccgcacaag cggaggaaca tgtggtttaa ttcgatgata

961 cgcgaggaac cttacccggg tttgaacgta gtctgaccgg agtggaaaca ctccttctag

1021 caatagcaga ttacgaggtg ctgcatggtt gtcgtcagct cgtgccgtga ggtgtcggct

1081 taagtgccat aacgagcgca acccttatca ctagttacta acaggtgaag ctgaggactc

1141 tggtgagact gccagcgtaa gctgtgagga aggtggggat gacgtcaaat cagcacggcc

1201 cttacatccg gggcgacaca cgtgttacaa tggcatggac aaagggcagc tacctggcga

1261 caggatgcta atctccaaac catgtctcag ttcggatcgg agtctgcaac tcgactccgt

1321 gaagctggat tcgctagtaa tcgcgcatca gccatggcgc ggtgaatacg ttcccgggcc

1381 ttgtacacac cgcccgtcaa gccatgggag ccgggggtac ctgaagtccg taaccgcaag

1441 gatcggccta gggtaaaact ggtgactggg gctaagtcgt aacaaggtaa cc

SEQ ID NO: 8 (Parabacteroides merdae 16S ribosomal RNA gene, partial sequence, strain: JCM 13405 - AB238929)

121 atgcaactta cctatcagag ggggatagcc cggcgaaagt cggattaata ccccataaaa

181 caggggttcc gcatgggaat atttgttaaa gattcatcgc tgatagatag gcatgcgttc

241 cattaggcag ttggcggggt aacggcccac caaaccgacg atggataggg gttctgagag

301 gaaggtcccc cacattggta ctgagacacg gaccaaactc ctacgggagg cagcagtgag

361 gaatattggt caatggccga gaggctgaac cagccaagtc gcgtgaagga agaaggatct

421 atggtttgta aacttctttt ataggggaat aaagtggagg acgtgtcctt ttttgtatgt

481 accctatgaa taagcatcgg ctaactccgt gccagcagcc gcggtaatac ggaggatgcg

541 agcgttatcc ggatttattg ggtttaaagg gtgcgtaggt ggtgatttaa gtcagcggtg

601 aaagtttgtg gctcaaccat aaaattgccg ttgaaactgg gttacttgag tgtgtttgag

661 gtaggcggaa tgcgtggtgt agcggtgaaa tgcatagata tcacgcagaa ctccgattgc

721 gaaggcagct tactaaacca taactgacac tgaagcacga aagcgtgggg atcaaacagg

781 attagatacc ctggtagtcc acgcagtaaa cgatgattac taggagtttg cgatacaatg

841 taagctctac agcgaaagcg ttaagtaatc cacctgggga gtacgccggc aacggtgaaa

901 ctcaaaggaa ttgacggggg cccgcacaag cggaggaaca tgtggtttaa ttcgatgata

961 cgcgaggaac cttacccggg tttgaacgta gtctgaccgg agtggaaaca ctccttctag

1021 caatagcaga ttacgaggtg ctgcatggtt gtcgtcagct cgtgccgtga ggtgtcggct

1081 taagtgccat aacgagcgca acccttatca ctagttacta acaggtgaag ctgaggactc

1141 tggtgagact gccagcgtaa gctgtgagga aggtggggat gacgtcaaat cagcacggcc

1201 cttacatccg gggcgacaca cgtgttacaa tggcatggac aaagggcagc tacctggcga

1261 caggatgcta atctccaaac catgtctcag ttcggatcgg agtctgcaac tcgactccgt

1321 gaagctggat tcgctagtaa tcgcgcatca gccatggcgc ggtgaatacg ttcccgggcc

1381 ttgtacacac cgcccgtcaa gccatgggag ccgggggtac ctgaagtccg taaccgcaag

1441 gatcggccta gggtaaaact ggtgactggg gctaagtcgt aacaaggtaa cc

- 44 038133

SEQ ID NO: 9 (consensus sequence 16S rRNA of Parabacteroides distasonis 755)

AMCCGGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTGCCTAT

CAGAGGGGGATAACCCGGCGAAAGTCGGACTAATACCGCATGAAGCAGGGATCCC GCATGGGAATATTTGCTAAAGATTCATCGCTGATAGATAGGCATGCGTTCCATTAGG CAGTTGGCGGGGTAACGGCCCACCAAACCGACGATGGATAGGGGTTCTGAGAGGAA GGTCCCCCACATTGGTACTGAGACACGGACCAAACTCCTACGGGAGGCAGCAGTGA GGAATATTGGTCAATGGGCGTGAGCCTGAACCAGCCAAGTCGCGTGAGGGATGAAG GTTCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGTGTCCCGTT TTGTATGTACCTTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATA CGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTT TTAAGTCAGCGGTGAAAGTCTGTGGCTCAACCATAGAATTGCCGTTGAAACTGGGA GGCTTGAGTATGTTTGAGGCAGGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGA TATCACGCAGAACCCCGATTGCGAAGGCAGCCTGCCAAGCCATTACTGACGCTGAT GCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCAGTAAA CGATGATCACTAGCTGTTTGCGATACACTGTAAGCGGCACAGCGAAAGCGTTAAGT GATCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCG GGTTTGAACGCATTCGGACMGAKGTGGAAACACATTTTCTAGCAATAGCCATTTGC GAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATA ACGAGCGCAACCCTTGCCACTAGTTAC TAACAGGTAAAGCTGAGGACTCTGGTGGG ACTGCCAGCGTAAGCTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTT ACATCCGGGGCGACACACGTGTTACAATGGCGTGGACAAAGGGAAGCCACCTGGCG ACAGGGAGCGAATCCCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCAACCCGAC TCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTT CCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGCCGGGGGTACCTGAAGTC CGTAACCGCGAGGATCGGCCTAGGGTAAAACTGGTGACTGGGGCTAAGTCGTACGG GG

SEQ ID NO: 10 (genomic sequence of strain 755) - see electronic sequence listing.

SEQ ID NO: 11 (consensus sequence 16S rRNA strain Megasphaera massiliensis MRX0029)

- 45 038133

TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTA AGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATA CGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCCTTCGGGCTTTCGCTGG AGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGAC GATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGG AGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATATGGGAC GAACAGGACATCGGTTAATACCCGGTGTCTTTGACGGTACCGTAAGAGAAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAA TTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGTCGGTCTTAAAAGTGCGGG GCTTAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCG GAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAA AGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACG GGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGAGGT ATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGC CGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGT GGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGCCTTGACATTGATTGCTACGG AAAGAGATTTCCGGTTCTTCTTCGGAAGACAAG AAAACAGGTGGTGCACGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCT TCTGTTGCCAGCACCTCGGGTGGGGACTCAGAAGAGACTGCCGCAGACAATGCGGA GGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGGCTTGGGCTACACACGT ACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGCAAACCCCAAA AACAGAGTCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCG CTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGAGGCAACCGCAAG

Primers used for qPCR (with SEQ ID NO in parentheses)

Name Direct sequence Reverse sequence ASTV GATCAAGATCATTGCTCCTC (12) TTGTCAAGAAAGGGTGTAAC (13) GAPDH GGTATCGTGGAAGGACTCATG (14) ATGCCAGTGAGCTTCCCGTTC (15) MAP2 CTCAGCACCGCTAACAGAGG (16) CATTGGCGCTTCTCTCCTC (17) Occlu Hill AAGAGGAATTTTGACACTGG (18) (K (\ 1 (i l \ (1 (1K \ (Ί 1 TS (| H) Ι.ΙΙ AAGTCACACTGGTGAAATCC (20) (K I 1 (, (1 (, ((- \ \ \ (| \ | (| (21) TJP2 CCCTCCCCTGGATCAGGAT (22) GCCATCAAACTCGTCCATCA (23) Willin CATTACCTGCTCTACGTTTG (24) AGATGGACATAAGATGAGGTG

- 46 038133

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Claims (16)

CLAIM 1. The use of a composition containing a bacterial strain of the genus Parabacteroides for the treatment or prevention of a neurodegenerative disease 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-induced parkinsonism; Alzheimer's disease, including Benson's syndrome; 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. 2. The use of a composition according to claim 1, wherein said composition is intended for the treatment or prevention of Parkinson's disease. 3. The use of a composition according to claim 1 or 2, characterized in that said composition is intended for the treatment or prevention of a neurodegenerative disease that occurs at an early age. 4. The use of a composition according to any of the preceding claims, wherein said composition is intended to prevent or delay the onset or progression of a neurodegenerative disease. 5. The use of a composition according to any of the preceding claims, wherein said bacterial strain is Parabacteroides distasonis. 6. The use of a composition according to any of the preceding claims, characterized in that said bacterial strain has a 16s rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to the 16s rRNA sequence of a bacterial strain Parabacteroides distasonis. 7. The use of a composition according to any one of claims 1 to 5, characterized in that said bacterial strain has a 16s rRNA sequence that is at least 95, 96, 97, 98, 99, 99.5 or 99.9% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9. 8. The use of a composition according to claim 7, wherein said 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: 9 , or characterized in that said bacterial strain has the 16s rRNA sequence represented by SEQ ID NO: 9. 9. The use of a composition according to claim 1, wherein said composition comprises a bacterial strain of the species Parabacteroides distasonis for the treatment or prevention of Parkinson's disease. 10. The use of a composition according to any of the preceding claims, wherein said composition is intended for oral administration. 11. Use of a composition according to any of the preceding claims, wherein said composition contains one or more pharmaceutically acceptable excipients or carriers. 12. The use of a composition according to any of the preceding claims, characterized in that said bacterial strain is lyophilized. 13. The use of a food supplement containing a bacterial strain of the genus Parabacteroides for the prevention of neurodegenerative disease 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; 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. 14. A method of treating or preventing a neurodegenerative disease 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; 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, comprising administering a composition containing a bacterial strain of the Parabacteroides genus to a patient in need thereof. 15. Application of a cell of the Parabacteroides distasonis strain deposited under accession number - 48 038133 NCIMB 42382, for the treatment or prevention of a neurodegenerative disease 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-induced parkinsonism; Alzheimer's disease, including Benson's syndrome; 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, comprising administering a composition containing a bacterial strain of the Parabacteroides genus to a patient in need thereof. 16. A cell according to claim 15, wherein the neurodegenerative disease is Parkinson's disease.