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EP3989989A1 - Intestinal microbiota bacteria and composition containing same for use in the prevention and/or treatment of diseases characterised by excess 2-hydroxyglutarate - Google Patents

Intestinal microbiota bacteria and composition containing same for use in the prevention and/or treatment of diseases characterised by excess 2-hydroxyglutarate

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Publication number
EP3989989A1
EP3989989A1 EP20739276.2A EP20739276A EP3989989A1 EP 3989989 A1 EP3989989 A1 EP 3989989A1 EP 20739276 A EP20739276 A EP 20739276A EP 3989989 A1 EP3989989 A1 EP 3989989A1
Authority
EP
European Patent Office
Prior art keywords
bacteria
composition
use according
genus
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20739276.2A
Other languages
German (de)
French (fr)
Inventor
Georges Rawadi
Sandrine CLAUS
Laure RINALDI
Katy Nicole LECORF
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Verb Biotics
Original Assignee
Ysopia Biosciences SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ysopia Biosciences SA filed Critical Ysopia Biosciences SA
Publication of EP3989989A1 publication Critical patent/EP3989989A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the invention relates to the treatment of pathologies characterized by the excess in humans or animals of 2-hydroxyglutarate, in particular diseases having a degenerative effect on cells such as neurons, motor nerve cells, cancers. brain, colon, kidney, blood, skin, liver, lymph, prostate, thyroid, stomach, breast, pancreas, pituitary, and 2-hydroxyglutaric aciduria.
  • Alzheimer's disease dementias, Parkinson's disease and associated disorders, prion diseases, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, lateral sclerosis amyotrophic.
  • Dementia is the most serious form of pathological aging of the brain. It causes an increasing impairment of memory and cognitive functions as well as behavioral disorders leading to a progressive loss of autonomy. It is the cause of disabilities and addictions in the elderly. Alzheimer's disease is a decline in cognitive faculties and memory. Nerve cells are gradually destroyed in areas of the brain related to memory and language. Over time, the affected person has more and more difficulty memorizing events, recognizing objects and faces, remembering the meanings of words and exercising judgment.
  • the treatments offered consist of treating certain symptoms of the patient with drugs (Donepezil, Rivastigmine, Galantamine, Memantine) or of taking charge of the patient in a non-pharmacological manner (speech therapy, physiotherapy, osteopathy, psychology, occupational therapy, psychomotor skills. treatments are not satisfactory because they do not prevent the progression of the disease, treat certain only occasional disorders.
  • Parkinson's disease affects the central nervous system responsible for progressive disorders such as slowed movements, tremors, stiffness and cognitive impairment. It is the second most common neurodegenerative disease, after Alzheimer's disease.
  • Huntington's disease is an inherited disease characterized by significant motor, cognitive and psychiatric disturbances, and progresses to loss of autonomy and ultimately death. We can classify these symptoms into three main families.
  • Prion diseases are diseases characterized in particular by degeneration of the central nervous system. They are also called subacute transmissible spongiform encephalopathies (TSSS). These diseases are caused by the accumulation in the brain of a normal but poorly conformed protein, the prion protein. These diseases are characterized by a rapid and fatal course.
  • TSSS subacute transmissible spongiform encephalopathies
  • Neuromuscular diseases are diseases that affect the motor nerve cells of the spinal cord or motor neurons (spinal atrophies, amyotrophic lateral sclerosis), the roots and nerves of the limbs (peripheral neuropathies), the junction between the nerve and the muscle (myasthenia gravis) and muscle (myopathies). They can affect the motor skills of the legs or arms but sometimes also other organs and functions which depend on the muscles (motor skills of the eyes, speech, swallowing, digestion, breathing, heart). The cause can be genetic or due to a malfunction of the immunity ("autoimmune" disease) which will cause damage to the nerves (dysimmune neuropathies), the neuromuscular junction (myasthenia gravis) or inflammation of the muscles (myositis). . There are other possible causes: drug or environmental toxicity, vitamin deficiency, endocrine or general diseases, infections.
  • Treatments are different depending on the cause of the disease. In genetic diseases, therapeutic trials are only in their early stages, no routine treatment exists. In the event of an abnormality in the metabolism of the cell, there are often drugs which aim to alleviate the consequences of this deficiency. In dysimmune diseases, there are many treatments that can be effective. However, undesirable side effects are known as haematological effects.
  • Spinocerebellar ataxia corresponds to autosomal dominant type 1 cerebellar ataxia. It is characterized by ataxia, progressive external ophthalmoplegia and other neurological manifestations.
  • the treatments offered consist of direct treatment of the cause of the disease by surgery, blood thinning drugs, antibiotics or steroids. Orphan drug treatments are also used. These treatments are not satisfactory because they are orphan drugs not developed for these indications, side effects can be various: eosinophilia, leukopenia, thrombocytopenia, diarrhea, rash and increased liver enzymes.
  • the other treatments can have the side effect of hemorrhages, digestive disorders such as nausea, vomiting, diarrhea, allergies, photosensitization.
  • Spinal amyotrophies or anterior spinal amyotrophies are a group of neuromuscular diseases characterized by progressive muscle weakness due to the degeneration and loss of the anterior motor neurons in the spinal cord and brainstem nuclei. These diseases are presented in four forms depending on the age of onset and the severity of the disease. Currently, the treatments proposed consist of a drug treatment (Nusinersen known as Spinraza ® ) which is not effective in all forms of these diseases and has respiratory and infectious side effects.
  • Nusinersen known as Spinraza ®
  • Amyotrophic lateral sclerosis or Charcot's disease, is a disease characterized by progressive degeneration of motor neurons in the cerebral cortex. It causes progressive paralysis of the entire skeletal musculature of the limbs, trunk and cephalic extremity.
  • 2-hydroxyglutarate causes abnormal myelination, disrupts the homeostasis of neuronal stem cells, increases the mortality of central nervous system cells.
  • the various brain cancers are also characterized by an excess production of 2-hydroxyglutarate in the sick person or animal.
  • 2-hydroxyglutarate is known for its status as a marker of neurodegenerative diseases as well as its role of oncometabolite in the development of brain tumors.
  • 2-Hydroxyglutarate acts to inhibit DNA repair.
  • the production of high levels of 2-hydroxyglutarate results in the inhibition of DNA repair pathways in cancer cells and therefore the accumulation of damaged DNA.
  • 2-Hydroxyglutarate is also known for its role as a marker of renal cell carcinomas and in tissues under oxygen limitation or hypoxic conditions such as hepatocellular carcinomas or colon carcinomas, so it has a role in cancers of the breast. kidney and colon.
  • 2-hydroxyglutarate Inhibition of DNA repair by 2-hydroxyglutarate has been reported in other types of cancer: blood, skin, liver, lymph, prostate, thyroid, 'stomach, breast, pancreas, pituitary gland (Ye, D., Guan, K.-L. & Xiong, Y. Metabolism, Activity, and Targeting of D- and L-2-Hydroxyglutarates. Trends in cancer 4, 151-165 (2018)).
  • 2-Hydroxyglutaric aciduria is a group of neurometabolic diseases with a broad clinical spectrum ranging from severe neonatal manifestations to progressive forms, and asymptomatic cases, characterized biochemically by high levels of 2-hydroxyglutaric acid in plasma , cerebrospinal fluid and urine.
  • L-2-hydroxyglutaric aciduria is characterized by psychomotor retardation, cerebellar ataxia and epilepsy, and D-2-hydroxyglutaric aciduria (see this term) by variable metabolic, neurological and dysmorphic manifestations.
  • neurons and muscle nerve cells early diagnosis, optimization of physical health, cognitive and well-being activities, screening and treatment of physical and mental comorbidities.
  • the main cancer treatments are chemotherapy and radiotherapy, which have limited efficacy allowing a low survival rate and very serious side effects such as hair loss, nausea and vomiting, diarrhea, reduced blood pressure.
  • the invention is aimed at the use of particular bacteria of the human intestinal microbiota, in particular bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus genus Massiliomicrobiota.
  • Parasutterella excrementihominis Bacteria of the genus Parasutterella have already been studied and described. This is particularly the case with Parasutterella excrementihominis and Parasutterella secunda, which have been observed in several studies without an explanation of causation. Parasutterella excrementihominis was first described in 2009 and Parasutterella secunda was first described in 2011. Other species were also studied. This is the case with Parasutterella mcl which has been associated with the abundance of certain metabolites in healthy mice.
  • Bacteria of the genus Negativibacillus have been described. This is the case with Negativibacillus massiliensis, which was described in 2016. Bacteria of the genus Negativibacillus have been characterized according to the description presented in “Negativibacillus massiliensis” gen. Nov., sp. Nov., isolated from human left colon, D. Spainboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017; 17: 36-38. No therapeutic use of bacteria of the genus Negativibacillus has ever been described or considered.
  • Massiliomicrobiota timonensis and Massiliomicrobiota escudieri are known.
  • Massiliomicrobiota timonensis was first described in 2016. Massiliomicrobiota escudieri was first described in 2018. Bacteria of the genus Massiliomicrobiota were characterized according to the description presented in “Massiliomicrobiota timonensis”, a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016; 13: 25-26 and in “Massiliomicrobiota escudieri sp. nov.
  • Massiliomicrobiota - bacteria of the genus Massiliomicrobiota, in particular Massiliomicrobiota timonensis and Massiliomicrobiota escudieri, and
  • the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or the treatment of at least one disease characterized by an excess of 2-hydroxyglutarate in humans or animals, in particular neurodegenerative diseases and cancers.
  • such bacteria when they are administered to a human being or an animal exhibiting a neurodegenerative disease or a cancer, are capable of acting on the 2-hydroxyglutarate produced in excess during these diseases.
  • bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota are preferably used in compositions.
  • a subject of the invention is therefore also the compositions comprising at least one bacterium of the Christensenellaceae family, preferably of the genus Christensenella, and / or a bacterium of the genus Parasutterella and / or a bacterium of the genus Negativibacillus and / or a bacterium of the genus Massiliomicrobiota. , for its use in the prevention or treatment of diseases characterized by the excess of 2-hydroxyglutarate in humans or animals, in particular in the prevention or treatment of neurodegenerative diseases and / or cancers.
  • Figure 1 demonstrates the antiproliferative effect of the supernatants of bacteria according to the invention, in particular 3 strains of C. minuta on a human colon adenocarcinoma cell line, the HTC-116 line, after 24 hours and 48 hours of treatment.
  • FIG. 2 demonstrates the antiproliferative effect of the supernatants of bacteria according to the invention, in particular 3 strains of C. minuta on a human hepatocarcinoma cell line, the HepG2 line after 24 hours and 48 hours of treatment.
  • disease characterized by the excess of 2-hydroxyglutarate or “disease characterized by an overproduction of 2-hydroxyglutarate” within the meaning of the invention is meant a disease of which at least one of the causes is the excess or overproduction of 2-hydroxyglutarate in the body of the sick person or animal. It may in particular be a neurodegenerative disease or cancer.
  • the term “marker” of a disease means a molecule or a substance, the dosage of which makes it possible to follow the course of said disease.
  • the subject of the invention is therefore a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment. at least one disease characterized by an excess of 2-hydroxyglutarate in humans or animals.
  • Bacteria of the genus Negativibacillus have been characterized according to the description presented in “Negativibacillus massiliensis” gen. Nov., sp. Nov., isolated from human left colon, D. Spainboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017; 17: 36-38 and in “Massiliomicrobiota escudieri sp. nov. isolated as part of a culturomics exploration of the gut microbiota of renal cancer patients ”, unpublished, Tidjani Alou, M., Derosa, L. and Zitvogel, L., submitted (ll-JUN-2018) U1015, Institut Gustave Roussy, 114 rue édouard vaillant, Villejuif 94800, France, Metropolitan.
  • Bacteria of the genus Massiliomicrobiota were characterized according to the description presented in “Massiliomicrobiota timonensis”, a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016; 13: 25-26.
  • the subject of the invention is a bacterium of the Christensenellaceae family, in particular of the genus Christensenella, and / or of bacteria of the genus Parasutterella and / or of bacteria of the genus Negativibacillus and / or of bacteria of the genus Massiliomicrobiota, for its use in prevention and / or treatment of at least one neurodegenerative disease and / or at least one cancer in humans or animals.
  • the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or the treatment of at least one neurodegenerative disease selected from Alzheimer's disease, dementias, Parkinson's disease and associated disorders, prion diseases, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal atrophy, amyotrophic lateral sclerosis.
  • a neurodegenerative disease selected from Alzheimer's disease, dementias, Parkinson's disease and associated disorders, prion diseases, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal atrophy, amyotrophic lateral sclerosis.
  • the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or or the treatment of at least one cancer of the brain and / or kidneys and / or liver and / or colon, in humans or animals.
  • a subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment of at least one cancer of the breast. brain and / or kidneys and / or liver and / or colon, in humans or animals.
  • the subject of the invention is a bacterium of the genus Parasutterella, for its use in the prevention and / or the treatment of at least one neurodegenerative disease, preferably chosen from Alzheimer's disease, dementias, disease. of Parkinson's disease and associated disorders, prion disease, neuromuscular disease, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, amyotrophic lateral sclerosis.
  • at least one neurodegenerative disease preferably chosen from Alzheimer's disease, dementias, disease. of Parkinson's disease and associated disorders, prion disease, neuromuscular disease, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, amyotrophic lateral sclerosis.
  • bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota when they are administered to a human being or an animal presenting neurodegenerative disease, are capable of acting on molecules produced in excess during neurodegenerative disease or cancer, in particular on 2-hydroxyglutarate.
  • the decrease in the amount of 2-hydroxyglutarate is a sign of the reduction of those diseases having a degenerative effect on neurons, that is, bacteria at the origin of this production are less stimulated.
  • the excessive production in question of the disease having a degenerative effect is slowed down and the system gradually returns to the norm.
  • a substrate for 2-hydroxyglutarate, glutarate decreases as 2-hydroxyglutarate increases.
  • a return to normal by a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
  • the decrease in the synthesis of 2-hydroxyglutarate is a sign of the reduction in the inhibition of DNA repair pathways, that is to say that the bacteria responsible for this production are less stimulated.
  • the excessive production involved in inhibiting DNA repair is slowed down and the system gradually returns to normal.
  • a substrate for 2-hydroxyglutarate, glutarate decreases as 2-hydroxyglutarate increases.
  • a return to normal by a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
  • 2-hydroxyglutaric aciduria the decrease in 2-hydroxyglutarate synthesis is a sign of the reduction in clinical symptoms associated with this disease.
  • a substrate for 2-hydroxyglutarate, glutarate decreases as 2-hydroxyglutarate increases.
  • a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
  • the bacteria useful according to the invention are administered to humans or animals in an amount effective for an action on 2-hydroxyglutarate, that is to say to reduce its production in the organism.
  • the bacteria or bacteria can be administered at a dose of 10 9 to 10 12 colony forming units (CFU) per day, regardless of the weight of the person or of the animal.
  • CFU colony forming units
  • it is a single dose, that is to say administered all at once or a dose before each meal, ie three times a day.
  • the bacteria or bacteria useful according to the invention are: - bacteria of the Christensenellaceae family, preferably of the Christensenella genus. It may in particular be Christensenella massiliensis, Christensenella timonensis and / or Christensenella minuta. According to a particularly suitable variant, it is Christensenella minuta.
  • These bacteria can be isolated from human stool, for example according to the protocols published by Morotomi et al. 2012 (Morotomi, M., Nagai, F. & Watanabe, Y. Description of Christensenella minuta gen. Nov., Sp.
  • Parasutterella excrementihominis and Parasutterella secundo are isolated from human stool, for example according to the protocols published by Nagai et al. 2009 (Parasutterella excrementihominis gen. Nov., Sp. Nov., A member of the family Alcaligenaceae isolated from human faeces Nagai F Morotomi M Sakon H Tanaka R, International Journal of Systematic and Evolutionary Microbiology, 2009) and Morotomi et al. 2011 (Parasutterella secundo sp. Nov., Isolated from human faeces and proposai of Sutterellaceae fam. Nov. In the order Burkholderiales, Morotomi M Nagai F Watanabe Y, International Journal of Systematic and Evolutionary Microbiology, 2011). These documents also describe the methods of culturing useful bacteria according to the invention.
  • Negativibacillus massiliensis a bacteria of the genus Negativibacillus, preferably Negativibacillus massiliensis.
  • These bacteria can be isolated from human stool, for example according to the protocols published by Fournier et al. 2016 ("Negativibacillus massiliensis" gen. Nov., Sp. Nov., Isolated from human left colon, Fournier P Ricaboni D Vitton V Raoult D Andrieu C Mailhe M, New Microbes and New Infections, 2016). This document also describes the methods of culturing useful bacteria according to the invention.
  • Massiliomicrobiota bacteria of the genus Massiliomicrobiota. It may be in particular Massiliomicrobiota timonensis and Massiliomicrobiota escudieri. According to a particularly suitable variant, it is Massiliomicrobiota timonensis.
  • Massiliomicrobiota timonensis These bacteria can be isolated from stool human, for example according to the protocol published by Ndongo et al. 2016 ("Massiliomicrobiota timonensis", a new bacterial species isolated from the human gut. Ndongo S Khelaifia S Fournier P Raoult D. New microbes and new infections, 2016 vol: 13 pp: 25-6). New Microbes and New Infections 13, 32-33 (2016)). This document also describes the methods of culturing useful bacteria according to the invention.
  • bacteria (s) useful according to the invention are preferably administered in a composition.
  • composition comprising bacteria according to the invention
  • a subject of the invention is therefore also a composition
  • a composition comprising at least one bacterium of the Christensenellaceae family and / or a bacterium of the genus Parasutterella and / or a bacterium of the genus Negativibacillus and / or a bacterium of the genus Massiliomicrobiota, in the prevention and / or or the treatment of diseases characterized by an excess of 2-hydroxyglutarate, in particular neurodegenerative diseases and / or cancers, in humans or animals.
  • the bacteria or bacteria are present in an effective amount in the composition allowing an effect on 2-hydroxyglutarate and on the disease or diseases to be treated, in particular neurodegenerative diseases and / or cancers, from which the persons or animals treated are affected.
  • the composition useful according to the invention comprises 10 6 to 10 12 colony-forming units (CFU) of bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria. of the genus Massiliomicrobiota per daily dose of composition.
  • CFU colony-forming units
  • the composition useful according to the invention can be in liquid form.
  • This medium can be, for example, Columbia agar medium for bacteria of the Christensenellaceae family, bacteria of the genus Negativibacillus, bacteria of the genus Massiliomicrobiota, or BTU medium for bacteria of the genus Parasutterella, anaerobic enriched with sheep blood, or an equivalent medium containing no derivative product of animal origin.
  • the composition useful according to the invention can be provided in solid form.
  • the bacteria can be present in lyophilized form, and can also include excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum , calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, sorbitol and / or glycerol.
  • excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum , calcium lactate,
  • compositions useful according to the invention can be in particular in the form of powder, microencapsulated powder, gelatin capsule, capsule, tablet, lozenge, granules, emulsion, suspension or suppository. According to a particularly suitable embodiment, they can be in a gastro-resistant form, such as a coated tablet containing microencapsulated bacteria.
  • compositions When the compositions are in solid form, they are preferably packaged in capsules or in a coating hermetic to light and to oxygen maintained at an ambient temperature of between 15 ° C and 40 ° C and a humidity of between 3% and 70%.
  • the bacteria can be used alive or inactivated, for example by heat, exposure to an appropriate pH, gamma rays or high pressure.
  • At least part of the bacteria consists of living bacteria, in particular at least 50% (by number), even more preferably at least 90% (by number).
  • the bacteria present in the composition useful according to the invention are at least 50% of living bacteria (by number), preferably for at least 90% of living bacteria (by number), even more preferably all alive.
  • compositions useful according to the invention can be administered orally, topically, respiratory (inhalation) or rectally.
  • compositions useful according to the invention, in addition to the bacteria useful according to the invention can comprise other compounds, such as:
  • At least one bacterium making it possible to create an anaerobic environment favorable to the bacteria present in the composition, such as at least one bacterium chosen from bacteria of the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and / or at least one other organism promoting the anaerobic conditions necessary for the survival of Christensenellaceae, such as at least one yeast chosen from Saccharomyces spp. or microorganisms of the Methanobacteriaceae family and / or
  • At least one bacterium associated with the ecosystem of bacteria present in the composition because they facilitate their survival in the intestine such as at least one bacterium chosen from bacteria of the phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, betaproteobacteria and Verrucomicrobia, and or
  • bacterium chosen from bacteria of the order of Burkholderiales, Clostridales, Verrucomicrobiales, Aeromonadales, Alteromonadales, ML615J-28, RF32, YS2, of the Clostridiaceae, Lachnospiraceae, Erysipelotrichaceae, Ruminococcaceae family, Bacteroidaceae, Enterococcaceae, Rikenéllaceae, Dehalobacteriaceae, Veillonellaceae, Loctobocilloceae and / or
  • Faecalibacterium chosen from bacteria of the genus Faecalibacterium, Akkermansia, Eubacterium, Sutterella, Burkholderia, Derxia, Brackiella, Oligella, Pelistega, Taylorella, Tetrathiobacter, Advenella, Alcicaligenes, Pigmentiphaga, Kerstersia, Achromoboctani, Bordetusimonella, Castellimonella, and Oscillospira, such as, for example, Faecalibacterium prausnitzii, Akkermansia muciniphila, Eubacterium halii, Oscillospira guilliermondii, Turicibacter sanguinis, Suterella parvirubra, Derxia gummosa, Brackiella oedipodis, Oligella urethrigen, Tetayaegauenalishauigenalis, tetayaegauenalisobacteria, Admiralishauigenalis, t
  • At least one prebiotic such as for example at least one prebiotic chosen from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and / or beta-caseins, and / or - at least one polyphenol such as for example at least one polyphenol chosen from quercetin, kaempferol, resveratrol, flavones (such as luteolin), flavan-3-ols (such as catechins), flavanones (such as narinenin) ), isoflavones, anthocyanidins, proanthocyanidins, and / or
  • prebiotic such as for example at least one prebiotic chosen from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and / or beta-caseins
  • polyphenol such as for example at least one
  • At least one pharmaceutical active principle preferably at least one pharmaceutical active principle exhibiting a therapeutic effect on the pathology (s) (diseases having a degenerative effect on neurons or muscle nerve cells and / or cancer (s) in particular) for which or which the bacteria present in the composition are used, such as for example:
  • Chemotherapy drugs including targeted therapies or biotherapies, hormone therapy or immunotherapy
  • the invention is now illustrated by examples of bacteria useful according to the invention, methods of culturing these bacteria, examples of compositions containing them and test results demonstrating the efficacy of bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota on 2-hydroxyglucarate and therefore on diseases induced by the excess of this molecule.
  • the Christensenella minuta bacteria can be cultured according to the operating protocol described below.
  • the Christensenella massiliensis bacteria can be cultured according to the operating protocol described below.
  • the composition of the meat filtrate is shown in Table 2.
  • the meat filtrate is prepared as follows.
  • Bacteria should be grown in anaerobic condition at 37 ° C.
  • the Christensenella timonensis bacteria can be cultured according to the same operating protocol as that described in Example 2 for Christensenella massiliensis.
  • the Parasutterella excrementihominis bacteria can be cultured according to the operating protocol described below.
  • BTU (Minced meat medium + Formate / fumarate) + 5% bovine serum
  • Ground beef (fat free) 500.0g) + distilled water (1000.0 ml) + IN NaOH (25.0 ml).
  • Use lean beef or horse meat Remove fat and connective tissue before chopping. Mix the meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, remove the fat from the surface and filter, keeping both: the meat particles and the filtrate. Add water to the filtrate for a final volume of 1000.0 ml, and then add: casitone (30.0g), yeast extract (5.0g), K2HPO4 (5.0 g), resazurin (l.Omg)
  • Boil under a nitrogen atmosphere add 0.5g / l of cystine and adjust the pH to 7.0.
  • test tubes For the preparation of the agar: use test tubes and put 15g of agar for 1000.01 of medium.
  • Haemin, Vitamin K1 or vitamin K3 if needed. Add 1000.0ml of medium after autoclaving: Haemin solution (10.0ml) + Vitaline Kl or K3 solution (0.2ml).
  • Haemin solution dissolve 50 mg of Haemin in 1 ml of 1 N NaOH, add 100 ml of distilled water and sterilize by filtration, store refrigerated.
  • Vitamin K1 solution dissolve 0.1 ml of vitamin K1 in 20 ml of 95% ethanol and sterilize by filtration. Store refrigerated in a brown bottle.
  • Vitamin K3 solution
  • the Parasutterella secunda bacteria can be cultured according to the operating protocol described below.
  • the Negativibacillus massiliensis bacteria can be cultured according to the operating protocol described below.
  • Example 7 Massiliomicrobiota timonensis
  • Massiliomicrobiota timonensis can be cultivated according to the operating protocol described in the following:
  • Example 8 Massiliomicrobiota escudieri
  • the Massiliomicrobiota escudieri bacteria can be cultured according to the same operating protocol as that described in Example 7 for Massiliomicrobiota timonensis.
  • Example 9 Composition of Christensenella minuta useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Christensenella minuta 10 9 CFU / mL in the anaerobic RCM culture medium described above, modified to contain no product of animal origin and enriched in 5% glycerol. .
  • Example 9 The composition of Example 9 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella minuta 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
  • RCB search cell bank
  • the frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • Example 10 Composition of Christensenella massiliensis useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Christensenella massiliensis 10 9 CFU / mL in the anaerobic carboxymethylcellulose culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. .
  • Example 10 The composition of Example 10 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella massiliensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • RCB search cell bank
  • Example 11 Composition of Christensenella timonensis useful according to the invention in liquid form
  • An example of a composition useful according to the invention in liquid form is a composition comprising Christensenella timonensis 10 9 CFU / mL in the anaerobic carboxymethylcellulose culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol.
  • the composition of Example 11 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella timonensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight bag. with oxygen.
  • Example 12 Composition of Parasutterella excrementihominis useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Parasutterella excrementihominis 10 9 CFU / mL in the anaerobic BTU culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. .
  • Example 12 The composition of Example 12 is obtained from an RCB (“research cell bank”) prepared on the basis of Parasutterella excrementihominis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight bag. with oxygen.
  • RCB search cell bank
  • the frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • Example 13 Composition of Parasutterella secundo useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Parasutterella secundo 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described above, modified to contain no product of animal origin and enriched in glycerol 5%.
  • Example 13 The composition of Example 13 is obtained from an RCB (“research cell bank”) prepared on the basis of Parasutterella secundo 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • RCB search cell bank
  • Example 14 Composition of Negativibacillus massiliensis useful according to the invention in liquid form
  • An example of a composition useful according to the invention in liquid form is a composition comprising Negativibacillus massiliensis 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described below. - top modified to contain no product of animal origin and enriched with 5% glycerol.
  • Example 14 The composition of Example 14 is obtained from an RCB (“research cell bank”) prepared on the basis of Negativibacillus massiliensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
  • RCB search cell bank
  • the frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • Example 15 Composition of Massiliomicrobiota timonensis useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Massiliomicrobiota timonensis 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described above, modified to contain no product of animal origin and enriched with glycerol 5%.
  • the composition of Example 15 is obtained from an RCB (“research cell bank”) prepared on the basis of Massiliomicrobiota timonensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
  • Example 16 Composition of Massiliomicrobiota escudieri useful according to the invention in liquid form
  • composition useful according to the invention in liquid form is a composition comprising Massiliomicrobiota escudieri 10 9 CFU / mL in the EG culture medium or else Columbia anaerobic described above modified to contain no product of animal origin and enriched with 5% glycerol.
  • Example 16 The composition of Example 16 is obtained from an RCB (“research cell bank”) prepared on the basis of Massiliomicrobiota escudieri 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
  • RCB search cell bank
  • the frozen composition must be warmed to room temperature until it returns to liquid form before use.
  • Example 17 Composition of at least one bacterium useful according to the invention in liquid form mixed with at least one other distinct bacterium useful according to the invention in liquid form
  • composition useful according to the invention in liquid form can be obtained by mixing at least one composition of Examples 9 to 16 with at least one composition distinct from Examples 9 to 16.
  • Example 18 Composition useful according to the invention in solid form
  • An example of a composition useful according to the invention in lyophilized form can be obtained by lyophilization of one of the compositions of Examples 9 to 17 in the frozen state.
  • the objective of this study is to demonstrate in vitro the effect of treating neurodegenerative diseases and bacterial cancers according to the invention.
  • the demonstration was carried out on 2-hydroxyglutarate, and its precursor, glutarate.
  • - Donors must not have taken antibiotics during the six months preceding the experiment and have no history of gastrointestinal disorders.
  • the donors were between 18 and 60 years old.
  • - Collection of fresh samples of their faeces is obtained in sterile plastic containers, kept in anaerobic vials containing one sachet of 2.51 of AnaeroGenTM from OxoidTM (O2 ⁇ 0.1%; CO2: 7-15%). These samples were brought to the laboratory within two hours of production.
  • - Faeces samples were diluted 1/5 (w / v) in phosphate buffered saline (IM) (PBS), pH 7.4. The suspension was homogenized in a stomacher for 120 seconds.
  • IM phosphate buffered saline
  • the basic nutrient medium was prepared from 2g / L of soybean tryptone broth, 2g / L of yeast extract, 0.1g / L of NaCl, 0.04g / L of K2HPO4, 0.01g / L of MgS0 3 .7H 2 0, 0.01g / L of CaCl 2 .6H 2 0, 2g / L NaHCOs, 0.5g / L of L-cystine HCl, 2m L / L of tween
  • the 20mL biofermenters contained 18mL of basic nutrient medium autoclaved (121 ° C for 15 minutes) and poured aseptically into sterile biofermenters. This system was left to stand overnight with oxygen-free nitrogen bubbling through the medium at a rate of 2 mL / min.
  • the pH was maintained between 6.7 and 6.9 using HCl or NaOH (0.5M).
  • the temperature of each biofermenter was controlled at 37 ° C and the contents of the vessel homogenized with a magnetic mixer
  • predigested proteins (0.35g) was added to the containers before inoculation with 2mL of fecal inocula at T0.
  • the predigested proteins were obtained according to the gastrointestinal digestion protocol adapted from that of Versantvoort et al (2005).
  • the mixture was mixed and filtered through a 5-kDa cutoff filter to remove macromolecules.
  • Relative peak area (peak area of a metabolite) / (peak area of internal standard x amount of sample).
  • the DNA contained in the samples was extracted using the NucleoSpin ® 96 Soil kit from Macherey-Nagel according to the manufacturer's instructions. - The total extracted DNA was then fragmented randomly into fragments of 350 bp and then used to construct a library using the NEBNext Ultra II kit by New England Biolabs according to the manufacturer's instructions.
  • the library was then sequenced using 2 x 150 bp paired-end sequencing on an Illumina HiSeq platform.
  • the abundance of bacteria was measured by creating a catalog of metagenomic species (MGS) from a reference catalog containing 22M genes. These AMSs were then associated to an appropriate taxonomic level. In the case of Christensenella, these were detected at the genus level and are therefore referenced in this experiment by Christensenella spp.
  • bacteria according to the invention are able to act by reducing the production of 2-hydroxyglutarate. They can therefore be used to prevent and / or treat neurodegenerative diseases and cancers.
  • HCT-116 cells colon adenocarcinoma
  • the objective of this study is to demonstrate in vitro the effect of bacteria according to the invention, in particular of C. minuta on the proliferation of tumor cells.
  • the effect of the supernatants has was evaluated after 24 hours and 48 hours of treatment of a human colon adenocarcinoma cell line, the HCT-116 line.
  • the operating protocol for the study is described below.
  • the HCT-116 cell line was maintained in culture in McCoy's 5A medium supplemented with 10% feta calf serum (Gibco) and 1% penicillin / stretomycin (Sigma-Aldrich) in a humid atmosphere incubator at 37 ° C. and 5% CO2.
  • the HCT-116 cells were seeded at a density of 10,000 cells / well in a total volume of 100 ⁇ l in a 96-well plate. After 24 h of incubation, the culture medium was removed from the adherent cells and new medium supplemented with 10% of C. minuta stationary phase supernatant was added (DSMZ: DSM22607, C. minuta 1, C. minuta 2 or C. minuta 3 and in GAM control (bacterial culture medium)). Each condition was made in 4 replicates. The cells were incubated for an additional 24 or 48 hours.
  • Proliferation assay Cell proliferation was determined using the CelITiter-GIo 2.0 assay kit (Promega). The measurements were carried out according to the manufacturer's instructions. Briefly, the plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min and an equal volume of CelITiter-GIo 2.0 reagent was added directly to the wells (100mI). The plates were stirred at 300 rpm for 2 min using a rotary shaker, then incubated at room temperature for 10 min. The reaction mixture was then transferred to a white-walled 96-well plate and the luminescent signal was measured using a microplate reader (FLUOstar Omega, BMG Labtech).
  • a microplate reader FLUOstar Omega, BMG Labtech
  • the supernatants of the various bacteria according to the invention tested induce a decrease in the proliferation of the tumor line HCT-116, making it possible to indicate a beneficial effect of the bacteria according to the invention in inhibiting tumor development.
  • the objective of this study is to demonstrate in vitro the effect of bacteria according to the invention, in particular of C. minuta on the proliferation of tumor cells.
  • the effect of the supernatants was evaluated after 24 hours and 48 hours of treatment of a human adenocarcinoma cell line, the HepG2 line.
  • the operating protocol for the study is described below.
  • the HepG2 cell line was maintained in culture in DMEM medium supplemented with 10% feta calf serum (Gibco) and 1% penicillin / stretomycin (Sigma-Aldrich) in a humid atmosphere incubator at 37 ° C and 5%. C02.
  • the cell treatment and the proliferation test is identical to the test previously described on the HCT-116 line.
  • Akt inhibitor VIII is used as a control for blocking cell proliferation.
  • the supernatants of the bacteria according to the invention tested induce a decrease in the proliferation of the tumor line HepG2, making it possible to indicate a beneficial effect of the bacteria according to the invention in inhibiting tumor development.

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Abstract

The subject matter of the invention is a human intestinal microbiota bacterium selected from bacteria in the Christensenellaceae family, bacteria of the genus Negativbacillus and bacteria of the genus Massiliomicrobiota, or a composition containing same, for use in the prevention and/or treatment of diseases characterised by excess 2-hydroxyglutarate in humans or animals.

Description

BACTERIES DU MICROBIOTE INTESTINAL ET COMPOSITION EN CONTENANT POUR SON UTILISATION DANS LA PREVENTION ET/OU LE TRAITEMENT DE MALADIES CARACTERISEES BACTERIA OF THE INTESTINAL MICROBIOTE AND COMPOSITION OF CONTAINER FOR USE IN THE PREVENTION AND / OR TREATMENT OF CHARACTERIZED DISEASES
PAR L'EXCES DE 2-HYDROXYGLUTARATE BY THE EXCESS OF 2-HYDROXYGLUTARATE
DOMAINE TECHNIQUE TECHNICAL AREA
L'invention concerne le traitement de pathologies caractérisées par l'excès chez l'être humain ou l'animal de 2-hydroxyglutarate, en particulier des maladies ayant un effet dégénératif sur des cellules telles que les neurones, les cellules nerveuses motrices, des cancers du cerveau, du colon, du rein, du sang, de la peau, du foie, de la lymphe, de la prostate, de la thyroïde, de l'estomac, du sein, du pancréas, de l'hypophyse, et l'acidurie 2-hydroxyglutarique. The invention relates to the treatment of pathologies characterized by the excess in humans or animals of 2-hydroxyglutarate, in particular diseases having a degenerative effect on cells such as neurons, motor nerve cells, cancers. brain, colon, kidney, blood, skin, liver, lymph, prostate, thyroid, stomach, breast, pancreas, pituitary, and 2-hydroxyglutaric aciduria.
ART ANTERIEUR PRIOR ART
Les maladies ayant un effet dégénératif sur les neurones sont des maladies chroniques invalidantes à évolution lente et discrète qui se caractérisent par la perte progressive de neurones dans des régions plus ou moins localisées du système nerveux, entraînant des complications cognitives, motrices ou perceptives. À terme, elles peuvent conduire à la mort. En effet, les neurones sont des cellules qui ne se divisent pas et ne se renouvellement pas d'elles-mêmes. Ainsi, l'endommagement ou la mort des neurones entraîne leur disparition définitive du corps humain ou animal. Or, la dégénérescence progressive et la mort des cellules nerveuses sont à l'origine de problèmes liés au mouvement (appelés ataxie) ou au fonctionnement mental (appelés démences), caractéristiques les maladies neurodégénératives. Diseases having a degenerative effect on neurons are disabling chronic diseases with a slow and discrete course which are characterized by the progressive loss of neurons in more or less localized regions of the nervous system, leading to cognitive, motor or perceptual complications. Ultimately, they can lead to death. Indeed, neurons are cells that do not divide and do not renew themselves. Thus, the damage or death of neurons leads to their permanent disappearance from the human or animal body. However, the progressive degeneration and death of nerve cells are the cause of problems related to movement (called ataxia) or mental functioning (called dementias), characteristic of neurodegenerative diseases.
Les maladies ayant un effet dégénératif sur les neurones sont généralement liées au vieillissement et touchent souvent les personnes de plus de 65 ans. On connaît en particulier la maladie d'Alzheimer, les démences, la maladie de Parkinson et les troubles associés, les maladies à prions, les maladies neuromusculaires, la maladie de Huntington, l'ataxie spinocérébelleuse, l'amyotrophie spinale progressive, la sclérose latérale amyotrophique. Diseases with a degenerative effect on neurons are generally linked to aging and often affect people over 65. We know in particular Alzheimer's disease, dementias, Parkinson's disease and associated disorders, prion diseases, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, lateral sclerosis amyotrophic.
La démence est la forme la plus grave de vieillissement cérébral pathologique. Elle provoque une altération croissante de la mémoire et des fonctions cognitives ainsi que des troubles du comportement conduisant une perte progressive d'autonomie. Elle est à l'origine de handicaps et de dépendances chez les personnes âgées. La maladie d'Alzheimer correspond à un déclin des facultés cognitives et de la mémoire. Les cellules nerveuses se détruisent progressivement dans les régions du cerveau liées à la mémoire et au langage. Avec le temps, la personne atteinte a de plus en plus de difficulté à mémoriser les événements, à reconnaître les objets et les visages, à se rappeler la signification des mots et à exercer son jugement. Dementia is the most serious form of pathological aging of the brain. It causes an increasing impairment of memory and cognitive functions as well as behavioral disorders leading to a progressive loss of autonomy. It is the cause of disabilities and addictions in the elderly. Alzheimer's disease is a decline in cognitive faculties and memory. Nerve cells are gradually destroyed in areas of the brain related to memory and language. Over time, the affected person has more and more difficulty memorizing events, recognizing objects and faces, remembering the meanings of words and exercising judgment.
Actuellement, les traitements proposés consistent à traiter certains symptômes du malade avec des médicaments (Donépézil, Rivastigmine, Galantamine, Mémantine) ou bien de prendre en charge le malade de manière non pharmacologique (orthophonie, kinésithérapie, ostéopathie, psychologie, ergothérapie, psychomotricité. Ces traitements ne sont pas satisfaisants car ils n'empêchent pas la progression de la maladie, traitent certains troubles seulement ponctuels. Currently, the treatments offered consist of treating certain symptoms of the patient with drugs (Donepezil, Rivastigmine, Galantamine, Memantine) or of taking charge of the patient in a non-pharmacological manner (speech therapy, physiotherapy, osteopathy, psychology, occupational therapy, psychomotor skills. treatments are not satisfactory because they do not prevent the progression of the disease, treat certain only occasional disorders.
La maladie de Parkinson affecte le système nerveux central responsable de troubles progressifs tels que des mouvements ralentis, des tremblements, une rigidité et troubles cognitifs. C'est la deuxième maladie neurodégénérative la plus fréquente, après la maladie d'Alzheimer. Parkinson's disease affects the central nervous system responsible for progressive disorders such as slowed movements, tremors, stiffness and cognitive impairment. It is the second most common neurodegenerative disease, after Alzheimer's disease.
Actuellement, les traitements proposés consistent en : Currently, the treatments offered consist of:
des traitements médicamenteux pour pallier le manque de dopamine en mimant l'action de la dopamine, en administrant une substance qui sera transformée en dopamine, en donnant une substance qui bloque la dégradation de la dopamine, drug treatments to compensate for the lack of dopamine by mimicking the action of dopamine, by administering a substance that will be transformed into dopamine, by giving a substance that blocks the degradation of dopamine,
des traitements chirurgicaux consistant à stimuler la fonction cérébrale profonde (implantation d'électrodes dans le cerveau). Ces traitements ne sont pas satisfaisants car ils ont de nombreux effets secondaires (nausées, vomissements, dyskinésies, troubles du comportement comme de nouvelles addictions) et ils améliorent la qualité de vie du patient mais n'arrêtent pas l'évolution de la maladie. surgical treatments consisting of stimulating deep brain function (implantation of electrodes in the brain). These treatments are not satisfactory because they have many side effects (nausea, vomiting, dyskinesias, behavioral problems such as new addictions) and they improve the patient's quality of life but do not stop the progression of the disease.
La maladie de Huntington est une maladie héréditaire qui se caractérise par d'importants troubles moteurs, cognitifs et psychiatriques, et évolue jusqu'à la perte d'autonomie et enfin la mort. On peut classer ces symptômes en trois grandes familles. Huntington's disease is an inherited disease characterized by significant motor, cognitive and psychiatric disturbances, and progresses to loss of autonomy and ultimately death. We can classify these symptoms into three main families.
Actuellement, les traitements proposés consistent à traiter les symptômes pour soulager le patient et ralentir sa dégradation physique et psychologique avec des médicaments psychotropes, des médicaments neuroleptiques et de la rééducation par kinésithérapie et orthophonie. Ces traitements ne sont pas satisfaisants car ils ne traitent pas de manière curative la maladie. Les maladies à prions, sont des maladies caractérisées notamment par une dégénérescence du système nerveux central. Elles sont également appelées les encéphalopathies subaiguës spongiformes transmissibles (ESST). Ces maladies sont dues à l'accumulation dans le cerveau d'une protéine normale mais mal conformée, la protéine prion. Ces maladies sont caractérisées par une évolution rapide et fatale. La plus connue est la maladie de Creutzfeldt- Jakob (MJC). Currently, the treatments proposed consist of treating the symptoms to relieve the patient and slow down his physical and psychological degradation with psychotropic drugs, neuroleptic drugs and rehabilitation through physiotherapy and speech therapy. These treatments are not satisfactory because they do not cure the disease in a curative way. Prion diseases are diseases characterized in particular by degeneration of the central nervous system. They are also called subacute transmissible spongiform encephalopathies (TSSS). These diseases are caused by the accumulation in the brain of a normal but poorly conformed protein, the prion protein. These diseases are characterized by a rapid and fatal course. The best known is Creutzfeldt-Jakob disease (CJD).
Il n'existe actuellement aucun traitement. There is currently no treatment.
Les maladies neuromusculaires sont des maladies qui atteignent les cellules nerveuses motrices de la moelle épinière ou motoneurones (amyotrophies spinales, sclérose latérale amyotrophique), les racines et les nerfs des membres (neuropathies périphériques), la jonction entre le nerf et le muscle (myasthénie) et le muscle (myopathies). Elles peuvent toucher la motricité des jambes ou des bras mais quelquefois aussi d'autres organes et fonctions qui dépendent des muscles (motricité des yeux, de la parole, de la déglutition, de la digestion, de la respiration, du cœur). La cause peut être génétique ou due à un mauvais fonctionnement de l'immunité (maladie « auto-immune ») qui va provoquer des lésions des nerfs (neuropathies dysimmunitaires), de la jonction neuromusculaire (myasthénie) ou une inflammation des muscles (myosites). Il existe d'autres causes possibles: toxicité médicamenteuse ou environnementale, carence vitaminique, maladies endocriniennes ou générales, infections. Neuromuscular diseases are diseases that affect the motor nerve cells of the spinal cord or motor neurons (spinal atrophies, amyotrophic lateral sclerosis), the roots and nerves of the limbs (peripheral neuropathies), the junction between the nerve and the muscle (myasthenia gravis) and muscle (myopathies). They can affect the motor skills of the legs or arms but sometimes also other organs and functions which depend on the muscles (motor skills of the eyes, speech, swallowing, digestion, breathing, heart). The cause can be genetic or due to a malfunction of the immunity ("autoimmune" disease) which will cause damage to the nerves (dysimmune neuropathies), the neuromuscular junction (myasthenia gravis) or inflammation of the muscles (myositis). . There are other possible causes: drug or environmental toxicity, vitamin deficiency, endocrine or general diseases, infections.
Les traitements sont différents selon la cause de la maladie. Dans les maladies génétiques, les essais thérapeutiques ne sont qu'à leur début, aucun traitement de routine n'existe. Lors d'une anomalie du métabolisme de la cellule, il existe souvent des médicaments qui visent à pallier les conséquences de cette déficience. Dans les maladies dysimmunitaires, il existe de nombreux traitements qui peuvent être efficaces. Cependant des effets secondaires indésirables sont connus comme des effets hématologiques. Treatments are different depending on the cause of the disease. In genetic diseases, therapeutic trials are only in their early stages, no routine treatment exists. In the event of an abnormality in the metabolism of the cell, there are often drugs which aim to alleviate the consequences of this deficiency. In dysimmune diseases, there are many treatments that can be effective. However, undesirable side effects are known as haematological effects.
L'ataxie spinocérébelleuse, correspond à une ataxie cérébelleuse autosomique dominante type 1. Elle est caractérisée par une ataxie, une ophtalmoplégie externe progressive et d'autres manifestations neurologiques. Spinocerebellar ataxia corresponds to autosomal dominant type 1 cerebellar ataxia. It is characterized by ataxia, progressive external ophthalmoplegia and other neurological manifestations.
Actuellement, les traitements proposés consistent en des traitements directs de la cause de la maladie par chirurgie, médicaments fluidifiants le sang, antibiotiques ou stéroïdes. Des traitements par médicaments orphelins sont aussi utilisés. Ces traitements ne sont pas satisfaisants car ils sont des médicaments orphelins non développés pour ces indications, les effets secondaires peuvent être divers : éosinophilie, leucopénie, thrombopénie, diarrhées, éruption cutanée et augmentation des enzymes hépatiques. Les autres traitements peuvent avoir pour effet indésirable des hémorragies, des troubles digestifs comme des nausées, vomissements, diarrhées, des allergies, une photosensibilisation. Currently, the treatments offered consist of direct treatment of the cause of the disease by surgery, blood thinning drugs, antibiotics or steroids. Orphan drug treatments are also used. These treatments are not satisfactory because they are orphan drugs not developed for these indications, side effects can be various: eosinophilia, leukopenia, thrombocytopenia, diarrhea, rash and increased liver enzymes. The other treatments can have the side effect of hemorrhages, digestive disorders such as nausea, vomiting, diarrhea, allergies, photosensitization.
Les amyotrophies spinales ou amyotrophies spinales antérieures sont un groupe de maladies neuromusculaires caractérisées par une faiblesse musculaire progressive due à la dégénérescence et la perte des motoneurones antérieurs de la moelle épinière et des noyaux du tronc cérébral. Ces maladies sont présentées sous quatre formes selon l'âge d'apparition et la sévérité de la maladie. Actuellement, les traitements proposés consistent en un traitement médicamenteux (Nusinersen connu sous le nom de Spinraza®) qui n'est pas efficace dans toutes les formes de ces maladies et compte des effets indésirables d'ordre respiratoire et infectieux. Spinal amyotrophies or anterior spinal amyotrophies are a group of neuromuscular diseases characterized by progressive muscle weakness due to the degeneration and loss of the anterior motor neurons in the spinal cord and brainstem nuclei. These diseases are presented in four forms depending on the age of onset and the severity of the disease. Currently, the treatments proposed consist of a drug treatment (Nusinersen known as Spinraza ® ) which is not effective in all forms of these diseases and has respiratory and infectious side effects.
La sclérose latérale amyotrophique, ou maladie de Charcot, est une maladie caractérisée par une dégénérescence progressive des motoneurones du cortex cérébral. Elle provoque une paralysie progressive de l'ensemble de la musculature squelettique des membres, du tronc et de l'extrémité céphalique. Amyotrophic lateral sclerosis, or Charcot's disease, is a disease characterized by progressive degeneration of motor neurons in the cerebral cortex. It causes progressive paralysis of the entire skeletal musculature of the limbs, trunk and cephalic extremity.
Seul un médicament est proposé Actuellement, pour traiter la sclérose latérale amyotrophique. Il s'agit du Riluzole® mais ce médicament n'est pas efficace et ne permet pas d'améliorer la qualité de vie et prolonge la survie de quelques mois seulement. Currently, only one drug is proposed to treat amyotrophic lateral sclerosis. It is Riluzole ® but this drug is not effective and does not improve the quality of life and prolongs survival by only a few months.
Toutes les maladies ayant un effet dégénératif sur les neurones sont caractérisées par une production en excès de 2-hydroxyglutarate par la personne ou l'animal malade (Gibson, K. M., Craigen, W., Herman, G. E. & Jakobs, C. D-2-Hydroxyglutaric Aciduria in a Newborn with Neurologicol Abnormolities: A New Neurometabolic Disorder? J. Inher. Metab. Dis 16, (1993) ; Ma, S. et al. L2hgdh Deficiency Accumulâtes I-2-Hydroxyglutarate with Progressive Leukoencephalopathy and Neurodegeneration. Mol. Cell. Biol. (2017). doi:10.1128/MCB.00492-16). All diseases having a degenerative effect on neurons are characterized by an excess production of 2-hydroxyglutarate by the sick person or animal (Gibson, KM, Craigen, W., Herman, GE & Jakobs, C. D-2 -Hydroxyglutaric Aciduria in a Newborn with Neurologicol Abnormolities: A New Neurometabolic Disorder? J. Inher. Metab. Dis 16, (1993); Ma, S. et al. L2hgdh Deficiency Accumulates I-2-Hydroxyglutarate with Progressive Leukoencephalopathy and Molodegeneration. . Cell. Biol. (2017). Doi: 10.1128 / MCB.00492-16).
L'accumulation du 2-hydroxyglutarate cause une myélinisation anormale, perturbe l'homéostasie des cellules souches neuronales, augmente la mortalité des cellules du système nerveux central. The accumulation of 2-hydroxyglutarate causes abnormal myelination, disrupts the homeostasis of neuronal stem cells, increases the mortality of central nervous system cells.
Les différents cancers du cerveau sont aussi caractérisés par une production en excès de 2- hydroxyglutarate chez la personne ou l'animal malade. En effet, le 2-hydroxyglutarate est connu pour son statut de marqueur des maladies neurodégénératives ainsi que son rôle d'oncométabolite dans le développement de tumeurs du cerveau. Le 2-hydroxyglutarate a une action d'inhibition de la réparation de l'ADN. La production de hauts niveaux de 2- hydroxyglutarate entraîne l'inhibition des voies de réparation de l'ADN dans des cellules cancéreuses et donc de l'accumulation d'ADN endommagé. The various brain cancers are also characterized by an excess production of 2-hydroxyglutarate in the sick person or animal. Indeed, 2-hydroxyglutarate is known for its status as a marker of neurodegenerative diseases as well as its role of oncometabolite in the development of brain tumors. 2-Hydroxyglutarate acts to inhibit DNA repair. The production of high levels of 2-hydroxyglutarate results in the inhibition of DNA repair pathways in cancer cells and therefore the accumulation of damaged DNA.
Le 2-hydroxyglutarate est aussi connu pour son rôle de marqueur de carcinomes de cellules rénales et dans des tissus sous limitation d'oxygène ou conditions d'hypoxie comme les carcinomes hépatocellulaires ou les carcinomes de côlon, il a donc un rôle dans les cancers du rein et du côlon. 2-Hydroxyglutarate is also known for its role as a marker of renal cell carcinomas and in tissues under oxygen limitation or hypoxic conditions such as hepatocellular carcinomas or colon carcinomas, so it has a role in cancers of the breast. kidney and colon.
L'inhibition de la réparation de l'ADN par le 2-hydroxyglutarate a été reportée dans d'autres types de cancer : du sang, de la peau, du foie, de la lymphe, de la prostate, de la thyroïde, de l'estomac, du sein, du pancréas, de l'hypophyse (Ye, D., Guan, K.-L. & Xiong, Y. Metabolism, Activity, and Targeting of D- and L-2-Hydroxyglutarates. Trends in cancer 4, 151-165 (2018)). L'acidurie 2-hydroxyglutarique est un groupe de maladies neurométaboliques avec un large spectre clinique allant de manifestations néonatales sévères à des formes progressives, et des cas asymptomatiques, caractérisée sur le plan biochimique par des taux élevés d'acide 2- hydroxyglutarique dans le plasma, le liquide céphalo-rachidien et les urines. Inhibition of DNA repair by 2-hydroxyglutarate has been reported in other types of cancer: blood, skin, liver, lymph, prostate, thyroid, 'stomach, breast, pancreas, pituitary gland (Ye, D., Guan, K.-L. & Xiong, Y. Metabolism, Activity, and Targeting of D- and L-2-Hydroxyglutarates. Trends in cancer 4, 151-165 (2018)). 2-Hydroxyglutaric aciduria is a group of neurometabolic diseases with a broad clinical spectrum ranging from severe neonatal manifestations to progressive forms, and asymptomatic cases, characterized biochemically by high levels of 2-hydroxyglutaric acid in plasma , cerebrospinal fluid and urine.
L'acidurie L-2-hydroxyglutarique est caractérisée par un retard psychomoteur, une ataxie cérébelleuse et une épilepsie, et l'acidurie D-2-hydroxyglutarique (voir ce terme) par des manifestations métaboliques, neurologiques et dysmorphiques variables. L-2-hydroxyglutaric aciduria is characterized by psychomotor retardation, cerebellar ataxia and epilepsy, and D-2-hydroxyglutaric aciduria (see this term) by variable metabolic, neurological and dysmorphic manifestations.
Les mutations sur le gène L2HGDH (14q22.1) ont été associées à l'acidurie L-2- hydroxyglutarique, et les mutations sur les gènes D2HGDH (2q37.3) et IDH2 (15q26.1) à l'acidurie D-2-hydroxyglutarique. Mutations in the L2HGDH gene (14q22.1) have been associated with L-2-hydroxyglutaric aciduria, and mutations in the D2HGDH (2q37.3) and IDH2 (15q26.1) genes with D-2 aciduria. -hydroxyglutaric.
Il n'existe aucun traitement curatif contre les maladies ayant un effet dégénératif sur les cellules telles que les neurones et les cellules nerveuses motrices, mais seulement des interventions permettant d'améliorer la prise en charge des patients atteints de maladies ayant un effet dégénératif sur les neurones et les cellules nerveuses musculaires : un diagnostic précoce, l'optimisation de la santé physique, des activités cognitives et de bien- être, le dépistage et le traitement de comorbidités physiques et psychiques. There is no curative treatment for diseases having a degenerative effect on cells such as neurons and motor nerve cells, but only interventions to improve the management of patients with diseases having a degenerative effect on them. neurons and muscle nerve cells: early diagnosis, optimization of physical health, cognitive and well-being activities, screening and treatment of physical and mental comorbidities.
Par ailleurs les principaux traitements des cancers sont la chimiothérapie et la radiothérapie, qui ont une efficacité limitée permettant un faible taux de survie et des effets secondaires très lourds tels que chute de cheveux, nausées et vomissements, diarrhées, baisse des globules blancs, globules rouges et plaquettes, lésions de la bouche, sensations d'engourdissement ou de fourmillement dans les mains ou pieds, troubles cutanés et syndrome main-pied, modification de la couleur et une fragilisation des ongles, douleurs musculaires et articulaires, troubles du cycle menstruel, troubles cardiaques, fatigue, réactions allergiques, altération de cellules saines par irradiation des tissus sains à côté de la tumeur, troubles sexuels, problèmes de fertilité, réaction inflammatoire, effets sur les cellules du sang. In addition, the main cancer treatments are chemotherapy and radiotherapy, which have limited efficacy allowing a low survival rate and very serious side effects such as hair loss, nausea and vomiting, diarrhea, reduced blood pressure. white blood cells, red blood cells and platelets, mouth lesions, numbness or tingling sensations in the hands or feet, skin disorders and hand-foot syndrome, change in color and weakening of the nails, muscle and joint pain, disorders menstrual cycle, heart problems, fatigue, allergic reactions, damage to healthy cells by irradiation of healthy tissue next to the tumor, sexual problems, fertility problems, inflammatory reaction, effects on blood cells.
Il n'existe également aucun traitement pour les aciduries L- ou D-2-hydroxyglutariques. Dans le cas de l'acidurie D-2-hydroxyglutarique, un contrôle de l'épilepsie est présent, le pronostic de vie dépend entièrement de la gravité du tableau clinique et de l'évolution de la maladie, donc on ne peut prévenir. De plus le pronostic de vie des aciduries L-2-hydroxyglutariques est mauvais pour ces patients, bien que la plupart atteignent l'âge adulte. There is also no treatment for L- or D-2-hydroxyglutaric aciduria. In the case of D-2-hydroxyglutaric aciduria, epilepsy control is present, the life prognosis depends entirely on the severity of the clinical picture and the course of the disease, so it cannot be prevented. In addition, the life prognosis for L-2-hydroxyglutaric aciduria is poor for these patients, although most reach adulthood.
Ainsi, il existe un besoin important pour un traitement efficace à la fois des maladies neurodégénératives et des cancers, en particulier des tumeurs cérébrales, rénales, du colon et intestinales, capable d'agir sur la synthèse du 2-hydroxyglutarate, qui soit facile à administrer et qui ne présente pas d'effets secondaires. Thus, there is a great need for an effective treatment of both neurodegenerative diseases and cancers, in particular brain, kidney, colon and intestinal tumors, capable of acting on the synthesis of 2-hydroxyglutarate, which is easy to manage. administer and which does not present side effects.
EXPOSE DE L'INVENTION DISCLOSURE OF THE INVENTION
Pour y répondre, l'invention vise l'utilisation de bactéries particulières du microbiote intestinal humain, en particulier des bactéries de la famille des Christensenellacées et/ou des bactéries du genre Parasutterella et/ou des bactéries du genre Negativibacillus et/ou des bactéries du genre Massiliomicrobiota. To answer this, the invention is aimed at the use of particular bacteria of the human intestinal microbiota, in particular bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus genus Massiliomicrobiota.
Des bactéries de la famille des Christensenellacées, notamment du genre Christensenella, ont déjà été étudiées et décrites. C'est le cas en particulier de Christensenella minuta, Christensenella massiliensis et Christensenella timonensis. Christensenella minuta en particulier a été décrite pour la première fois en 2012. En 2014, une étude a montré qu'il s'agissait du taxon le plus héritable dans une cohorte de jumeaux britanniques et que leur présence est associée à un indice de masse corporel faible. Cette corrélation entre Christensenella minuta et l'indice de masse corporel faible a ensuite été observée dans une dizaine d'études parues depuis 2014 dans des populations géographiquement diverses. Enfin, la demande de brevet US 2018/255819 décrit l'utilisation des bactéries de la famille des Christensenellacées dans le traitement de l'obésité. Des bactéries du genre Parasutterella, ont déjà été étudiées et décrites. C'est le cas en particulier de Parasutterella excrementihominis et Parasutterella secunda, qui ont été observées dans plusieurs études, sans explication de causalité. Parasutterella excrementihominis a été décrite pour la première fois en 2009 et Parasutterella secunda a été décrite pour la première fois en 2011. D'autres espèces ont aussi été étudiées. C'est le cas de Parasutterella mcl qui a été associée à l'abondance de certains métabolites dans des souris saines. Bacteria of the Christensenellaceae family, in particular of the genus Christensenella, have already been studied and described. This is the case in particular with Christensenella minuta, Christensenella massiliensis and Christensenella timonensis. Christensenella minuta in particular was first described in 2012. In 2014, a study showed that it was the most heritable taxon in a cohort of British twins and that their presence is associated with a body mass index. low. This correlation between Christensenella minuta and the low body mass index was then observed in around ten studies published since 2014 in geographically diverse populations. Finally, patent application US 2018/255819 describes the use of bacteria of the Christensenellaceae family in the treatment of obesity. Bacteria of the genus Parasutterella have already been studied and described. This is particularly the case with Parasutterella excrementihominis and Parasutterella secunda, which have been observed in several studies without an explanation of causation. Parasutterella excrementihominis was first described in 2009 and Parasutterella secunda was first described in 2011. Other species were also studied. This is the case with Parasutterella mcl which has been associated with the abundance of certain metabolites in healthy mice.
Des bactéries du genre Negativibacillus ont été décrites. C'est le cas Negativibacillus massiliensis qui a été décrite en 2016. Les bactéries du genre Negativibacillus ont été caractérisées selon la description présentée dans « Negativibacillus massiliensis » gen. Nov., sp. Nov., isolated from human left colon, D. Ricaboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017; 17 : 36-38. Aucune utilisation thérapeutique des bactéries du genre Negativibacillus n'a jamais été décrite ni envisagée. Bacteria of the genus Negativibacillus have been described. This is the case with Negativibacillus massiliensis, which was described in 2016. Bacteria of the genus Negativibacillus have been characterized according to the description presented in “Negativibacillus massiliensis” gen. Nov., sp. Nov., isolated from human left colon, D. Ricaboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017; 17: 36-38. No therapeutic use of bacteria of the genus Negativibacillus has ever been described or considered.
Les bactéries du genre Massiliomicrobiota, ont été peu étudiées. On connaît notamment Massiliomicrobiota timonensis et Massiliomicrobiota escudieri. Bacteria of the genus Massiliomicrobiota have been little studied. In particular, Massiliomicrobiota timonensis and Massiliomicrobiota escudieri are known.
Massiliomicrobiota timonensis a été décrite pour la première fois en 2016. Massiliomicrobiota escudieri a été décrite pour la première fois en 2018. Les bactéries du genre Massiliomicrobiota ont été caractérisées selon la description présentée dans « Massiliomicrobiota timonensis », a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016 ; 13 : 25-26 et dans « Massiliomicrobiota escudieri sp. nov. isolated as part of a culturomics exploration of the gut microbiota of rénal cancer patients », unpublished, Tidjani Alou,M., Derosa,L. and Zitvogel,L., submitted (ll-JUN-2018) U1015, Institut Gustave Roussy, 114 rue edouard vaillant, Villejuif 94800, France, Metropolitan. Aucune utilisation thérapeutique des bactéries du genre Massiliomicrobiota n'a jamais été décrite ni envisagée. Massiliomicrobiota timonensis was first described in 2016. Massiliomicrobiota escudieri was first described in 2018. Bacteria of the genus Massiliomicrobiota were characterized according to the description presented in “Massiliomicrobiota timonensis”, a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016; 13: 25-26 and in “Massiliomicrobiota escudieri sp. nov. isolated as part of a culturomics exploration of the gut microbiota of renal cancer patients ”, unpublished, Tidjani Alou, M., Derosa, L. and Zitvogel, L., submitted (ll-JUN-2018) U1015, Institut Gustave Roussy, 114 rue edouard vaillant, Villejuif 94800, France, Metropolitan. No therapeutic use of bacteria of the genus Massiliomicrobiota has ever been described or considered.
De façon surprenante, et selon l'invention : Surprisingly, and according to the invention:
- les bactéries de la famille des Christensenellacées, notamment du genre Christensenella, et notamment Christensenella minuta, Christensenella massiliensis et Christensenella timonensis, - bacteria of the Christensenellaceae family, in particular of the genus Christensenella, and in particular Christensenella minuta, Christensenella massiliensis and Christensenella timonensis,
- les bactéries du genre Parasutterella, en particulier Parasutterella excrementihominis et Parasutterella secunda, - les bactéries du genre Negativibacillus, en particulier Negativibacillus massiliensis, - bacteria of the genus Parasutterella, in particular Parasutterella excrementihominis and Parasutterella secunda, - bacteria of the genus Negativibacillus, in particular Negativibacillus massiliensis,
- les bactéries du genre Massiliomicrobiota, en particulier Massiliomicrobiota timonensis et Massiliomicrobiota escudieri, et - bacteria of the genus Massiliomicrobiota, in particular Massiliomicrobiota timonensis and Massiliomicrobiota escudieri, and
- les mélanges d'au moins deux de ces bactéries, - mixtures of at least two of these bacteria,
lorsqu'elles sont administrées à l'homme ou à l'animal, sont capables d'agir sur le 2- hydroxyglutarate à l'origine des maladies neurodégénératives et de cancers. when administered to humans or animals, are capable of acting on 2-hydroxyglutarate which causes neurodegenerative diseases and cancers.
C'est pourquoi, l'invention a pour objet une bactérie de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie caractérisée par l'excès de 2-hydroxyglutarate chez l'être humain ou l'animal, en particulier de maladies neurodégénératives et de cancers. This is why the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or the treatment of at least one disease characterized by an excess of 2-hydroxyglutarate in humans or animals, in particular neurodegenerative diseases and cancers.
Avantageusement, de telles bactéries, lorsqu'elles sont administrées à un être humain ou un animal présentant une maladie neurodégénérative ou un cancer, sont capables d'agir sur le 2-hydroxyglutarate produit en excès lors de ces maladies. Advantageously, such bacteria, when they are administered to a human being or an animal exhibiting a neurodegenerative disease or a cancer, are capable of acting on the 2-hydroxyglutarate produced in excess during these diseases.
Pour son utilisation comme dans la prévention ou le traitement de maladies caractérisées par l'excès de 2-hydroxyglutarate chez l'être humain ou l'animal, en particulier dans la prévention ou le traitement de maladies neurodégénératives et/ou de cancers, les bactéries de la famille des Christensenellacées et/ou les bactéries du genre Parasutterella et/ou les bactéries du genre Negativibacillus et/ou les bactéries du genre Massiliomicrobiota, sont préférentiellement utilisées dans des compositions. L'invention a donc également pour objet les compositions comprenant au moins une bactérie de la famille des Christensenellacées, préférentiellement du genre Christensenella, et/ou une bactérie du genre Parasutterella et/ou une bactérie du genre Negativibacillus et/ou une bactérie du genre Massiliomicrobiota, pour son utilisation dans la prévention ou le traitement de maladies caractérisées par l'excès de 2-hydroxyglutarate chez l'être humain ou l'animal, en particulier dans la prévention ou le traitement de maladies neurodégénératives et/ou de cancers. For its use as in the prevention or treatment of diseases characterized by the excess of 2-hydroxyglutarate in humans or animals, in particular in the prevention or treatment of neurodegenerative diseases and / or cancers, bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, are preferably used in compositions. A subject of the invention is therefore also the compositions comprising at least one bacterium of the Christensenellaceae family, preferably of the genus Christensenella, and / or a bacterium of the genus Parasutterella and / or a bacterium of the genus Negativibacillus and / or a bacterium of the genus Massiliomicrobiota. , for its use in the prevention or treatment of diseases characterized by the excess of 2-hydroxyglutarate in humans or animals, in particular in the prevention or treatment of neurodegenerative diseases and / or cancers.
D'autres caractéristiques et avantages ressortiront de la description en détails de l'invention qui va suivre. Other characteristics and advantages will emerge from the detailed description of the invention which follows.
BREVE DESCRIPTION DES DESSINS BRIEF DESCRIPTION OF THE DRAWINGS
La Figure 1 démontre l'effet antiprolifératif des surnageants de bactéries selon l'invention, en particulier 3 souches de C. minuta sur une lignée cellulaire humaine d'adénocarcinome du colon, la lignée HTC-116, après 24h et 48h de traitement. La Figure 2 démontre l'effet antiprolifératif des surnageants de bactéries selon l'invention, en particulier 3 souches de C. minuta sur une lignée cellulaire d'hépatocarcinome humain, la lignée HepG2 après 24h et 48h de traitement. Figure 1 demonstrates the antiproliferative effect of the supernatants of bacteria according to the invention, in particular 3 strains of C. minuta on a human colon adenocarcinoma cell line, the HTC-116 line, after 24 hours and 48 hours of treatment. FIG. 2 demonstrates the antiproliferative effect of the supernatants of bacteria according to the invention, in particular 3 strains of C. minuta on a human hepatocarcinoma cell line, the HepG2 line after 24 hours and 48 hours of treatment.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
Définitions Definitions
Par « excès » ou « hyperproduction » de 2-hydroxyglutarate au sens de l'invention on entend une production excessive de 2-hydroxyglutarate par rapport à la production chez une personne ou un animal sain sans pathologie. By “excess” or “hyperproduction” of 2-hydroxyglutarate within the meaning of the invention is meant an excessive production of 2-hydroxyglutarate relative to the production in a healthy person or animal without pathology.
Par « maladie caractérisée par l'excès de 2-hydroxyglutarate » ou « maladie caractérisée par une hyperproduction de 2-hydroxyglutarate » au sens de l'invention on entend une maladie dont au moins une des causes est l'excès ou l'hyperproduction de 2-hydroxyglutarate dans l'organisme de la personne ou de l'animal malade. Il peut s'agir notamment d'une maladie neurodégénérative ou d'un cancer. By “disease characterized by the excess of 2-hydroxyglutarate” or “disease characterized by an overproduction of 2-hydroxyglutarate” within the meaning of the invention is meant a disease of which at least one of the causes is the excess or overproduction of 2-hydroxyglutarate in the body of the sick person or animal. It may in particular be a neurodegenerative disease or cancer.
Par « marqueur » d'une maladie au sens de l'invention on entend une molécule ou une substance dont le dosage permet de suivre l'évolution de ladite maladie. For the purposes of the invention, the term “marker” of a disease means a molecule or a substance, the dosage of which makes it possible to follow the course of said disease.
Bactéries selon l'invention Bacteria according to the invention
L'invention a donc pour objet une bactérie de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie caractérisée par l'excès de 2-hydroxyglutarate chez l'être humain ou l'animal. The subject of the invention is therefore a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment. at least one disease characterized by an excess of 2-hydroxyglutarate in humans or animals.
Les bactéries du genre Negativibacillus ont été caractérisées selon la description présentée dans « Negativibacillus massiliensis » gen. Nov., sp. Nov., isolated from human left colon, D. Ricaboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017 ; 17 : 36-38 et dans « Massiliomicrobiota escudieri sp. nov. isolated as part of a culturomics exploration of the gut microbiota of rénal cancer patients », unpublished, Tidjani Alou,M., Derosa,L. and Zitvogel,L., submitted (ll-JUN-2018) U1015, Institut Gustave Roussy, 114 rue édouard vaillant, Villejuif 94800, France, Metropolitan. Bacteria of the genus Negativibacillus have been characterized according to the description presented in “Negativibacillus massiliensis” gen. Nov., sp. Nov., isolated from human left colon, D. Ricaboni, M. Mailhe, V. Vitton, C. Andrieu, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2017; 17: 36-38 and in “Massiliomicrobiota escudieri sp. nov. isolated as part of a culturomics exploration of the gut microbiota of renal cancer patients ”, unpublished, Tidjani Alou, M., Derosa, L. and Zitvogel, L., submitted (ll-JUN-2018) U1015, Institut Gustave Roussy, 114 rue édouard vaillant, Villejuif 94800, France, Metropolitan.
Les bactéries du genre Massiliomicrobiota ont été caractérisées selon la description présentée dans « Massiliomicrobiota timonensis », a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016 ; 13 : 25-26. Bacteria of the genus Massiliomicrobiota were characterized according to the description presented in “Massiliomicrobiota timonensis”, a new bacterial species isolated from the human gut, S. Ndongo, S. Khelaifia, P.-E. Fournier, D. Raoult, New Microbe and New Infect 2016; 13: 25-26.
En particulier l'invention a pour objet une bactérie de la famille des Christensenellacées, notamment du genre Christensenella, et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative et/ou d'au moins un cancer chez l'être humain ou l'animal. In particular, the subject of the invention is a bacterium of the Christensenellaceae family, in particular of the genus Christensenella, and / or of bacteria of the genus Parasutterella and / or of bacteria of the genus Negativibacillus and / or of bacteria of the genus Massiliomicrobiota, for its use in prevention and / or treatment of at least one neurodegenerative disease and / or at least one cancer in humans or animals.
Selon une variante l'invention a pour objet une bactérie de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative choisie parmi la maladie d'Alzheimer, les démences, la maladie de Parkinson et les troubles associés, les maladies à prions, les maladies neuromusculaires, la maladie de Huntington, l'ataxie spinocérébelleuse, l'amyotrophie spinale progressive, la sclérose latérale amyotrophique. According to one variant, the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or the treatment of at least one neurodegenerative disease selected from Alzheimer's disease, dementias, Parkinson's disease and associated disorders, prion diseases, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal atrophy, amyotrophic lateral sclerosis.
Selon une autre variante l'invention a pour objet une bactérie de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins un cancer du cerveau et/ou des reins et/ou du foie et/ou du côlon, chez l'être humain ou l'animal. According to another variant, the subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or or the treatment of at least one cancer of the brain and / or kidneys and / or liver and / or colon, in humans or animals.
Préférentiellement, l'invention a pour objet une bactérie de la famille des Christensenellacées et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins un cancer du cerveau et/ou des reins et/ou du foie et/ou du côlon, chez l'être humain ou l'animal. Preferably, a subject of the invention is a bacterium of the Christensenellaceae family and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment of at least one cancer of the breast. brain and / or kidneys and / or liver and / or colon, in humans or animals.
Selon un autre variante, l'invention a pour objet une bactérie du genre Parasutterella, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative, préférentiellement choisie parmi la maladie d'Alzheimer, les démences, la maladie de Parkinson et les troubles associés, les maladies à prions, les maladies neuromusculaires, la maladie de Huntington, l'ataxie spinocérébelleuse, l'amyotrophie spinale progressive, la sclérose latérale amyotrophique. According to another variant, the subject of the invention is a bacterium of the genus Parasutterella, for its use in the prevention and / or the treatment of at least one neurodegenerative disease, preferably chosen from Alzheimer's disease, dementias, disease. of Parkinson's disease and associated disorders, prion disease, neuromuscular disease, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, amyotrophic lateral sclerosis.
Selon l'invention, les bactéries de la famille des Christensenellacées et/ou les bactéries du genre Parasutterella et/ou les bactéries du genre Negativibacillus et/ou les bactéries du genre Massiliomicrobiota, lorsqu'elles sont administrées à un être humain ou un animal présentant une maladie neurodégénérative, sont capables d'agir sur les molécules produites en excès lors d'une maladie neurodégénérative ou d'un cancer, en particulier sur le 2- hydroxyglutarate. According to the invention, bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota, when they are administered to a human being or an animal presenting neurodegenerative disease, are capable of acting on molecules produced in excess during neurodegenerative disease or cancer, in particular on 2-hydroxyglutarate.
Dans les maladies ayant un effet dégénératif sur les neurones, la diminution de la quantité de 2-hydroxyglutarate est le signe de la réduction de ces maladies ayant un effet dégénératif sur les neurones, c'est-à-dire que les bactéries à l'origine de cette production sont moins stimulées. Dès lors, la production excessive en cause de la maladie ayant un effet dégénératif est ralentie et le système retourne progressivement à la norme. En particulier, un substrat du 2-hydroxyglutarate, le glutarate, diminue lorsque le 2-hydroxyglutarate augmente. Ainsi un retour à la normale par une diminution du 2-hydroxyglutarate est accompagné d'une augmentation du glutarate. In diseases having a degenerative effect on neurons, the decrease in the amount of 2-hydroxyglutarate is a sign of the reduction of those diseases having a degenerative effect on neurons, that is, bacteria at the origin of this production are less stimulated. As a result, the excessive production in question of the disease having a degenerative effect is slowed down and the system gradually returns to the norm. In particular, a substrate for 2-hydroxyglutarate, glutarate, decreases as 2-hydroxyglutarate increases. Thus a return to normal by a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
Dans les cancers, la diminution de synthèse du 2-hydroxyglutarate est le signe de la réduction de l'inhibition des voies de réparation de l'ADN, c'est-à-dire que les bactéries à l'origine de cette production sont moins stimulées. Dès lors, la production excessive en cause de l'inhibition de réparation de l'ADN est ralentie et le système retourne progressivement à la normal. En particulier, un substrat du 2-hydroxyglutarate, le glutarate, diminue lorsque le 2- hydroxyglutarate augmente. Ainsi un retour à la normale par une diminution du 2- hydroxyglutarate est accompagné d'une augmentation du glutarate. In cancers, the decrease in the synthesis of 2-hydroxyglutarate is a sign of the reduction in the inhibition of DNA repair pathways, that is to say that the bacteria responsible for this production are less stimulated. As a result, the excessive production involved in inhibiting DNA repair is slowed down and the system gradually returns to normal. In particular, a substrate for 2-hydroxyglutarate, glutarate, decreases as 2-hydroxyglutarate increases. Thus a return to normal by a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
Dans les aciduries 2-hydroxyglutariques, la diminution de synthèse du 2-hydroxyglutarate est le signe de la réduction des symptômes cliniques liés à cette maladie. En particulier, un substrat du 2-hydroxyglutarate, le glutarate, diminue lorsque le 2-hydroxyglutarate augmente. Ainsi une diminution du 2-hydroxyglutarate est accompagnée d'une augmentation du glutarate. In 2-hydroxyglutaric aciduria, the decrease in 2-hydroxyglutarate synthesis is a sign of the reduction in clinical symptoms associated with this disease. In particular, a substrate for 2-hydroxyglutarate, glutarate, decreases as 2-hydroxyglutarate increases. Thus a decrease in 2-hydroxyglutarate is accompanied by an increase in glutarate.
Là où les bactéries utiles selon l'invention, sont administrées à des êtres humains ou des animaux dans une quantité efficace pour une action sur le 2-hydroxyglutarate, c'est-à-dire pour diminuer sa production dans l'organisme. Selon un mode de réalisation adapté, la ou les bactéries peuvent être administrées à raison d'une dose de 109 à 1012 unités formant des colonies (CFU) par jour, quel que soit le poids de la personne ou de l'animal. Préférentiellement, il s'agit d'une dose unique, c'est-à-dire administrée en une seule fois ou une dose avant chaque repas soit trois fois par jour. Where the bacteria useful according to the invention are administered to humans or animals in an amount effective for an action on 2-hydroxyglutarate, that is to say to reduce its production in the organism. According to a suitable embodiment, the bacteria or bacteria can be administered at a dose of 10 9 to 10 12 colony forming units (CFU) per day, regardless of the weight of the person or of the animal. Preferably, it is a single dose, that is to say administered all at once or a dose before each meal, ie three times a day.
La ou les bactéries utiles selon l'invention sont : - des bactéries de la famille des Christensenellacées, préférentiellement du genre Christensenella. Il peut s'agir en particulier de Christensenella massiliensis, Christensenella timonensis et/ou Christensenella minuta. Selon une variante particulièrement adaptée, il s'agit de Christensenella minuta. Ces bactéries peuvent être isolées à partir de selles humaines par exemple selon les protocoles publiés par Morotomi et al. 2012 (Morotomi, M., Nagai, F. & Watanabe, Y. Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposai of Christensenellaceae fam. nov. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 62, 144-149 (2012)) et NDongo et al. 2016 (Ndongo, S., Dubourg, G., Khelaifia, S., Fournier, P. E. & Raoult, D. Christensenella timonensis, a new bacterial species isolated from the human gut. New Microbes and New Infections 13, 32-33 (2016)). Ces documents décrivent également les méthodes de culture des bactéries utiles selon l'invention. The bacteria or bacteria useful according to the invention are: - bacteria of the Christensenellaceae family, preferably of the Christensenella genus. It may in particular be Christensenella massiliensis, Christensenella timonensis and / or Christensenella minuta. According to a particularly suitable variant, it is Christensenella minuta. These bacteria can be isolated from human stool, for example according to the protocols published by Morotomi et al. 2012 (Morotomi, M., Nagai, F. & Watanabe, Y. Description of Christensenella minuta gen. Nov., Sp. Nov., Isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposai of Christensenellaceae fam . nov. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 62, 144-149 (2012)) and NDongo et al. 2016 (Ndongo, S., Dubourg, G., Khelaifia, S., Fournier, PE & Raoult, D. Christensenella timonensis, a new bacterial species isolated from the human gut. New Microbes and New Infections 13, 32-33 (2016 )). These documents also describe the methods of culturing useful bacteria according to the invention.
- des bactéries du genre Parasutterella. Il peut s'agir en particulier de Parasutterella excrementihominis et Parasutterella secundo. Ces bactéries peuvent être isolées à partir de selles humaines par exemple selon les protocoles publiés par Nagai et al. 2009 (Parasutterella excrementihominis gen. nov., sp. nov., a member of the family Alcaligenaceae isolated from human faeces Nagai F Morotomi M Sakon H Tanaka R, International Journal of Systematic and Evolutionary Microbiology, 2009) et Morotomi et al. 2011 (Parasutterella secundo sp. nov., isolated from human faeces and proposai of Sutterellaceae fam. nov. in the order Burkholderiales, Morotomi M Nagai F Watanabe Y, International Journal of Systematic and Evolutionary Microbiology, 2011). Ces documents décrivent également les méthodes de culture des bactéries utiles selon l'invention. - bacteria of the genus Parasutterella. It may in particular be Parasutterella excrementihominis and Parasutterella secundo. These bacteria can be isolated from human stool, for example according to the protocols published by Nagai et al. 2009 (Parasutterella excrementihominis gen. Nov., Sp. Nov., A member of the family Alcaligenaceae isolated from human faeces Nagai F Morotomi M Sakon H Tanaka R, International Journal of Systematic and Evolutionary Microbiology, 2009) and Morotomi et al. 2011 (Parasutterella secundo sp. Nov., Isolated from human faeces and proposai of Sutterellaceae fam. Nov. In the order Burkholderiales, Morotomi M Nagai F Watanabe Y, International Journal of Systematic and Evolutionary Microbiology, 2011). These documents also describe the methods of culturing useful bacteria according to the invention.
- des bactéries du genre Negativibacillus, préférentiellement Negativibacillus massiliensis. Ces bactéries peuvent être isolées à partir de selles humaines par exemple selon les protocoles publiés par Fournier et al. 2016 ("Negativibacillus massiliensis" gen. nov., sp. nov., isolated from human left colon, Fournier P Ricaboni D Vitton V Raoult D Andrieu C Mailhe M, New Microbes and New Infections, 2016). Ce document décrit également les méthodes de culture des bactéries utiles selon l'invention. - bacteria of the genus Negativibacillus, preferably Negativibacillus massiliensis. These bacteria can be isolated from human stool, for example according to the protocols published by Fournier et al. 2016 ("Negativibacillus massiliensis" gen. Nov., Sp. Nov., Isolated from human left colon, Fournier P Ricaboni D Vitton V Raoult D Andrieu C Mailhe M, New Microbes and New Infections, 2016). This document also describes the methods of culturing useful bacteria according to the invention.
- des bactéries du genre Massiliomicrobiota. Il peut s'agir en particulier de Massiliomicrobiota timonensis et Massiliomicrobiota escudieri. Selon une variante particulièrement adaptée, il s'agit de Massiliomicrobiota timonensis. Ces bactéries peuvent être isolées à partir de selles humaines par exemple selon le protocole publié par Ndongo et al. 2016 ("Massiliomicrobiota timonensis", a new bacterial species isolated from the human gut. Ndongo S Khelaifia S Fournier P Raoult D. New microbes and new infections, 2016 vol: 13 pp: 25-6). New Microbes and New Infections 13, 32-33 (2016)). Ce document décrit également les méthodes de culture des bactéries utiles selon l'invention. - bacteria of the genus Massiliomicrobiota. It may be in particular Massiliomicrobiota timonensis and Massiliomicrobiota escudieri. According to a particularly suitable variant, it is Massiliomicrobiota timonensis. These bacteria can be isolated from stool human, for example according to the protocol published by Ndongo et al. 2016 ("Massiliomicrobiota timonensis", a new bacterial species isolated from the human gut. Ndongo S Khelaifia S Fournier P Raoult D. New microbes and new infections, 2016 vol: 13 pp: 25-6). New Microbes and New Infections 13, 32-33 (2016)). This document also describes the methods of culturing useful bacteria according to the invention.
- et les mélanges d'au moins deux de ces bactéries. - and mixtures of at least two of these bacteria.
La ou les bactéries utiles selon l'invention, pour leur utilisation précédemment décrite, sont préférentiellement administrées dans une composition. The bacteria (s) useful according to the invention, for their use described above, are preferably administered in a composition.
Composition comprenant des bactéries selon l'invention Composition comprising bacteria according to the invention
L'invention a donc également pour objet une composition comprenant au moins une bactérie de la famille des Christensenellacées et/ou une bactérie du genre Parasutterella et/ou une bactérie du genre Negativibacillus et/ou une bactérie du genre Massiliomicrobiota, dans la prévention et/ou le traitement de maladies caractérisées par l'excès de 2-hydroxyglutarate, en particulier de maladies neurodégénératives et/ou de cancers, chez l'être humain ou l'animal. La ou les bactéries sont présentes en une quantité efficace dans la composition permettant un effet sur le 2-hydroxyglutarate et sur la ou les maladies à traiter, en particulier les maladies neurodégénératives et/ou cancers, dont sont atteints les personnes ou les animaux traités. A subject of the invention is therefore also a composition comprising at least one bacterium of the Christensenellaceae family and / or a bacterium of the genus Parasutterella and / or a bacterium of the genus Negativibacillus and / or a bacterium of the genus Massiliomicrobiota, in the prevention and / or or the treatment of diseases characterized by an excess of 2-hydroxyglutarate, in particular neurodegenerative diseases and / or cancers, in humans or animals. The bacteria or bacteria are present in an effective amount in the composition allowing an effect on 2-hydroxyglutarate and on the disease or diseases to be treated, in particular neurodegenerative diseases and / or cancers, from which the persons or animals treated are affected.
Préférentiellement, la composition utile selon l'invention comprend 106 à 1012 unités formant des colonies (CFU) de bactéries de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota par dose quotidienne à administrer de composition. Préférentiellement, cela correspond à une dose quotidienne de bactéries à administrer, quel que soit le poids de la personne ou de l'animal. De façon préférée, cette dose est administrée en une seule fois. La composition utile selon l'invention peut être sous forme liquide. Elle peut notamment comprendre des bactéries de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota et un milieu de culture desdites bactéries qui permet de les conserver. Ce milieu peut être par exemple le milieu Columbia agar pour les bactéries de la famille des Christensenellacées, les bactéries du genre Negativibacillus, les bactéries du genre Massiliomicrobiota, ou le milieu BTU pour les bactéries du genre Parasutterella, anaérobique enrichi en sang de mouton, ou un milieu équivalent ne contenant pas de produit dérivé d'origine animale. Preferably, the composition useful according to the invention comprises 10 6 to 10 12 colony-forming units (CFU) of bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria. of the genus Massiliomicrobiota per daily dose of composition. Preferably, this corresponds to a daily dose of bacteria to be administered, regardless of the weight of the person or the animal. Preferably, this dose is administered all at once. The composition useful according to the invention can be in liquid form. It may in particular comprise bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota and a culture medium for said bacteria which makes it possible to preserve them. This medium can be, for example, Columbia agar medium for bacteria of the Christensenellaceae family, bacteria of the genus Negativibacillus, bacteria of the genus Massiliomicrobiota, or BTU medium for bacteria of the genus Parasutterella, anaerobic enriched with sheep blood, or an equivalent medium containing no derivative product of animal origin.
Selon une variante, la composition utile selon l'invention peut se présenter sous forme solide. Dans ce cas les bactéries peuvent être présentes sous forme lyophilisée, et peuvent comprendre également des excipients tels que par exemple la cellulose microcrystalline, le lactose, le saccharose, le fructose, le lévulose, les amidons, le stachyose, le raffinose, l'amylum, le lactate de calcium, le sulfate de magnésium, le citrate de sodium, le calcium stéarate, la polyvinylpyrrolidone, la maltodextrine, les galactooligosaccharides, les fructooligosaccharides, les pectines, les béta-glucans, les lactoglobulines, les isomaltooligosaccharides, les polydextroses, le sorbitol et/ou le glycérol. According to one variant, the composition useful according to the invention can be provided in solid form. In this case, the bacteria can be present in lyophilized form, and can also include excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum , calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, sorbitol and / or glycerol.
Les compositions utiles selon l'invention peuvent se présenter en particulier sous forme de poudre, de poudre microencapsulée, de gélule, de capsule, de comprimé, de pastille, de granulés, d'émulsion, de suspension ou de suppositoire. Selon un mode de réalisation particulièrement adapté, elles peuvent se présenter sous une forme gastro-résistante, telles qu'un comprimé enrobé contenant des bactéries microencapsulées. The compositions useful according to the invention can be in particular in the form of powder, microencapsulated powder, gelatin capsule, capsule, tablet, lozenge, granules, emulsion, suspension or suppository. According to a particularly suitable embodiment, they can be in a gastro-resistant form, such as a coated tablet containing microencapsulated bacteria.
Lorsque les compositions sont sous forme solide, elles sont préférentiellement conditionnées dans des capsules ou dans un enrobage hermétique à la lumière et à l'oxygène maintenu à une température ambiante comprise entre 15°C et 40°C et un taux d'humidité compris entre 3% et 70%. When the compositions are in solid form, they are preferably packaged in capsules or in a coating hermetic to light and to oxygen maintained at an ambient temperature of between 15 ° C and 40 ° C and a humidity of between 3% and 70%.
Les bactéries peuvent être utilisées vivantes ou inactivées par exemple par la chaleur, l'exposition à un pH approprié, aux rayons gamma ou à la mise sous haute pression. The bacteria can be used alive or inactivated, for example by heat, exposure to an appropriate pH, gamma rays or high pressure.
Elles peuvent être toutes vivantes ou toutes inactivées. They can be all alive or all inactivated.
Préférentiellement, au moins une partie des bactéries est constituée par des bactéries vivantes, en particulier au moins 50% (en nombre), encore plus préférentiellement au moins 90% (en nombre). Preferably, at least part of the bacteria consists of living bacteria, in particular at least 50% (by number), even more preferably at least 90% (by number).
Ainsi, selon un mode de réalisation adapté, les bactéries présentes dans la composition utile selon l'invention sont pour au moins 50% des bactéries vivantes (en nombre), préférentiellement pour au moins 90% des bactéries vivantes (en nombre), encore plus préférentiellement toutes vivantes. Thus, according to a suitable embodiment, the bacteria present in the composition useful according to the invention are at least 50% of living bacteria (by number), preferably for at least 90% of living bacteria (by number), even more preferably all alive.
Les bactéries utiles selon l'invention, et en particulier les compositions l'incluant, peuvent être administrées par voie orale, topique, respiratoire (inhalation) ou rectale. Les compositions utiles selon l'invention, en plus des bactéries utiles selon l'invention peuvent comprendre d'autres composés, tels que : The bacteria useful according to the invention, and in particular the compositions including it, can be administered orally, topically, respiratory (inhalation) or rectally. The compositions useful according to the invention, in addition to the bacteria useful according to the invention can comprise other compounds, such as:
- au moins un probiotique, et/ou - at least one probiotic, and / or
- au moins une bactérie permettant de créer un environnement anaérobique favorable aux bactéries présentes dans la composition telle qu'au moins une bactérie choisie parmi les bactéries du genre Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. et/ou au moins un autre organisme favorisant les conditions anaérobiques nécessaires à la survie des Christensenellacées telle qu'au moins une levure choisie parmi des Saccharomyces spp. ou des micro-organismes de la famille des Methanobacteriaceae et/ou - at least one bacterium making it possible to create an anaerobic environment favorable to the bacteria present in the composition, such as at least one bacterium chosen from bacteria of the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and / or at least one other organism promoting the anaerobic conditions necessary for the survival of Christensenellaceae, such as at least one yeast chosen from Saccharomyces spp. or microorganisms of the Methanobacteriaceae family and / or
- au moins une bactérie associée à l'écosystème des bactéries présentes dans la composition car elles facilitent leur survie dans l'intestin telle qu'au moins une bactérie choisie parmi les bactéries du phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, betaproteobacteria et Verrucomicrobia, et/ou - at least one bacterium associated with the ecosystem of bacteria present in the composition because they facilitate their survival in the intestine, such as at least one bacterium chosen from bacteria of the phylum Firmicutes, Bacteroidetes, Actinobacteria, Tenericutes, betaproteobacteria and Verrucomicrobia, and or
- au moins une bactérie choisie parmi les bactéries de l'ordre des Burkholderiales, Clostridales, des Verrucomicrobiales, des Aeromonadales, des Alteromonadales, ML615J-28, RF32, YS2, de la famille des Clostridiacées, des Lachnospiracées, des Erysipelotrichacées, des Ruminococcacées, des Bacteroidacées, des Enterococcacées, des Rikenéllacées, des Dehalobactériacées, des Veillonellacées, des Loctobocillocées et/ou - at least one bacterium chosen from bacteria of the order of Burkholderiales, Clostridales, Verrucomicrobiales, Aeromonadales, Alteromonadales, ML615J-28, RF32, YS2, of the Clostridiaceae, Lachnospiraceae, Erysipelotrichaceae, Ruminococcaceae family, Bacteroidaceae, Enterococcaceae, Rikenéllaceae, Dehalobacteriaceae, Veillonellaceae, Loctobocilloceae and / or
- au moins une bactérie choisie parmi les bactéries du genre Faecalibacterium, Akkermansia, Eubacterium, Sutterella, Burkholderia, Derxia, Brackiella, Oligella, Pelistega, Taylorella, Tetrathiobacter, Advenella, Alcicaligenes, Pigmentiphaga, Kerstersia, Achromobocter, Bordetella, Castellaniella, Pusillimonas, Turicibacter et Oscillospira telle que par exemple Faecalibacterium prausnitzii, Akkermansia muciniphila, Eubacterium halii, Oscillospira guilliermondii, Turicibacter sanguinis, Suterella parvirubra, Derxia gummosa, Brackiella oedipodis, Oligella urethralis, Pelistega europea, Taylorella eguigenitalis, Tetrathiobacter kashmirensis, Advenella incenata, Alcaligenes faecalis, Pigmentiphaga kullae, Kerstersia gyiorum, Achromobacter xylosoxidans, Bordetella pertussis, Castellaniella defragrans, Pusillimonas noertemannii et/ou - at least one bacterium chosen from bacteria of the genus Faecalibacterium, Akkermansia, Eubacterium, Sutterella, Burkholderia, Derxia, Brackiella, Oligella, Pelistega, Taylorella, Tetrathiobacter, Advenella, Alcicaligenes, Pigmentiphaga, Kerstersia, Achromoboctani, Bordetusimonella, Castellimonella, and Oscillospira, such as, for example, Faecalibacterium prausnitzii, Akkermansia muciniphila, Eubacterium halii, Oscillospira guilliermondii, Turicibacter sanguinis, Suterella parvirubra, Derxia gummosa, Brackiella oedipodis, Oligella urethrigen, Tetayaegauenalishauigenalis, tetayaegauenalisobacteria, Admiralishauigenalis, tayloraeushauenalis, Brackiella oedipodis, Oligella urethrigen, tetayaehauenalishauigenalis, taylorashauigenalis, taylorashau-genalis, eg, taylorhauenalis, eg, taylashigenalis, eg, taylorashauhau-genalis, eg, Brackiella oedipodis, Oligella urethrigen, tayloraegauenalis, eg, taylorashauhauenalis, egmiralis, Egmirella eg. , Kerstersia gyiorum, Achromobacter xylosoxidans, Bordetella pertussis, Castellaniella defragrans, Pusillimonas noertemannii and / or
- au moins un prébiotique tel que par exemple au moins un prébiotique choisi parmi les galactooligosaccharides, les fructooligosaccharides, les inulines, les arabinoxylans, les béta- glucanes, les lactoglobulines et/ou les béta-caséines, et/ou - au moins un polyphénol tel que par exemple au moins un polyphénol choisi parmi la quercetin, le kaempferol, le resvératrol, les flavones (comme la lutéoline), les flavan-3-ols (comme les catéchines), les flavanones (comme la narinénine), les isoflavones, les anthocyanidines, les proanthocyanidines, et/ou - at least one prebiotic such as for example at least one prebiotic chosen from galactooligosaccharides, fructooligosaccharides, inulins, arabinoxylans, beta-glucans, lactoglobulins and / or beta-caseins, and / or - at least one polyphenol such as for example at least one polyphenol chosen from quercetin, kaempferol, resveratrol, flavones (such as luteolin), flavan-3-ols (such as catechins), flavanones (such as narinenin) ), isoflavones, anthocyanidins, proanthocyanidins, and / or
- au moins un minéral et/ou au moins une vitamine et/ou au moins un agent nutritionnel, et/ou - at least one mineral and / or at least one vitamin and / or at least one nutritional agent, and / or
- au moins un principe actif pharmaceutique, préférentiellement au moins un principe actif pharmaceutique présentant un effet thérapeutique sur la ou les pathologies (maladies ayant un effet dégénératif sur les neurones ou cellules nerveuses musculaires et/ou cancer(s) en particulier) pour laquelle ou lesquelles les bactéries présentes dans la composition sont utilisées, tel que par exemple : - at least one pharmaceutical active principle, preferably at least one pharmaceutical active principle exhibiting a therapeutic effect on the pathology (s) (diseases having a degenerative effect on neurons or muscle nerve cells and / or cancer (s) in particular) for which or which the bacteria present in the composition are used, such as for example:
Les médicaments contre l'effet dégénératif des neurones et cellules nerveuses musculaires Donépézil, Rivastigmine, Galantamine, Mémantine, Riluzole, Nusinersen, des psychotropes, fluidifiants, neuroleptiques Medicines against the degenerative effect of neurons and muscle nerve cells Donepezil, Rivastigmine, Galantamine, Memantine, Riluzole, Nusinersen, psychotropic drugs, thinners, neuroleptics
Les médicaments de chimiothérapies dont les thérapies ciblées ou biothérapies, l'hormonothérapie ou l'immunothérapie Chemotherapy drugs including targeted therapies or biotherapies, hormone therapy or immunotherapy
L'invention est à présent illustrée par des exemples de bactéries utiles selon l'invention, de procédés de cultures de ces bactéries, des exemples de compositions les contenant et des résultats d'essais démontrant l'efficacité des bactéries de la famille des Christensenellacées et/ou de bactéries du genre Parasutterella et/ou de bactéries du genre Negativibacillus et/ou de bactéries du genre Massiliomicrobiota sur le 2-hydroxyglucarate et par conséquent sur les maladies induites par l'excès de cette molécule. The invention is now illustrated by examples of bacteria useful according to the invention, methods of culturing these bacteria, examples of compositions containing them and test results demonstrating the efficacy of bacteria of the Christensenellaceae family and / or bacteria of the genus Parasutterella and / or bacteria of the genus Negativibacillus and / or bacteria of the genus Massiliomicrobiota on 2-hydroxyglucarate and therefore on diseases induced by the excess of this molecule.
EXEMPLES EXAMPLES
Exemple 1 : Christensenella minuta. Example 1: Christensenella minuta.
Les bactéries Christensenella minuta peuvent-être cultivées selon le protocole opératoire décrit en suivant. The Christensenella minuta bacteria can be cultured according to the operating protocol described below.
1/ Dissoudre un milieu RCM ("Reinforced Clostridial Medium" milieu clostridiale renforcé) déshydraté dans de l'eau distillée 1 / Dissolve a dehydrated RCM ("Reinforced Clostridial Medium") medium in distilled water
2/ Ajouter 0,5 mL/L de solution de résazurine-Na (0,1% p/v) 2 / Add 0.5 mL / L of resazurin-Na solution (0.1% w / v)
3/Porter à ébullition et refroidir à température ambiante tout en injectant un mélange gazeux à 80% de N2 et à 20% de C02 4/Etaler le milieu sous la même atmosphère gazeuse dans des tubes de type Hungate anoxiques ou dans des flacons de sérum puis autoclaver 3 / Bring to the boil and cool to room temperature while injecting a gas mixture of 80% N 2 and 20% C0 2 4 / Spread the medium under the same gas atmosphere in anoxic Hungate-type tubes or in vials of serum then autoclave
5/Avant utilisation, ajoutez 1,0 g de carbonate de sodium par litre à partir d'une solution mère anoxique stérile préparée avec un mélange gazeux à 80% de ISh et à 20% de CO2 5 / Before use, add 1.0 g of sodium carbonate per liter from a sterile anoxic stock solution prepared with a gas mixture of 80% ISh and 20% CO2
6/ Vérifier le pH du milieu après autoclavage et ajuster le pH entre 7,3 et 7,5, en utilisant une solution mère anoxique stérile de bicarbonate de sodium (5% p / v) préparée dans une atmosphère gazeuse à 80% de N2 et à 20% de CO2. 6 / Check the pH of the medium after autoclaving and adjust the pH between 7.3 and 7.5, using a sterile anoxic stock solution of sodium bicarbonate (5% w / v) prepared in a gaseous atmosphere at 80% N2 and 20% CO2.
Exemple 2 : Christensenella massiliensis Example 2: Christensenella massiliensis
Les bactéries Christensenella massiliensis peuvent-être cultivées selon le protocole opératoire décrit en suivant. The Christensenella massiliensis bacteria can be cultured according to the operating protocol described below.
1/Préparer un milieu carboxyméthylcellulose (N2/ CO2) en suivant les instructions suivantes fournies par DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), présentées dans le Tableau 1. 1 / Prepare a carboxymethylcellulose (N2 / CO2) medium by following the following instructions provided by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), presented in Table 1.
[Tableau 1] [Table 1]
2/Dissoudre les différents constituants listés dans le tableau ci-dessus, sauf la cystéine, les carbohydrates et le carbonate. 2 / Dissolve the different constituents listed in the table above, except cysteine, carbohydrates and carbonate.
3/Faire bouillir le milieu pendant 1 min, puis le laisser refroidir à la température ambiante sous une atmosphère gazeuse à 80% de N2 et à 20% de CO2. 3 / Boil the medium for 1 min, then let it cool to ambient temperature under a gaseous atmosphere at 80% N2 and 20% CO2.
4/ Ajouter 0,5 g/L de L-cystéine-HCI x H2O et le verser sous la même atmosphère gazeuse dans des tubes de type Hungate (pour les souches exigeant des particules de viande, introduire celles-ci en premier dans le tube, utiliser 1 partie de particules de viande pour 4 ou 5 parties de liquide). 4 / Add 0.5 g / L of L-cysteine-HCl x H2O and pour it under the same gaseous atmosphere into Hungate type tubes (for strains requiring meat particles, introduce these first in the tube, use 1 part of meat particles for 4 or 5 parts of liquid).
5/ Autoclavage à 121°C pendant 20 min. 5 / Autoclaving at 121 ° C for 20 min.
6/ Après autoclavage, ajouter le glucose, le cellobiose, le maltose et l'amidon provenant de solutions mères anoxiques stériles préparées à 100% de gaz N2 et de carbonate à partir d'une solution mère anoxique stérile préparée sous des mélanges gazeux à 80% de N2 et 20% de C02. 6 / After autoclaving, add the glucose, cellobiose, maltose and starch from sterile anoxic stock solutions prepared with 100% N 2 gas and carbonate from a sterile anoxic stock solution prepared in gas mixtures at 80% of N 2 and 20% of C0 2 .
7/ Ajuster le pH du milieu à 7, si nécessaire. 7 / Adjust the pH of the medium to 7, if necessary.
La composition du filtrat de viande est présentée dans le Tableau 2. The composition of the meat filtrate is shown in Table 2.
[Tableau 2] [Table 2]
Le filtrat de viande est préparé comme suit. The meat filtrate is prepared as follows.
a/Utiliser du bœuf maigre ou de la viande de cheval. a / Use lean beef or horse meat.
b/Enlever la graisse et le tissu conjonctif avant de hacher. b / Remove fat and connective tissue before chopping.
c/ Mélanger la viande, l'eau et NaOH, puis faire bouillir pendant 15 min sous agitation. c / Mix the meat, water and NaOH, then boil for 15 min with stirring.
d/ Laisser refroidir à la température ambiante, retirer la graisse de la surface et filtrer, en retenant les particules de viande et le filtrat. d / Let cool to room temperature, remove the fat from the surface and filter, retaining the meat particles and the filtrate.
e/Ajouter au filtrat de l'eau jusqu'à un volume final de 1000,0 ml. e / Add water to the filtrate to a final volume of 1000.0 ml.
Les bactéries doivent être cultivées en condition anaérobie à 37°C. Bacteria should be grown in anaerobic condition at 37 ° C.
Exemple 3 : Christensenella timonensis Example 3: Christensenella timonensis
Les bactéries Christensenella timonensis peuvent-être cultivées selon le même protocole opératoire que celui décrit à l'exemple 2 pour Christensenella massiliensis. The Christensenella timonensis bacteria can be cultured according to the same operating protocol as that described in Example 2 for Christensenella massiliensis.
Exemple 4 : Parasutterella excrementihominis Example 4: Parasutterella excrementihominis
Les bactéries Parasutterella excrementihominis peuvent-être cultivées selon le protocole opératoire décrit en suivant. The Parasutterella excrementihominis bacteria can be cultured according to the operating protocol described below.
BTU = (Milieu viande hachée + Formiate/fumarate) + 5% sérum bovin BTU = (Minced meat medium + Formate / fumarate) + 5% bovine serum
Milieu viande hachée Minced meat medium
Bœuf haché (sans gras) (500.0g) + eau distillée (1000.0 ml) + NaOH IN (25.0 ml). Utiliser de la viande maigre de bœuf ou cheval. Enlever le gras et le tissu conjonctif avant d'hacher. Mélanger la viande, l'eau et le NaOH, puis faire bouillir pendant 15 min sous agitation. Faire refroidir à température ambiante, retirer le gras de la surface et filtrer en gardant les deux : les particules de viande et le filtrat. Ajouter de l'eau au filtrat pour un volume final de 1000.0 ml, et ajouter ensuite : casitone (30.0g), extrait de levure (5.0g), K2HPO4 (5.0 g), resazurine (l.Omg) Ground beef (fat free) (500.0g) + distilled water (1000.0 ml) + IN NaOH (25.0 ml). Use lean beef or horse meat. Remove fat and connective tissue before chopping. Mix the meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, remove the fat from the surface and filter, keeping both: the meat particles and the filtrate. Add water to the filtrate for a final volume of 1000.0 ml, and then add: casitone (30.0g), yeast extract (5.0g), K2HPO4 (5.0 g), resazurin (l.Omg)
Faire bouillir sous atmosphère Nitrogen, ajouter 0.5g/l de cystine et ajuster le pH à 7.0. Répartir sous anaérobie 7ml de milieu dans des tubes Hungate contenant les particules de viande (utiliser une part de particules de viande pour 4 à 5 parts de liquide). Autoclaver à 121°C pendant 30min. Boil under a nitrogen atmosphere, add 0.5g / l of cystine and adjust the pH to 7.0. Distribute 7ml of medium under anaerobic conditions in Hungate tubes containing the meat particles (use one part meat particles for 4 to 5 parts liquid). Autoclave at 121 ° C for 30min.
Pour la préparation de l'agar : utiliser des éprouvettes et y mettre 15g d'agar pour 1000.01 de milieu. For the preparation of the agar: use test tubes and put 15g of agar for 1000.01 of medium.
Dans certains cas, il est possible d'ajouter Haemin, Vitamine Kl ou vitamine K3 si besoin. Additionner 1000.0ml de milieu après autoclavage : solution Haemin (10.0ml) + Solution vitaline Kl ou K3 (0.2ml). In some cases, it is possible to add Haemin, Vitamin K1 or vitamin K3 if needed. Add 1000.0ml of medium after autoclaving: Haemin solution (10.0ml) + Vitaline Kl or K3 solution (0.2ml).
Solution Haemin : dissoudre 50mg d'Haemin dans 1 ml de 1 N NaOH, ajouter 100ml d'eau distillée et stériliser par filtration, stocker réfrigérée. Haemin solution: dissolve 50 mg of Haemin in 1 ml of 1 N NaOH, add 100 ml of distilled water and sterilize by filtration, store refrigerated.
Solution de vitamine Kl : dissoudre 0.1ml de vitamine Kl dans 20 ml d'éthanol à 95% et stériliser par filtration. Stocker réfrigérée dans une bouteille brune. Vitamin K1 solution: dissolve 0.1 ml of vitamin K1 in 20 ml of 95% ethanol and sterilize by filtration. Store refrigerated in a brown bottle.
Solution de vitamine K3 : Vitamin K3 solution:
Dissoudre 5 mg/ml de vitamine K3 dans 10 ml d'éthanol 95% et stériliser par filtration. Dissolve 5 mg / ml of vitamin K3 in 10 ml of 95% ethanol and sterilize by filtration.
Stocker réfrigérée dans une bouteille brune. Store refrigerated in a brown bottle.
Formiate/fumarate solution Formate / fumarate solution
Mélanger Na-formiate (6.0 g) + Na-fumarate (6.0g) + eau distillée (100.0 ml). Stériliser par filtration. Ajouter 30 microlitre par ml de milieu 78 avant inoculation. Mix Na-formate (6.0 g) + Na-fumarate (6.0g) + distilled water (100.0 ml). Sterilize by filtration. Add 30 microliter per ml of medium 78 before inoculation.
Exemple 5 : Parasutterella secunda, Example 5: Parasutterella secunda,
Les bactéries Parasutterella secunda peuvent-être cultivées selon le protocole opératoire décrit en suivant. The Parasutterella secunda bacteria can be cultured according to the operating protocol described below.
Milieu EG : EG medium:
- Ajouter 0,2g de L-cystine à 50mL de HCl à IN et mélanger vigoureusement. Puis ajouter : 2,4g de poudre Lab-Lemco, 10,0g de peptone protéose No3, 5,0g d'extrait de levure, 4,0g de Na2HPC>4, 1,5g de glucose, 0,5 g d'amidon soluble, 15,0g d'agar, de 0,5g de L-cystéine HCl H2O puis de l'eau distillée pour atteindre un volume de 950, OmL. - Add 0.2g of L-cystine to 50mL of IN HCl and mix vigorously. Then add: 2.4g of Lab-Lemco powder, 10.0g of peptone proteose No3, 5.0g of yeast extract, 4.0g of Na2HPC> 4, 1.5g of glucose, 0.5g of soluble starch, 15.0g of agar, 0.5g of L-cysteine HCl H2O then distilled water to reach a volume of 950, OmL .
- Ajuster le pH à 7, 6-7, 8. - Adjust the pH to 7, 6-7, 8.
- Autoclaver et laisser refroidir à 50°C, puis ajouter 50,0 mL de sang de cheval de façon aseptique. - Autoclave and cool to 50 ° C, then add 50.0 mL of horse blood aseptically.
-Mélanger vigoureusement et distribuer dans les contenants stériles adéquates (boîtes de pétri ou tubes stériles). -Mix vigorously and dispense in the appropriate sterile containers (petri dishes or sterile tubes).
Ou Milieu agar sang Columbia avec 5% de sang de cheval : Or Columbia blood agar medium with 5% horse blood:
- Préparer la base agar sang Columbia (Oxoid CM331) selon les directives, stériliser et refroidir à 45°C. - Prepare Columbia blood agar base (Oxoid CM331) according to instructions, sterilize and cool to 45 ° C.
- ajouter 50,0 mL de sang de cheval défibrinaté de façon aseptique. - add 50.0 mL of aseptically defibrinated horse blood.
- mélanger et disperser rapidement dans les contenants stériles adéquates (boîtes de pétri ou tubes stériles). - mix and disperse quickly in the appropriate sterile containers (petri dishes or sterile tubes).
Exemple 6 : Neqativibacillus massiliensis Example 6: Neqativibacillus massiliensis
Les bactéries Negativibacillus massiliensis peuvent-être cultivées selon le protocole opératoire décrit en suivant. The Negativibacillus massiliensis bacteria can be cultured according to the operating protocol described below.
Espèce obtenue par croissance sur milieu agar Columbia (bioMérieux, Marcy G Etoile, France) avec 5% de sang de mouton sous atmosphère anaérobique (anaeroGEN, Oxoid, Dardilly, France) après 14 jours d'enrichissement d'un échantillon frais de colon gauche placé dans une bouteille en culture dans du sang (Becton Dickinson, Pont de Claix, France) avec 5 mL de sang de mouton (bioMérieux) et 5 mL de 0.2pm de rumen filtré (Thermo Fisher Scientific, Villebon- sur-Yvette, France) à 37°C. Puis 5 jours d'incubation anaérobique sur milieu agar Columbia enrichi de 5% de sang de mouton Species obtained by growth on Columbia agar medium (bioMérieux, Marcy G Etoile, France) with 5% sheep blood under anaerobic atmosphere (anaeroGEN, Oxoid, Dardilly, France) after 14 days of enrichment of a fresh sample of left colon placed in a bottle cultured in blood (Becton Dickinson, Pont de Claix, France) with 5 mL of sheep blood (bioMérieux) and 5 mL of 0.2 μm of filtered rumen (Thermo Fisher Scientific, Villebon-sur-Yvette, France) ) at 37 ° C. Then 5 days of anaerobic incubation on Columbia agar medium enriched with 5% sheep blood
Exemple 7 : Massiliomicrobiota timonensis Example 7: Massiliomicrobiota timonensis
Massiliomicrobiota timonensis peut être cultivée selon le protocole opératoire décrit en suivant : Massiliomicrobiota timonensis can be cultivated according to the operating protocol described in the following:
- milieu agar Columbia bioMérieux, Marcy l'Etoile, France) enrichi à 5% de sang de mouton Columbia agar - Columbia agar medium bioMérieux, Marcy l'Etoile, France) enriched with 5% Columbia agar sheep blood
- à 37°C - atmosphère anaérobique générée par AnaeroGen (bioMérieux). - at 37 ° C - anaerobic atmosphere generated by AnaeroGen (bioMérieux).
- 72 heures d'incubation - 72 hours of incubation
Exemple 8 : Massiliomicrobiota escudieri Example 8: Massiliomicrobiota escudieri
Les bactéries Massiliomicrobiota escudieri peuvent-être cultivées selon le même protocole opératoire que celui décrit à l'exemple 7 pour Massiliomicrobiota timonensis. The Massiliomicrobiota escudieri bacteria can be cultured according to the same operating protocol as that described in Example 7 for Massiliomicrobiota timonensis.
Exemple 9 : Composition de Christensenella minuta utile selon l'invention sous forme liquide Example 9: Composition of Christensenella minuta useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Christensenella minuta 109 CFU/mL dans le milieu de culture RCM anaérobie décrit ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. An example of a composition useful according to the invention in liquid form is a composition comprising Christensenella minuta 10 9 CFU / mL in the anaerobic RCM culture medium described above, modified to contain no product of animal origin and enriched in 5% glycerol. .
La composition de l'exemple 9 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Christensenella minuta 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. The composition of Example 9 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella minuta 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 10 : Composition de Christensenella massiliensis utile selon l'invention sous forme liquide Example 10 Composition of Christensenella massiliensis useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Christensenella massiliensis 109 CFU/mL dans le milieu de culture carboxyméthylcellulose anaérobie décrit ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. An example of a composition useful according to the invention in liquid form is a composition comprising Christensenella massiliensis 10 9 CFU / mL in the anaerobic carboxymethylcellulose culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. .
La composition de l'exemple 10 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Christensenella massiliensis 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The composition of Example 10 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella massiliensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 11 : Composition de Christensenella timonensis utile selon l'invention sous forme liquide Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Christensenella timonensis 109 CFU/mL dans le milieu de culture carboxyméthylcellulose anaérobie décrit ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. La composition de l'exemple 11 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Christensenella timonensis 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. Example 11 Composition of Christensenella timonensis useful according to the invention in liquid form An example of a composition useful according to the invention in liquid form is a composition comprising Christensenella timonensis 10 9 CFU / mL in the anaerobic carboxymethylcellulose culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. . The composition of Example 11 is obtained from an RCB (“research cell bank”) prepared on the basis of Christensenella timonensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight bag. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. Exemple 12 : Composition de Parasutterella excrementihominis utile selon l'invention sous forme liquide The frozen composition must be warmed to room temperature until it returns to liquid form before use. Example 12: Composition of Parasutterella excrementihominis useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Parasutterella excrementihominis 109 CFU/mL dans le milieu de culture BTU anaérobie décrit ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. An example of a composition useful according to the invention in liquid form is a composition comprising Parasutterella excrementihominis 10 9 CFU / mL in the anaerobic BTU culture medium described above modified to contain no product of animal origin and enriched in 5% glycerol. .
La composition de l'exemple 12 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Parasutterella excrementihominis 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. The composition of Example 12 is obtained from an RCB (“research cell bank”) prepared on the basis of Parasutterella excrementihominis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight bag. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 13 : Composition de Parasutterella secundo utile selon l'invention sous forme liquide Example 13: Composition of Parasutterella secundo useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Parasutterella secundo 109 CFU/mL dans le milieu de culture EG ou bien Columbia anaérobie décrits ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. An example of a composition useful according to the invention in liquid form is a composition comprising Parasutterella secundo 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described above, modified to contain no product of animal origin and enriched in glycerol 5%.
La composition de l'exemple 13 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Parasutterella secundo 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The composition of Example 13 is obtained from an RCB (“research cell bank”) prepared on the basis of Parasutterella secundo 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 14 : Composition de Negativibacillus massiliensis utile selon l'invention sous forme liquide Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Negativibacillus massiliensis 109 CFU/mL dans le milieu de culture EG ou bien Columbia anaérobie décrits ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. Example 14 Composition of Negativibacillus massiliensis useful according to the invention in liquid form An example of a composition useful according to the invention in liquid form is a composition comprising Negativibacillus massiliensis 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described below. - top modified to contain no product of animal origin and enriched with 5% glycerol.
La composition de l'exemple 14 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Negativibacillus massiliensis 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. The composition of Example 14 is obtained from an RCB (“research cell bank”) prepared on the basis of Negativibacillus massiliensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 15 : Composition de Massiliomicrobiota timonensis utile selon l'invention sous forme liquide Example 15: Composition of Massiliomicrobiota timonensis useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Massiliomicrobiota timonensis 109 CFU/mL dans le milieu de culture EG ou bien Columbia anaérobie décrits ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. La composition de l'exemple 15 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Massiliomicrobiota timonensis 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. An example of a composition useful according to the invention in liquid form is a composition comprising Massiliomicrobiota timonensis 10 9 CFU / mL in the EG or anaerobic Columbia culture medium described above, modified to contain no product of animal origin and enriched with glycerol 5%. The composition of Example 15 is obtained from an RCB (“research cell bank”) prepared on the basis of Massiliomicrobiota timonensis 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. Exemple 16 : Composition de Massiliomicrobiota escudieri utile selon l'invention sous forme liquide The frozen composition must be warmed to room temperature until it returns to liquid form before use. Example 16: Composition of Massiliomicrobiota escudieri useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide est une composition comprenant Massiliomicrobiota escudieri 109 CFU/mL dans le milieu de culture EG ou bien Columbia anaérobie décrits ci-dessus modifié pour ne contenir aucun produit d'origine animale et enrichi en glycérol 5%. An example of a composition useful according to the invention in liquid form is a composition comprising Massiliomicrobiota escudieri 10 9 CFU / mL in the EG culture medium or else Columbia anaerobic described above modified to contain no product of animal origin and enriched with 5% glycerol.
La composition de l'exemple 16 est obtenue à partir d'une RCB («research cell bank » banque de cellules de recherche) préparée à base de Massiliomicrobiota escudieri 1010 CFU/ mL puis conservée congelée à -20°C dans un sachet hermétique à l'oxygène. The composition of Example 16 is obtained from an RCB (“research cell bank”) prepared on the basis of Massiliomicrobiota escudieri 10 10 CFU / mL then stored frozen at -20 ° C in an airtight sachet. with oxygen.
La composition congelée doit être réchauffée à température ambiante jusqu'à retrouver une forme liquide avant utilisation. The frozen composition must be warmed to room temperature until it returns to liquid form before use.
Exemple 17 : Composition d'au moins une bactérie utile selon l'invention sous forme liquide en mélange avec au moins une autre bactérie distincte utile selon l'invention sous forme liquide Example 17 Composition of at least one bacterium useful according to the invention in liquid form mixed with at least one other distinct bacterium useful according to the invention in liquid form
Un exemple de composition utile selon l'invention sous forme liquide peut être obtenu par mélange d'au moins une composition des exemples 9 à 16 avec au moins une composition distincte des exemples de 9 à 16. An example of a composition useful according to the invention in liquid form can be obtained by mixing at least one composition of Examples 9 to 16 with at least one composition distinct from Examples 9 to 16.
Exemple 18 : Composition utile selon l'invention sous forme solide Un exemple de composition utile selon l'invention sous forme lyophilisée peut être obtenu par lyophilisation de l'une des compositions des exemples 9 à 17 à l'état congelé. Example 18 Composition useful according to the invention in solid form An example of a composition useful according to the invention in lyophilized form can be obtained by lyophilization of one of the compositions of Examples 9 to 17 in the frozen state.
Démonstration in vitro de l'effet de traitement des maladies caractérisées par l'excès de 2- hydroxyglucarate, en particulier les maladies neurodégénératives et cancers In vitro demonstration of the treatment effect of diseases characterized by excess 2-hydroxyglucarate, in particular neurodegenerative diseases and cancers
L'objectif de cette étude est de démontrer in vitro l'effet de traitement des maladies neurodégénératives et des cancers de bactéries selon l'invention. La démonstration a été réalisée sur le 2-hydroxyglutarate, et son précurseur, le glutarate. The objective of this study is to demonstrate in vitro the effect of treating neurodegenerative diseases and bacterial cancers according to the invention. The demonstration was carried out on 2-hydroxyglutarate, and its precursor, glutarate.
Le protocole opératoire de l'étude est décrit en suivant. The operating protocol of the study is described below.
1/ Protocole de fermentation à partir de fèces d'origine humaine contenant du Christensenella spp., du Parasutterella spp., du Negativibacillus spp., du Massiliomicrobiota spp. : 1 / Fermentation protocol from faeces of human origin containing Christensenella spp., Parasutterella spp., Negativibacillus spp., Massiliomicrobiota spp. :
- Les donneurs ne devaient pas avoir pris d'antibiotiques durant les six mois précédents l'expérience et n'avoir aucun historique de désordres gastro-intestinaux. Les donneurs étaient âgés entre 18 et 60 ans. - La collecte des échantillons frais de leur fèces est obtenue dans des contenants stériles en plastique, conservés dans des flacons anaérobies contenant un sachet de 2,51 d'AnaeroGenTM d'OxoidTM (O2 <0.1%; CO2: 7-15%). Ces échantillons ont été apportés au laboratoire dans les deux heures après leur production. - Les échantillons de fèces ont été dilués au 1/5 (poids/volumes) dans une solution saline tamponnée au phosphate (IM) (PBS), pH 7,4. La suspension a été homogénéisée dans un stomacher pendant 120 secondes. - Donors must not have taken antibiotics during the six months preceding the experiment and have no history of gastrointestinal disorders. The donors were between 18 and 60 years old. - Collection of fresh samples of their faeces is obtained in sterile plastic containers, kept in anaerobic vials containing one sachet of 2.51 of AnaeroGenTM from OxoidTM (O2 <0.1%; CO2: 7-15%). These samples were brought to the laboratory within two hours of production. - Faeces samples were diluted 1/5 (w / v) in phosphate buffered saline (IM) (PBS), pH 7.4. The suspension was homogenized in a stomacher for 120 seconds.
- Milieu nutritif de base: le milieu nutritif de base a été préparé à partir de 2g/L de bouillon de tryptone de soja, 2g/L d'extrait de levure, 0,lg/L de NaCI, 0,04g/L de K2HPO4, 0,01g/L de MgS03.7H20, 0,01g/L de CaCI2.6H20, 2g/L NaHCOs, 0,5g/L de L-cystine HCl, 2m L/L de tween- Basic nutrient medium: the basic nutrient medium was prepared from 2g / L of soybean tryptone broth, 2g / L of yeast extract, 0.1g / L of NaCl, 0.04g / L of K2HPO4, 0.01g / L of MgS0 3 .7H 2 0, 0.01g / L of CaCl 2 .6H 2 0, 2g / L NaHCOs, 0.5g / L of L-cystine HCl, 2m L / L of tween
80, 10pL/L de vitamine Kl, 0,05g/L d'hème, 0,05g/L de sels biliaires, 4ml/L de résazarin (pH7) 80, 10pL / L of vitamin Kl, 0.05g / L of heme, 0.05g / L of bile salts, 4ml / L of resazarin (pH7)
- Fermentation en biofermenteur : Les biofermenteurs de 20mL de contenance contenaient 18mL de milieu nutritif de base autoclavé (121°C pendant 15 minutes) et versé aseptiquement dans les biofermenteurs stériles. Ce système a été laissé au repos toute la nuit avec un bullage d'azote sans oxygène à travers le milieu à un taux de 2mL/min. Le pH était maintenu entre 6,7 et 6,9 en utilisant du HCl ou NaOH (0,5M). La température de chaque biofermenteur était contrôlée à 37°C et le contenu du récipient homogénéisé avec un mélangeur magnétique - Fermentation in a biofermenter: The 20mL biofermenters contained 18mL of basic nutrient medium autoclaved (121 ° C for 15 minutes) and poured aseptically into sterile biofermenters. This system was left to stand overnight with oxygen-free nitrogen bubbling through the medium at a rate of 2 mL / min. The pH was maintained between 6.7 and 6.9 using HCl or NaOH (0.5M). The temperature of each biofermenter was controlled at 37 ° C and the contents of the vessel homogenized with a magnetic mixer
- un mélange de protéines prédigérées (0,35g) a été ajouté dans les récipients avant l'inoculation avec 2mL d'inocula fécal à T0. Les protéines prédigérées ont été obtenues selon le protocole de digestion gastro-intestinale adapté de celui de Versantvoort et al (2005). - a mixture of predigested proteins (0.35g) was added to the containers before inoculation with 2mL of fecal inocula at T0. The predigested proteins were obtained according to the gastrointestinal digestion protocol adapted from that of Versantvoort et al (2005).
- les échantillons ont été collectés avant la fermentation (T0) et après 48 heures de fermentation (T48), et congelés à -80°C jusqu'aux analyses. - the samples were collected before fermentation (T0) and after 48 hours of fermentation (T48), and frozen at -80 ° C until analyzes.
2/ Quantification des 2-hydroxyglutarate et glutarate. 50 pL d'échantillons collectés et conservés à -80°C ont été mélangés avec 20 pL d'eau Milli-Q contenant des standards internes. 2 / Quantification of 2-hydroxyglutarate and glutarate. 50 µL of samples collected and stored at -80 ° C were mixed with 20 µL of Milli-Q water containing internal standards.
- Le mélange a été mélangé et filtré à travers un filtre de seuil 5-kDa pour retirer les macromolécules. - Les métabolites ont été détectés par analyses en électrophorèse capillaire-spectrométrie de masse à temps de vol (CE-TOFMS). La limite de détection des pics a été déterminée sur la base du ratio signal/bruit, S/N=3. - The mixture was mixed and filtered through a 5-kDa cutoff filter to remove macromolecules. - The metabolites were detected by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) analyzes. The peak detection limit was determined based on the signal to noise ratio, S / N = 3.
Aire relative du pic = (aire du pic d'un métabolite)/(aire du pic du standard interne x quantité d'échantillon). Relative peak area = (peak area of a metabolite) / (peak area of internal standard x amount of sample).
3/ Quantification de Christensenella spp., de Parasutterella spp., de Negativibacillus spp., de Massiliomicrobiota spp. 3 / Quantification of Christensenella spp., Parasutterella spp., Negativibacillus spp., Massiliomicrobiota spp.
- L'ADN contenu dans les échantillons a été extrait en utilisant le kit NucleoSpin®96 Soil de Macherey-Nagel selon les instructions du fabricant. - L'ADN extrait total a ensuite été fragmenté aléatoirement en fragments de 350 bp puis utilisé pour construire une librairie en utilisant le kit NEBNext Ultra II par New England Biolabs selon les instructions du fabricant. - The DNA contained in the samples was extracted using the NucleoSpin ® 96 Soil kit from Macherey-Nagel according to the manufacturer's instructions. - The total extracted DNA was then fragmented randomly into fragments of 350 bp and then used to construct a library using the NEBNext Ultra II kit by New England Biolabs according to the manufacturer's instructions.
- La librairie a ensuite été séquencée en utilisant du séquençage paired-end de 2 x 150 bp sur une plateforme Illumina HiSeq. - L'abondance des bactéries a été mesurée en créant un catalogue d'espèces métagénomiques (MGS) à partir d'un catalogue de référence contenant 22M de gènes. Ces MGS ont ensuite été associées à un niveau taxonomique adapté. Dans le cas des Christensenella, celles-ci ont été détectées au niveau du genre et sont donc référencées dans cette expérience par Christensenella spp. La quantité relative de 2-hydroxyglutarate et l'abondance relative de Christensenella spp., de Parasutterella spp., de Negativibacillus spp., de Massiliomicrobiota spp ont été analysées et corrélées, obtenant une régression linéaire de R=-0,42, R=-0,41, R=-0,31, R=-0,26 (n=18). - The library was then sequenced using 2 x 150 bp paired-end sequencing on an Illumina HiSeq platform. - The abundance of bacteria was measured by creating a catalog of metagenomic species (MGS) from a reference catalog containing 22M genes. These AMSs were then associated to an appropriate taxonomic level. In the case of Christensenella, these were detected at the genus level and are therefore referenced in this experiment by Christensenella spp. The relative amount of 2-hydroxyglutarate and the relative abundance of Christensenella spp., Parasutterella spp., Negativibacillus spp., Massiliomicrobiota spp were analyzed and correlated, obtaining a linear regression of R = -0.42, R = -0.41, R = -0.31, R = -0.26 (n = 18).
La quantité relative de glutarate et l'abondance relative de Christensenella spp., de Parasutterella spp., de Negativibacillus spp., de Massiliomicrobiota spp ont été analysées et corrélées, obtenant une régression linéaire de R=0,41, R=0,43, R=0,51, R=0,36 (n=18). The relative amount of glutarate and the relative abundance of Christensenella spp., Parasutterella spp., Negativibacillus spp., Massiliomicrobiota spp were analyzed and correlated, obtaining a linear regression of R = 0.41, R = 0.43 , R = 0.51, R = 0.36 (n = 18).
Les résultats sont présentés dans le Tableau 3. The results are shown in Table 3.
[Tableau B] [Table B]
On constate une corrélation négative entre les bactéries selon l'invention et le 2- hydroxyglutarate ainsi qu'une corrélation positive entre les bactéries selon l'invention et le glutarate, ce qui démontre un effet protecteur des bactéries du genre Massiliomicrobiota contre les maladies neurodégénératives et les cancers. There is a negative correlation between the bacteria according to the invention and 2-hydroxyglutarate as well as a positive correlation between the bacteria according to the invention and glutarate, which demonstrates a protective effect of bacteria of the genus Massiliomicrobiota against neurodegenerative diseases and cancers.
Ainsi les bactéries selon l'invention sont capables d'agir en diminuant la production de 2- hydroxyglutarate. Elles peuvent donc être utilisées pour prévenir et/ou traiter les maladies neurodégénératives et les cancers. Thus the bacteria according to the invention are able to act by reducing the production of 2-hydroxyglutarate. They can therefore be used to prevent and / or treat neurodegenerative diseases and cancers.
Démonstration in vitro de l'effet antiprolifératif sur des cellules tumorales, en particulier des cellules HCT-116 (adénocarcinome du colon). L'objectif de cette étude est de démontrer in vitro l'effet de bactéries selon l'invention, en particulier de C. minuta sur la prolifération de cellules tumorales. L'effet des surnageants a été évalué après 24h et 48h de traitement d'une lignée cellulaire humaine d'adénocarcinome du côlon, la lignée HCT-116. Le protocole opératoire de l'étude est décrit ci-après. In vitro demonstration of the antiproliferative effect on tumor cells, in particular HCT-116 cells (colon adenocarcinoma). The objective of this study is to demonstrate in vitro the effect of bacteria according to the invention, in particular of C. minuta on the proliferation of tumor cells. The effect of the supernatants has was evaluated after 24 hours and 48 hours of treatment of a human colon adenocarcinoma cell line, the HCT-116 line. The operating protocol for the study is described below.
Culture cellulaire Cellular culture
La lignée cellulaire HCT-116 a été maintenue en culture dans du milieu McCoy's 5A supplémenté avec 10% de sérum de veau fêtai (Gibco) et 1% de pénicilline/stretomycine (Sigma-Aldrich) dans un incubateur en atmosphère humide à 37°C et 5% C02. The HCT-116 cell line was maintained in culture in McCoy's 5A medium supplemented with 10% feta calf serum (Gibco) and 1% penicillin / stretomycin (Sigma-Aldrich) in a humid atmosphere incubator at 37 ° C. and 5% CO2.
Traitement cellulaire Cellular treatment
Les cellules HCT-116 ont été ensemencées à une densité de 10000 cellules/puits dans un volume total de 100 pl en plaque 96-puits. Après 24h d'incubation, le milieu de culture a été retiré des cellules adhérentes et du nouveau milieu supplémenté avec 10% de surnageant en phase stationnaire de C. minuta a été ajouté (DSMZ: DSM22607, C. minuta 1, C. minuta 2 ou C. minuta 3 et en contrôle GAM (milieu de culture bactérien)). Chaque condition a été faite en 4 réplicats. Les cellules ont été incubées pendant 24h ou 48h supplémentaires. The HCT-116 cells were seeded at a density of 10,000 cells / well in a total volume of 100 μl in a 96-well plate. After 24 h of incubation, the culture medium was removed from the adherent cells and new medium supplemented with 10% of C. minuta stationary phase supernatant was added (DSMZ: DSM22607, C. minuta 1, C. minuta 2 or C. minuta 3 and in GAM control (bacterial culture medium)). Each condition was made in 4 replicates. The cells were incubated for an additional 24 or 48 hours.
Test de prolifération La prolifération cellulaire a été déterminée par l'utilisation du kit CelITiter-GIo 2.0 assay (Promega). Les mesures ont été réalisées selon les instructions du fabricant. Brièvement, les plaques ont été retirées de l'incubateur et laissées s'équilibrer à température ambiante pendant 30 min et un volume égal de réactif CelITiter-GIo 2.0 a été ajouté directement aux puits (100 mI). Les plaques ont été mises sous agitation à 300 tr/min pendant 2 min en utilisant un agitateur rotatif, puis incubées à température ambiante pendant 10 min. Le mélange réactionnel a ensuite été transféré dans une plaque à 96 puits à paroi blanche et le signal luminescent a été mesuré à l'aide d'un lecteur de microplaques (FLUOstar Oméga, BMG Labtech). Proliferation assay Cell proliferation was determined using the CelITiter-GIo 2.0 assay kit (Promega). The measurements were carried out according to the manufacturer's instructions. Briefly, the plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min and an equal volume of CelITiter-GIo 2.0 reagent was added directly to the wells (100mI). The plates were stirred at 300 rpm for 2 min using a rotary shaker, then incubated at room temperature for 10 min. The reaction mixture was then transferred to a white-walled 96-well plate and the luminescent signal was measured using a microplate reader (FLUOstar Omega, BMG Labtech).
Résultats Les surnageants des différentes souches bactériennes de l'espèce C. minuta DSMZ: DSM22607, C. minuta 1, C. minuta 2 et C. minuta 3 réduisent significativement la prolifération des cellules traitées pendant 24h et 48h, par rapport au contrôle "GAM", correspondant aux cellules traitées avec le milieu de culture bactérien (GAM vs DSMZ: DSM22607, C. minuta 2, C. minuta 2 p<0,0001, GAM vs C. minuta 1 p=0.0002 à 24h et GAM vs DSMZ: DSM22607 p=0.0002, GAM vs C. minuta 1 p=0.0016, GAM vs C. minuta 2 et C. minuta 3 p<0.0001 à 48h, Dunnett's multiple comparisons test). L'inhibiteur VIII d'Akt est utilisé comme contrôle positif du blocage de la prolifération cellulaire. Results The supernatants of the different bacterial strains of the species C. minuta DSMZ: DSM22607, C. minuta 1, C. minuta 2 and C. minuta 3 significantly reduce the proliferation of cells treated for 24 hours and 48 hours, compared to the "GAM control"", corresponding to cells treated with the bacterial culture medium (GAM vs DSMZ: DSM22607, C. minuta 2, C. minuta 2 p <0.0001, GAM vs C. minuta 1 p = 0.0002 at 24 h and GAM vs DSMZ: DSM22607 p = 0.0002, GAM vs C. minuta 1 p = 0.0016, GAM vs C. minuta 2 and C. minuta 3 p <0.0001 at 48h, Dunnett's multiple comparisons test). Akt inhibitor VIII is used as a positive control for blocking cell proliferation.
Ainsi, les surnageants des différentes bactéries selon l'invention testés induisent une diminution de la prolifération de la lignée tumorale HCT-116, permettant d'indiquer un effet bénéfique des bactéries selon l'invention dans l'inhibition du développement tumoral. Thus, the supernatants of the various bacteria according to the invention tested induce a decrease in the proliferation of the tumor line HCT-116, making it possible to indicate a beneficial effect of the bacteria according to the invention in inhibiting tumor development.
Démonstration in vitro de l'effet antiprolifératif sur des cellules tumorales, en particulier des cellules HepG2 (hépatocarcinome). In vitro demonstration of the antiproliferative effect on tumor cells, in particular HepG2 cells (hepatocarcinoma).
L'objectif de cette étude est de démontrer in vitro l'effet de bactéries selon l'invention, en particulier de C. minuta sur la prolifération de cellules tumorales. L'effet des surnageants a été évalué après 24h et 48h de traitement d'une lignée cellulaire humaine d'adénocarcinome, la lignée HepG2. Le protocole opératoire de l'étude est décrit ci-après. The objective of this study is to demonstrate in vitro the effect of bacteria according to the invention, in particular of C. minuta on the proliferation of tumor cells. The effect of the supernatants was evaluated after 24 hours and 48 hours of treatment of a human adenocarcinoma cell line, the HepG2 line. The operating protocol for the study is described below.
Culture cellulaire Cellular culture
La lignée cellulaire HepG2 a été maintenue en culture dans du milieu DMEM supplémenté avec 10% de sérum de veau fêtai (Gibco) et 1% de pénicilline/stretomycine (Sigma-Aldrich) dans un incubateur en atmosphère humide à 37°C et 5% C02. The HepG2 cell line was maintained in culture in DMEM medium supplemented with 10% feta calf serum (Gibco) and 1% penicillin / stretomycin (Sigma-Aldrich) in a humid atmosphere incubator at 37 ° C and 5%. C02.
Le traitement cellulaire et le test de prolifération est identique à l'essai précédemment décrit sur la lignée HCT-116. The cell treatment and the proliferation test is identical to the test previously described on the HCT-116 line.
Résultats Les surnageants des bactéries selon l'invention réduisent significativement la prolifération des cellules traitées à 24h et l'ensemble des souches de C. minuta DSMZ: DSM22607, C. minuta 1, C. minuta 2 et C. minuta 3 à 48h, par rapport au contrôle "GAM", correspondant aux cellules traitées avec le milieu de culture bactérien (GAM vs DSMZ: DSM22607 or C. minuta 3, p =0.0003 et GAM vs C. minuta 2 p=0.0008 à 24h / GAM vs DSMZ: DSM22607, C. minuta 2, C. minuta 3 p<0,0001 et GAM vs C. minuta 1 p=0.0024 à 48h, Dunnett's multiple comparisons test). L'inhibiteur VIII d'Akt est utilisé comme contrôle du blocage de la prolifération cellulaire. Ainsi, les surnageant des bactéries selon l'invention testés induisent une diminution de la prolifération de la lignée tumorale HepG2, permettant d'indiquer un effet bénéfique des bactéries selon l'invention dans l'inhibition du développement tumoral. Results The supernatants of the bacteria according to the invention significantly reduce the proliferation of cells treated at 24 h and all of the C. minuta DSMZ strains: DSM22607, C. minuta 1, C. minuta 2 and C. minuta 3 at 48 h, by compared to the "GAM" control, corresponding to cells treated with the bacterial culture medium (GAM vs DSMZ: DSM22607 or C. minuta 3, p = 0.0003 and GAM vs C. minuta 2 p = 0.0008 at 24h / GAM vs DSMZ: DSM22607 , C. minuta 2, C. minuta 3 p <0.0001 and GAM vs C. minuta 1 p = 0.0024 at 48h, Dunnett's multiple comparisons test). Akt inhibitor VIII is used as a control for blocking cell proliferation. Thus, the supernatants of the bacteria according to the invention tested induce a decrease in the proliferation of the tumor line HepG2, making it possible to indicate a beneficial effect of the bacteria according to the invention in inhibiting tumor development.

Claims

REVENDICATIONS
[Revendication 1] [Bactérie du microbiote intestinal humain choisie parmi les bactéries de la famille des Christensenellacées, les bactéries du genre Negativibacillus et les bactéries du genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement d'au moins une maladie caractérisée par l'excès de 2-hydroxyglutarate chez l'être humain ou l'animal. [Claim 1] [Bacteria of the human intestinal microbiota selected from bacteria of the family Christensenellaceae, bacteria of the genus Negativibacillus and bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment of at least one characterized disease by excess of 2-hydroxyglutarate in humans or animals.
[Revendication 2] Bactérie pour son utilisation selon la revendication 1, dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative et/ou d'au moins un cancer chez l'être humain ou l'animal. [Claim 2] A bacterium for its use according to claim 1, in the prevention and / or treatment of at least one neurodegenerative disease and / or at least one cancer in humans or animals.
[Revendication 3] Bactérie pour son utilisation selon la précédente revendication, dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative choisie parmi la maladie d'Alzheimer, les démences, la maladie de Parkinson et les troubles associés, les maladies à prions, les maladies neuromusculaires, la maladie de Huntington, l'ataxie spinocérébelleuse, l'amyotrophie spinale progressive, la sclérose latérale amyotrophique. [Claim 3] Bacterium for its use according to the preceding claim, in the prevention and / or treatment of at least one neurodegenerative disease selected from Alzheimer's disease, dementias, Parkinson's disease and associated disorders, diseases with prions, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, amyotrophic lateral sclerosis.
[Revendication 4] Bactérie pour son utilisation selon l'une des revendications 2 ou 3, dans la prévention et/ou le traitement d'au moins un cancer du cerveau et/ou des reins et/ou du foie et/ou du côlon, chez l'être humain ou l'animal. [Claim 4] Bacterium for its use according to one of claims 2 or 3, in the prevention and / or treatment of at least one cancer of the brain and / or of the kidneys and / or of the liver and / or of the colon, in humans or animals.
[Revendication 5] Bactérie pour son utilisation selon l'une des précédentes revendications, caractérisée en ce que ladite bactérie est une bactérie du genre [Claim 5] Bacteria for its use according to one of the preceding claims, characterized in that said bacterium is a bacterium of the genus
Christensenella. Christensenella.
[Revendication 6] Bactérie pour son utilisation selon l'une des précédentes revendications, caractérisée en ce que ladite bactérie est choisie parmi Christensenella massiliensis, Christensenella timonensis et Christensenella minuta, Negativibacillus massiliensis, et Massiliomicrobiota timonensis. [Claim 6] Bacteria for its use according to one of the preceding claims, characterized in that said bacterium is chosen from Christensenella massiliensis, Christensenella timonensis and Christensenella minuta, Negativibacillus massiliensis, and Massiliomicrobiota timonensis.
[Revendication 7] Composition comprenant au moins une bactérie du microbiote intestinal humain choisie parmi les bactéries de la famille des Christensenellacées, les bactéries du genre Negativibacillus et les bactéries genre Massiliomicrobiota, pour son utilisation dans la prévention et/ou le traitement de maladies caractérisées par l'excès de 2- hydroxyglutarate chez l'être humain ou l'animal. [Claim 7] A composition comprising at least one bacterium of the human intestinal microbiota chosen from bacteria of the family Christensenellaceae, bacteria of the genus Negativibacillus and bacteria of the genus Massiliomicrobiota, for its use in the prevention and / or treatment of diseases characterized by excess of 2-hydroxyglutarate in humans or animals.
[Revendication s] Composition pour son utilisation selon la revendication 7, dans la prévention et/ou le traitement de maladies neurodégénératives et/ou d'au moins un cancer chez l'être humain ou l'animal. [Claim s] A composition for its use according to claim 7, in the prevention and / or treatment of neurodegenerative diseases and / or at least one cancer in humans or animals.
[Revendication 9] Composition pour son utilisation selon la précédente revendication, dans la prévention et/ou le traitement d'au moins une maladie neurodégénérative choisie parmi la maladie d'Alzheimer, les démences, la maladie de Parkinson et les troubles associés, les maladies à prions, les maladies neuromusculaires, la maladie de Huntington, l'ataxie spinocérébelleuse, l'amyotrophie spinale progressive, la sclérose latérale amyotrophique. [Claim 9] A composition for its use according to the preceding claim, in the prevention and / or treatment of at least one neurodegenerative disease chosen from Alzheimer's disease, dementias, Parkinson's disease and associated disorders, diseases with prions, neuromuscular diseases, Huntington's disease, spinocerebellar ataxia, progressive spinal muscular atrophy, amyotrophic lateral sclerosis.
[Revendication 10] Composition pour son utilisation selon l'une des revendications 8 ou 9, dans la prévention et/ou le traitement d'au moins un cancer du cerveau et/ou des reins et/ou du foie et/ou du côlon, chez l'être humain ou l'animal. [Claim 10] Composition for its use according to one of claims 8 or 9, in the prevention and / or treatment of at least one cancer of the brain and / or of the kidneys and / or of the liver and / or of the colon, in humans or animals.
[Revendication 11] Composition pour son utilisation selon l'une des revendications 7 à 10, caractérisée en ce qu'elle comprend au moins une bactérie du genre Christensenella. [Claim 11] Composition for its use according to one of claims 7 to 10, characterized in that it comprises at least one bacterium of the genus Christensenella.
[Revendication 12] Composition pour son utilisation selon l'une des revendications 7 à 10, caractérisée en ce qu'elle comprend au moins une bactérie choisie parmi Christensenella massiliensis, Christensenella timonensis et Christensenella minuta, Negativibacillus massiliensis et Massiliomicrobiota timonensis, ou un mélange d'au moins deux de ces bactéries. [Claim 12] Composition for its use according to one of claims 7 to 10, characterized in that it comprises at least one bacterium chosen from Christensenella massiliensis, Christensenella timonensis and Christensenella minuta, Negativibacillus massiliensis and Massiliomicrobiota timonensis, or a mixture of 'at least two of these bacteria.
[Revendication 13] Composition pour son utilisation selon la revendication 7 à 11, caractérisée en ce qu'elle se présente sous forme liquide. [Claim 13] A composition for its use according to claim 7 to 11, characterized in that it is provided in liquid form.
[Revendication 14] Composition pour son utilisation selon la revendication 7 à 11, caractérisée en ce qu'elle se présente sous forme solide. [Claim 14] Composition for its use according to claim 7 to 11, characterized in that it is provided in solid form.
[Revendication 15] Composition pour son utilisation selon la revendication 14, caractérisée en ce que les bactéries sont présentes sous forme lyophilisée. [Claim 15] A composition for its use according to claim 14, characterized in that the bacteria are present in lyophilized form.
[Revendication 16] Composition pour son utilisation selon l'une des revendications 7 à 15, caractérisée en ce que les bactéries présentes sont pour au moins 50% des bactéries vivantes (en nombre). [Claim 16] Composition for its use according to one of claims 7 to 15, characterized in that at least 50% of the bacteria present are living bacteria (by number).
[Revendication 17] Composition pour son utilisation selon l'une des revendications 7 à 16, caractérisée en ce que les bactéries présentes sont pour au moins 90% des bactéries vivantes (en nombre). [Claim 17] Composition for its use according to one of claims 7 to 16, characterized in that at least 90% of the bacteria present are living bacteria (by number).
[Revendication 18] Composition pour son utilisation selon l'une des revendications 7 à 17, par voie orale, rectale, inhalée. [Claim 18] A composition for its use according to one of claims 7 to 17, orally, rectally, inhaled.
[Revendication 19] Composition pour son utilisation selon l'une des revendications 7 à 18, caractérisée en ce qu'elle se présente sous forme de poudre, de poudre microencapsulée, de gélule, de capsule, de comprimé, de pastille, de granulés, d'émulsion, de suspension ou de suppositoire. [Claim 19] Composition for its use according to one of claims 7 to 18, characterized in that it is in the form of powder, microencapsulated powder, capsule, capsule, tablet, lozenge, granules, emulsion, suspension or suppository.
[Revendication 20] Composition pour son utilisation selon l'une des revendications 7 à 19, caractérisée en ce qu'elle se présente sous une forme gastro-résistante. [Claim 20] Composition for its use according to one of claims 7 to 19, characterized in that it is in a gastro-resistant form.
[Revendication 21] Composition pour son utilisation selon l'une des revendications 7 à 20, caractérisée en ce qu'elle comprend au moins un probiotique et/ou au moins un prébiotique. [Revendication 22] Composition pour son utilisation selon l'une des revendications 7 à 21, caractérisée en ce qu'elle comprend également : [Claim 21] Composition for its use according to one of claims 7 to 20, characterized in that it comprises at least one probiotic and / or at least one prebiotic. [Claim 22] Composition for its use according to one of claims 7 to 21, characterized in that it also comprises:
- au moins un polyphénol, et/ou - at least one polyphenol, and / or
- au moins un minéral et/ou au moins une vitamine et/ou au moins un agent nutritionnel, et/ou - at least one mineral and / or at least one vitamin and / or at least one nutritional agent, and / or
- au moins un autre principe actif pharmaceutique. - at least one other active pharmaceutical principle.
EP20739276.2A 2019-06-25 2020-06-24 Intestinal microbiota bacteria and composition containing same for use in the prevention and/or treatment of diseases characterised by excess 2-hydroxyglutarate Pending EP3989989A1 (en)

Applications Claiming Priority (2)

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FR1906845A FR3097739B1 (en) 2019-06-25 2019-06-25 Bacteria of the intestinal microbiota and composition containing them for use in the prevention and/or treatment of diseases characterized by excess 2-hydroxyglutarate
PCT/EP2020/067637 WO2020260358A1 (en) 2019-06-25 2020-06-24 Intestinal microbiota bacteria and composition containing same for use in the prevention and/or treatment of diseases characterised by excess 2-hydroxyglutarate

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EP3989989A1 true EP3989989A1 (en) 2022-05-04

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EP (1) EP3989989A1 (en)
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020182916A1 (en) * 2019-03-11 2020-09-17 Lnc Therapeutics Bacterium of the christensenellaceae family for the prevention and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancers

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3541400A2 (en) * 2016-11-18 2019-09-25 Sanford Burnham Prebys Medical Discovery Institute Gut microbiota and treatment of cancer
FR3063646B1 (en) * 2017-03-10 2021-07-30 International Nutrition Res Company BACTERIA SPECIFIC FOR THEIR USE AS A MEDICINAL PRODUCT, IN PARTICULAR FOR THE CONTROL OF OVERWEIGHT, OBESITY, CARDIOMETABOLIC DISEASES AND INFLAMMATORY DISEASES OF THE INTESTINE

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020182916A1 (en) * 2019-03-11 2020-09-17 Lnc Therapeutics Bacterium of the christensenellaceae family for the prevention and/or treatment of chronic inflammatory diseases and/or inflammatory gastrointestinal diseases and/or cancers

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FR3097739B1 (en) 2022-03-04
FR3097739A1 (en) 2021-01-01
US20230330158A1 (en) 2023-10-19

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