WO2023012540A1 - Protéine ou ses compositions pour une utilisation dans le traitement du microbiote - Google Patents

Protéine ou ses compositions pour une utilisation dans le traitement du microbiote Download PDF

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WO2023012540A1
WO2023012540A1 PCT/IB2022/055889 IB2022055889W WO2023012540A1 WO 2023012540 A1 WO2023012540 A1 WO 2023012540A1 IB 2022055889 W IB2022055889 W IB 2022055889W WO 2023012540 A1 WO2023012540 A1 WO 2023012540A1
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protein
diseases
microbiota
family
intestinal
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Vincenzo ROMANO SPICA
Fabrizio Michetti
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Genes S.R.L.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1738—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4727—Calcium binding proteins, e.g. calmodulin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • the presentinvention relatesto a protein belongingtothe S100 family for use in the treatment or amelioration of diseases such as ulcerative colitis,Crohn's disease,IBD (Inflammatory BowelDisease), neurological disorders,cardiovascular diseases,oncological diseases, kidney diseases,inflammatory diseases or diseaseslinked to immune system disorderssuch asallergies,rheumatoidarthritisorautoimmune diseases, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal and/or oral and/or pharyngeal and/or pulmonary and/or cutaneous and/or genital microbiota,whetherin humansoranimals; a composition comprising a protein belonging to the S100 family for use in the treatment or amelioration ofdiseasessuch asulcerativecolitis,Crohn'sdisease,IBD, neurological disorders,cardiovascular diseases,oncological diseases, renaland inflammatory diseasesor diseaseslinked to disordersofthe immune system such asallergies,rheumatoid arthritisorautoimmune diseases by beneficial
  • Theintestinalmicrobiotaist hecommunity ofmicro-organismsin the digestive tract,comprising mainly bacteria, as well as yeasts, parasites and viruses.When these communities live in balance, a condition called eubiosisisestablished.Thisisvery importantbecause it allows the different components ofthe intestinalmicrobiota to be functionally effective and above allto be synchronised with both each other and the other components of the intestinal ecosystem.
  • the microbiota is the set of microorganisms that characterise a particular biological fluid, be it human, environmental or animal.
  • changes in these species and this biodiversity have been associated with health or disease conditions (from diabetes and metabolic syndrome to cancer and neurodegenerative disorders, to simple well-being/wellness).
  • an intestine-brain axis associated with neurodegenerative diseases possibly influenced by the microbiota.
  • Other axes with other systems or organs have been described, including intestine-heart, -kidney, -liver, -thyroid, -immune systems.
  • This biodiversity is also influenced by lifestyles such as diet or physical activity.
  • probiotics and prebiotics in the known art have in many cases proved quite effective in modulating or stabilising the microbiota, but they are only successful in these activities after prolonged use over time, are rather expensive, and their effects are very different from one individual to another to whom they are administered.
  • the S100 proteins are a family of low molecular weight proteins that are present in vertebrates and are characterised by two calcium binding sites with a helix-loop -helix (EF-hand) structure. At least 21 types of S100 proteins have been found to exist.
  • S100 derives from the fact that these proteins, and particularly S100B protein, are 100% soluble in ammonium sulphate at neutral pH.
  • S100 proteins have a homodimer structure, that is they are two identical polypeptides bound together by non-covalent bonds. Although S100 proteins are structurally similar to calmodulins, they differ from these in that they are cell-specific, and are expressed in particular cells at different levels depending on environmental factors. In contrast, calmodulins are ubiquitous and universal Ca 2+ receptors, present in many cells.
  • S100 proteins are also distinguished from defensins, which are a family of proteins responsible for defending an organism against attack by potential pathogens and are a family of proteins with a highly conserved structure in mammals, insects and plants.
  • Defensins are structurally short peptides, around 29-34 amino acids long, of an amphipathic nature, which are able to insert themselves into cell membranes.
  • S100B is a commonly studied protein in the central nervous system and can be released in biological fluids, including milk, blood, saliva or urine, and can also be identified in faeces.
  • S100B protein has been quite well studied in the central nervous system and marginally in faeces, the role of this protein in the intestines has been little studied, except with regard to enteroglial cells.
  • S100B protein might have a non-pathological but beneficial physiological role in the intestinal lumen, being present in healthy individuals and less so in those who are ill [in this case with Crohn's disease, and/or IBD (Inflammatory Bowel Disease)].
  • the bioinformatic study has for the first time provided a method for evaluating the possibility of demonstrating an interaction between a protein present in the lumen and the set of bacterial proteins generated by the microbiota (proteome or rather metaproteome) “in silico’ ⁇
  • This scientific study (Orsini et al., 2020), which was also carried out with the involvement of the present inventors, demonstrated a role for S100B protein “in silico” and a difference in the interaction capacity of proteomes in healthy individuals compared to those who were ill.
  • S100B protein is present in milk specifically to modulate growth of the microbiota in the newborn and allow a harmonious development of the intestinal microflora.
  • proteins of the S100 family and in particular S100B protein, play a role in beneficially harmonising, stabilising, modulating, and regulating the intestinal and/or oral and/or pharyngeal and/or pulmonary and/or cutaneous and/or genital microbiota, in humans or animals, and that this is very useful in the treatment of various diseases.
  • a first object of the present invention is a protein belonging to the S100 family for use in the treatment or amelioration of diseases such as ulcerative colitis, Crohn's disease, IBD, neurological disorders, cardiovascular diseases, oncological diseases, kidney diseases, inflammatory diseases or immune system disorders such as allergies, rheumatoid arthritis or autoimmune diseases, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal and/or oral and/or pharyngeal and/or pulmonary and/or cutaneous and/or genital microbiota, whether in humans or animals.
  • diseases such as ulcerative colitis, Crohn's disease, IBD, neurological disorders, cardiovascular diseases, oncological diseases, kidney diseases, inflammatory diseases or immune system disorders such as allergies, rheumatoid arthritis or autoimmune diseases
  • diseases such as ulcerative colitis, Crohn's disease, IBD, neurological disorders, cardiovascular diseases, oncological diseases, kidney diseases, inflammatory diseases or diseases linked to disorders of the immune system, such as allergies, rheumatoid arthritis or autoimmune diseases
  • a further object of the present invention is a composition comprising a protein belonging to the S100 family, in particular S100B, in which the composition is a pharmaceutical, a dietary supplement, a medical device.
  • It is also an object of the present invention to provide a diagnostic kit for assessing the presence of S100B protein in the microbiota or intestinal lumen comprising an ELISA system which includes antibody and a detection system.
  • compositions and/or formulations of the invention may be prepared according to methods in the known art.
  • diseases such as ulcerative colitis, Crohn's disease, IBD, neurological disorders, cardiovascular diseases, oncological diseases, kidney diseases, inflammatory diseases or diseases linked to disorders of the immune system, such as allergies, rheumatoid arthritis or autoimmune diseases
  • a protein belonging to the S100 family typically means a protein preferably chosen from the group comprising S100A1 protein, S100A2 protein, S100A3 protein, S100A4 protein, S100A5 protein, S100A6 protein, S100A7 protein, S100A8 protein, S100A9 protein, S100A10 protein, S100A11 protein, S100A12 protein, S100A13 protein, S100A14 protein, S100A15 protein, S100A16 protein, S100B protein, S100G protein, S100P protein, S100Z protein; this group of proteins is illustrative and is not limited to the proteins mentioned.
  • the protein is S100B protein and/or its epitope and/or its functional and/or structural domain.
  • a further object of the present invention is S100B protein for use in the treatment of disorders of the intestinal microbiota.
  • the disorder of the intestinal microbiota is associated with Crohn's disease or IBD.
  • S100B protein is commercially available and can be derived from both animal and, surprisingly, also plant sources.
  • S100B protein may be either the pure protein or the protein obtained from extracts or by recombinant methodology preferably using bacteria, yeast or other cells, or by genetic engineering techniques, preferably gene therapy techniques or gene vectors enabling its expression in vivo, for example in the intestinal mucosa, and/or by the in vivo introduction of the DNA and/or RNA gene sequence of S100B, preferably through the use of liposomes.
  • S100B protein may be obtained from plant leaves or fruits of specific plants or from fungi or marine animal/plant organisms as revealed by bioinformatic analysis, chosen from, but not limited to, Spinacia oleracea; Artocarpus heterophyllus; Adansonia digitata; Carpinus fangiana; Durio zibethinus; Herrania umbratical; Salvia splendens; Helianthus annuus; Dendrobium catenatum; Musa acuminata; Olea europaea; Laccaria bicolor; Fistulina hepatica; Limulus polyphemus.
  • bioinformatic analysis chosen from, but not limited to, Spinacia oleracea; Artocarpus heterophyllus; Adansonia digitata; Carpinus fangiana; Durio zibethinus; Herrania umbratical; Salvia splendens; Helianthus annuus; Dendrobium catenatum; Musa acuminata; Olea europa
  • harmonisation of the intestinal microbiota is meant the function of beneficially balancing the intestinal microbiota, through a protein of the S100 family, as listed above.
  • stabilisation of the intestinal microbiota is meant the function of beneficially maintaining a stable intestinal microbiota, through a protein of the S100 family, as listed above.
  • modulation of the intestinal microbiota is meant the ability of a protein of the S100 family, as listed above, to harmoniously and beneficially alter the intestinal microbiota.
  • regulation of the intestinal microbiota, we mean the ability of a protein of the S100 family, as listed above, to make the composition of the intestinal microbiota regular in a beneficial sense.
  • diseases such as ulcerative colitis, Crohn's disease, IBD, neurological disorders, cardiovascular diseases, oncological diseases, kidney diseases, inflammatory diseases or diseases linked to disorders of the immune system, such as allergies, rheumatoid arthritis or autoimmune diseases
  • composition is typically meant a pharmaceutical-type composition, a dietary supplement or a medical device.
  • composition comprising a protein belonging to the S100 family, in particular S100B, in which the composition is a pharmaceutical, a dietary supplement or a medical device.
  • composition according to the present invention comprises S100B protein and/or its epitope and/or its functional and/or structural domain.
  • the composition according to the present invention comprises a protein of the S100 family and at least one physiologically acceptable excipient and in addition a prebiotic and/or a probiotic.
  • proteins of the S100 family can act as an enhancer of the activity of prebiotics and/or probiotics.
  • the at least one pharmaceutically acceptable excipient of the composition is chosen from the group comprising: diluents, binders, colouring agents, sweeteners, disinte grants, controlled release compounds, waxes, oils, paraffins, talc, titanium dioxide, cellulose.
  • the composition is in a form chosen from the group consisting preferably of an enema, a rectal and/or vaginal douche, a pessary, a suppository, a cream, an ointment, a lotion, an eyewash, or an oral formulation such as, for example, an aerosol, a mouthwash, a syrup, a capsule, a tablet, an orally dispersible tablet, a sachet, a powder, a granulate, or a solution in beverages, such as preferably thirst quenchers, energy drinks, fruit juices, or in milk and milk derivatives, including fermented derivatives such as yoghurt or the like or other ferments or fermented products.
  • an oral formulation such as, for example, an aerosol, a mouthwash, a syrup, a capsule, a tablet, an orally dispersible tablet, a sachet, a powder, a granulate, or a solution in beverages, such as preferably thirst quenchers, energy drinks
  • the ferment or fermented product according to the present invention comprises a protein of the S100 family, in particular S100B protein.
  • the fermented product covered by the present invention may be obtained by a method known in the art, by the various methods used in a small-scale, industrial or food industry context, such as Kombucha (https://it.wikipedia.org/wiki/ Kombucha) comprising the following steps:
  • a tea infusion typically 1-5 L
  • slightly sweetened typically between 2 and 10%, preferably 5%, e.g. sucrose or glucose or fructose,
  • a fermenter vessel to which mature Kombucha (typically between 3 and 15%, preferably 5%) and mother ferment (typically between 2 and 7%, preferably 5%) will be added
  • a preferred aspect of the invention is a mixture comprising a protein belonging to the S100 family, in particular S100B, and the most common physiologically acceptable excipients.
  • a preferred aspect of the present invention is a protein belonging to the S100 family, especially S100B protein, said composition comprising in particular:
  • S100B protein in an amount by weight between approximately 300 ng and 3 g, preferably between approximately 0.5 mg and approximately 50 mg, more preferably between approximately 1 mg and approximately 5 mg.
  • It is a further object of the present invention to provide a diagnostic kit for assessing the presence of S100B protein in the microbiota or intestinal lumen comprising an ELISA system including antibody and a detection system.
  • Said dosing regimen comprises the administration of a composition including S100B protein in tablet form 1-2 times a day, at least 5-8 hours apart, after meals, for a period of at least 10-30 days.
  • x concentration of S100B protein preferably in ng/ml
  • Indicators that can be used to assess the beneficial effect on the microbiota include the analysis of species and their relative representation (OTU), as well as biodiversity indices such as the beta diversity or alpha diversity value according to Shannon. More simply, the assay of particular species such as Lactobacilli or Bacteroides or Clostridia, or analysis of the F/B ratio in the species present in the microbiota, can also help in following the action of S100B on the intestinal microbiota, as well as on other microflora.
  • OTU relative representation
  • biodiversity indices such as the beta diversity or alpha diversity value according to Shannon.
  • the assay of particular species such as Lactobacilli or Bacteroides or Clostridia, or analysis of the F/B ratio in the species present in the microbiota, can also help in following the action of S100B on the intestinal microbiota, as well as on other microflora.
  • the protein used according to the invention is S100B.
  • S100B protein was administered orally to animals, faeces were collected and changes in the intestinal microbiota analysed. Specifically, 5 mg aliquots of purified S100B protein (Sigma Aldrich) were resuspended in sterile water at a final concentration of 1 milligram per millilitre, and 150 microlitres of this preparation was administered per os to three mice, 5 days per week, for a period of 4 weeks, according to methods known to those skilled in the art. Stool samples were collected over time and DNA extracted for microbiota analysis using standard protocols (Next Generation Sequencing) based on the gene sequence analysis of rDNA (16S) regions and analysed using standard bioinformatics methods (Illumina platform) according to procedures known to those skilled in the art.
  • Next Generation Sequencing Next Generation Sequencing
  • Faeces from different animals were collected over time and the biodiversity of the microbiota was assessed in relation to the concentration of the protein. Specifically, faeces from 36 animals were collected and the presence of S100B was measured by means of an enzyme-linked immunosorbent assay (ELISA), according to methods known to those skilled in the art. In parallel, DNA was extracted from the same faeces and microbiota analysis was carried out according to the methods known and reported above in Example 1.
  • ELISA enzyme-linked immunosorbent assay
  • the values of the biodiversity indices for the microbiota as observed in the different samples analysed are directly proportional to the values of SIOOB.
  • S100B increases, there is also a proportional increase in biodiversity.
  • the amounts of S100B protein present in faeces are indicative of the biodiversity of the intestinal microbiota, indicating the association between higher amounts of SIOOB and a favourable increase in harmonisation, modulation and stabilisation of the microbiota.
  • sequences were downloaded from a bioinformatics database and server for metagenomic analysis (MG-RAST) and compared with other sequences downloaded from other databases (NCBI) and from individuals suffering from other chronic/degenerative diseases in which the intestinal microbiota plays a part, as known to those skilled in the art.
  • F/B Firmicutes/Bacteroides
  • Clostridia Among the microorganisms found in the microbiota of patients with IBD and which have been associated with inflammatory damage are the Clostridia.
  • Clostridia Among the microorganisms found in the microbiota of patients with IBD and which have been associated with inflammatory damage are the Clostridia.
  • a further method of assessing the beneficial effect of S100B on the microbiota was to use mice genetically modified for the protein, and in particular Knock Out mice, that is mice from which the protein gene had been deleted from the animal genome. In the absence of S100B such animals show low levels of biodiversity in comparison with animals expressing the protein at high levels, and develop a microbiota with characteristics that tend to an increased risk for the development of IBD.
  • the harmonious structure of the intestinal microbiota has a significant beneficial role in protecting against various other diseases, including cardiovascular diseases, cancer, and neurological disorders.
  • This mechanism is nowadays explained through the gut-brain, gut- heart, gut-kidney, gut-thyroid axes, according to which knowledge, imbalances in the biodiversity of the microbiota are also reflected on other distant organs, through various hypothesised mechanisms, including the production of metabolites, the induction of inflammatory processes, and interaction with the enteric nervous system. Attempts are therefore also being made to treat such diseases through the introduction of specific beneficial microbial species, called probiotics, or substances that act as prebiotics by entering the gut directly to modify the biodiversity of the microflora.
  • S100B The ability of S100B to interfere with the structure of the microbiota, favouring harmonious biodiversity, therefore also has beneficial, preventive and curative implications for various other diseases that may benefit from harmonisation of the intestinal microbiota.
  • S100B was administered to three volunteer individuals and the effects on the microbiota were assessed after a period of time.
  • fruits of the species Durio zibethinus were chosen as the source of protein on the basis of bioinformatic analysis, and in particular material that had undergone sublimation according to known freezing and drying methods in order to maximise the preservation of protein structures and material stability.
  • the presence of S100B was verified by ELISA tests. This plant material was administered in the morning on an empty stomach according to the dosage of 5 grams per day for a total of 12 days.
  • Faecal samples were collected from each of the treated individuals by taking faecal material with a flocked swab, immediately placed in transport medium and kept at 4°C, according to known protocols for studying the microbiota. These samples were taken before the start of treatment and at the end of administration. DNA extraction and microbiota analysis by 16S gene sequencing for NGS was performed on these samples, according to known protocols.
  • Spinacia oleracea has also been found among the various plants that produce S100B;, the administration of this, for example in the form of powder obtained by sublimation after freezing and drying or of lipoprotein derivatives such as thylakoids, has been tested and surprisingly shown to produce precisely the various beneficial effects induced by S100B protein on the microbiota, in vitro or in vivo, and in particular harmonic rearrangement and beneficial synchro-modulation of the intestinal microbiota, increased production of isobutyrate and isovalerate, suppression of C. perfringens, and antineoplastic activity in mouse models fed with powder, characterised by the presence of S100B, obtained from this plant by sublimation processes.
  • An intestinal-renal axis has also been described among the various axes through which the microbiota acts on different organs.
  • the metabolic activity of the intestinal microbiota is modulated by biodiversity, but the expansion of certain bacterial families that produce uricases and uraemic toxins such as indole and p-cresyl has also been associated with kidney diseases such as chronic kidney disease, kidney failure, diabetic kidney disease and various clinical conditions (e.g. Chronic Kidney Disease, Diabetes Kidney Disease, end-stage kidney disease, Type 2 Diabetes).
  • Harmonisation of the microbiota through the administration of S100B can be achieved not only using plant-derived products, but also recombinant DNA techniques. These techniques can be applied through various methods in genetic engineering and gene therapy, through the administration of the protein sequence or a portion thereof in the form of RNA, DNA or through laboratory-built genetic vectors that can be transferred directly onto prokaryotic cells in vivo or in vitro, such as bacteria or eukaryotic cells such as yeast or insect cells. Such cells capable of producing S100B can then be used for expressing and purifying S100B protein for further use, or directly to produce it in vivo as forms of engineered probiotics, for example enterobacteria such as lactobacilli or Escherichia coli.
  • enterobacteria such as lactobacilli or Escherichia coli.
  • S100B protein in this example a genetic construct enabling the expression of S100B protein in a bacterial cell has been developed.
  • the gene encoding the human S100B protein (e.g. using nm_006272.3 and xm_017028424.2 transcription) was synthesised and cloned into a vector (e.g. PET28A, between the NDEI and SACI sites) by inserting a stop codon according to procedures known to those skilled in the art.
  • S100B protein was generated with an 8-histidine N-terminal tail and a cut site for thrombin protease in order to facilitate its subsequent purification, according to protocols known from biochemistry and molecular biology.
  • the S100B-pET28a vector was introduced (by means of heat shock) into the E. coli Shuffle strain, the clones were selected and expanded, demonstrating the production of S100B which could be purified by chromatography and was capable of reacting to specific antibodies (western blot), showing a band between 15 KDa and 10 KDa on electrophoresis, which corresponds to the theoretical weight of approximately 13 KDa for S100B protein.
  • the S100B protein was also very pure (over 95%), and this was further improved by subsequent techniques.
  • S100B protein was successfully expressed in an organism by means of genetic engineering techniques and affinity- purified, achieving a satisfactory yield (approximately 12 mg of protein per litre of bacterial culture).
  • a recombinant S100B protein and also a recombinant E. coli that is a bacterium belonging to a species of Enterob acteriaceae naturally present in the intestinal microbiota, but capable of producing S100B protein following the introduction of the gene by genetic engineering methods, were obtained.
  • Giaca Another fruit found to be positive for S100B is Giaca, from a plant belonging to the Rosidae species, Artocarpus heterophy llus.
  • S100B protein was found at concentrations ranging from 4 to over 48 ng/ml in products obtained from dried or freeze-dried or sublimated fruits.
  • the protein for example for the species Durio zibethinus, a plant and several fruits, both fresh and vacuum- packed, were obtained directly by importing them from their native lands, but the protein was not always present, neither had it retained intact and recognisable its three-dimensional structure of the protein domains of interest.
  • this species for example, it was found in the fresh fruit at concentrations of approximately 3.40 ng/ml and also in the leaves at approximately 4.37 ng/ml, but not in the peel or seed; moreover the protein was particularly enriched in sublimated fruit preparations, reaching as high as 27 ng/ml, but not in all derivatives obtained by drying or freeze-drying.
  • the protein was also observed in amounts exceeding 4 ng/ml in preparations obtained by freeze-drying as well as by sublimation.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une protéine appartenant à la famille S100 destinée à être utilisée dans le traitement ou le soulagement de maladies telles que la rectocolite hémorragique, la maladie de Crohn, les MICI, les troubles neurologiques, les maladies cardiovasculaires, les maladies oncologiques, les maladies rénales, les maladies inflammatoires ou les maladies liées à des troubles du système immunitaire, par l'harmonisation et/ou la stabilisation et/ou la modulation et/ou la régulation bénéfiques du microbiote intestinal et/ou buccal et/ou pharyngé et/ou pulmonaire et/ou cutané et/ou génital, chez les humains ou les animaux ; une composition comprenant une protéine appartenant à la famille S100 destinée à être utilisée dans le traitement ou le soulagement de maladies telles que la rectocolite hémorragique, la maladie de Crohn, les MICI, les troubles neurologiques, les maladies cardiovasculaires, les maladies oncologiques, les maladies rénales, les maladies inflammatoires ou les maladies liées à des troubles du système immunitaire, par l'harmonisation et/ou la stabilisation et/ou la modulation et/ou la régulation bénéfiques du microbiote intestinal ; un kit de diagnostic permettant d'évaluer la présence de la protéine S100B dans le microbiote intestinal ; un régime de dosage pour une protéine appartenant à la famille S100, en particulier la protéine S100B ; un procédé permettant d'identifier si une protéine de la famille S100 ou une autre protéine ou un autre peptide est utile en tant qu'harmonisateur et/ou stabilisant et/ou modulateur et/ou régulateur du microbiote intestinal.
PCT/IB2022/055889 2021-08-04 2022-06-24 Protéine ou ses compositions pour une utilisation dans le traitement du microbiote WO2023012540A1 (fr)

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Citations (1)

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WO2020069130A2 (fr) * 2018-09-27 2020-04-02 The Scripps Research Institute Remodelage chimiothérapeutique du microbiome intestinal

Patent Citations (1)

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WO2020069130A2 (fr) * 2018-09-27 2020-04-02 The Scripps Research Institute Remodelage chimiothérapeutique du microbiome intestinal

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